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Stem Cell Therapy Market Evaluation of Industry Trends, Growth Drivers and Forecast To 2025 NeighborWebSJ – NeighborWebSJ

Stem Cell Therapy Market: Snapshot

Of late, there has been an increasing awareness regarding the therapeutic potential of stem cells for management of diseases which is boosting the growth of the stem cell therapy market. The development of advanced genome based cell analysis techniques, identification of new stem cell lines, increasing investments in research and development as well as infrastructure development for the processing and banking of stem cell are encouraging the growth of the global stem cell therapy market.

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One of the key factors boosting the growth of this market is the limitations of traditional organ transplantation such as the risk of infection, rejection, and immunosuppression risk. Another drawback of conventional organ transplantation is that doctors have to depend on organ donors completely. All these issues can be eliminated, by the application of stem cell therapy. Another factor which is helping the growth in this market is the growing pipeline and development of drugs for emerging applications. Increased research studies aiming to widen the scope of stem cell will also fuel the growth of the market. Scientists are constantly engaged in trying to find out novel methods for creating human stem cells in response to the growing demand for stem cell production to be used for disease management.

It is estimated that the dermatology application will contribute significantly the growth of the global stem cell therapy market. This is because stem cell therapy can help decrease the after effects of general treatments for burns such as infections, scars, and adhesion. The increasing number of patients suffering from diabetes and growing cases of trauma surgery will fuel the adoption of stem cell therapy in the dermatology segment.

Global Stem Cell Therapy Market: Overview

Also called regenerative medicine, stem cell therapy encourages the reparative response of damaged, diseased, or dysfunctional tissue via the use of stem cells and their derivatives. Replacing the practice of organ transplantations, stem cell therapies have eliminated the dependence on availability of donors. Bone marrow transplant is perhaps the most commonly employed stem cell therapy.

Osteoarthritis, cerebral palsy, heart failure, multiple sclerosis and even hearing loss could be treated using stem cell therapies. Doctors have successfully performed stem cell transplants that significantly aid patients fight cancers such as leukemia and other blood-related diseases.

Global Stem Cell Therapy Market: Key Trends

The key factors influencing the growth of the global stem cell therapy market are increasing funds in the development of new stem lines, the advent of advanced genomic procedures used in stem cell analysis, and greater emphasis on human embryonic stem cells. As the traditional organ transplantations are associated with limitations such as infection, rejection, and immunosuppression along with high reliance on organ donors, the demand for stem cell therapy is likely to soar. The growing deployment of stem cells in the treatment of wounds and damaged skin, scarring, and grafts is another prominent catalyst of the market.

On the contrary, inadequate infrastructural facilities coupled with ethical issues related to embryonic stem cells might impede the growth of the market. However, the ongoing research for the manipulation of stem cells from cord blood cells, bone marrow, and skin for the treatment of ailments including cardiovascular and diabetes will open up new doors for the advancement of the market.

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Global Stem Cell Therapy Market: Market Potential

A number of new studies, research projects, and development of novel therapies have come forth in the global market for stem cell therapy. Several of these treatments are in the pipeline, while many others have received approvals by regulatory bodies.

In March 2017, Belgian biotech company TiGenix announced that its cardiac stem cell therapy, AlloCSC-01 has successfully reached its phase I/II with positive results. Subsequently, it has been approved by the U.S. FDA. If this therapy is well- received by the market, nearly 1.9 million AMI patients could be treated through this stem cell therapy.

Another significant development is the granting of a patent to Israel-based Kadimastem Ltd. for its novel stem-cell based technology to be used in the treatment of multiple sclerosis (MS) and other similar conditions of the nervous system. The companys technology used for producing supporting cells in the central nervous system, taken from human stem cells such as myelin-producing cells is also covered in the patent.

Global Stem Cell Therapy Market: Regional Outlook

The global market for stem cell therapy can be segmented into Asia Pacific, North America, Latin America, Europe, and the Middle East and Africa. North America emerged as the leading regional market, triggered by the rising incidence of chronic health conditions and government support. Europe also displays significant growth potential, as the benefits of this therapy are increasingly acknowledged.

Asia Pacific is slated for maximum growth, thanks to the massive patient pool, bulk of investments in stem cell therapy projects, and the increasing recognition of growth opportunities in countries such as China, Japan, and India by the leading market players.

Global Stem Cell Therapy Market: Competitive Analysis

Several firms are adopting strategies such as mergers and acquisitions, collaborations, and partnerships, apart from product development with a view to attain a strong foothold in the global market for stem cell therapy.

Some of the major companies operating in the global market for stem cell therapy are RTI Surgical, Inc., MEDIPOST Co., Ltd., Osiris Therapeutics, Inc., NuVasive, Inc., Pharmicell Co., Ltd., Anterogen Co., Ltd., JCR Pharmaceuticals Co., Ltd., and Holostem Terapie Avanzate S.r.l.

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Stem Cell Therapy Market Evaluation of Industry Trends, Growth Drivers and Forecast To 2025 NeighborWebSJ - NeighborWebSJ

Injectable hydrogel with MSNs/microRNA-21-5p delivery enables both immunomodification and enhanced angiogenesis for myocardial infarction therapy in…

INTRODUCTION

Myocardial infarction (MI) remains one of the leading causes of death worldwide. The inflammatory response caused by MI sets the stage for fibrous tissue and often progresses to chronic heart failure (1), resulting in a more than 50% 5-year mortality after MI (2). An immunomodulation strategy, which prevents an excessive inflammatory response, can be beneficial to reduce scar tissue formation. Immunomodulation alone can likely prevent ongoing damage but fails to restore the compromised heart function. Promoting angiogenesis in the infarct area has the potential to reperfuse and salvage the surviving ischemic myocardium (3). Therefore, we hypothesize that long-term improvements in heart function after MI can be achieved by the combination of resolving inflammation and promoting angiogenesis in the infarct area.

Various therapeutics, such as cell transplant, exosomes, and nucleic acids, have been explored to treat MI and restore cardiac function, with varying degrees of success. Cell transplantation could enhance the functions of the infarcted heart (4), but only cardiomyocytes derived from pluripotent stem cells have been shown to engraft and generate functional myocardium (5). Limitations in cell sources, potential immune responses, and rigorous regulations hinder the clinical translation of cell-based therapies. Several studies have shown that cell-derived exosomes may be effective in treating cardiovascular diseases (6). However, there are obvious variations in exosomes resulting from multiple factors such as cell phenotype, preparation procedure, and exosome storage conditions (7). MicroRNAs (miRNA) are appealing genetic tools to stimulate cardiac performance, as they could regulate the levels of multiple genes simultaneously. Recently, it has been suggested that the cardiovascular system is regulated via a miRNA network (8). High-throughput screening work revealed that miRNAs, particularly microRNA-21-5p (miR-21-5p), are highly expressed in endothelial cells and stimulate angiogenesis by targeting antiangiogenic genes (9). miRNAs have a unique capacity to simultaneously promote the secretion of multiple endogenous molecules that might enhance vessel regeneration in the ischemic tissue. Negatively charged miRNAs typically cannot cross the cell membrane without a transfection agent. In addition, miRNAs are relatively unstable and can be degraded rapidly in vivo (10). Thus, vectors that protect and deliver miRNAs into cells are crucial to improve the efficacy of miRNA therapy.

Mesoporous silica nanoparticles (MSNs) have been developed as a promising vector for miRNA delivery because of their many excellent properties, such as good biocompatibility and high transfection efficiency. Moreover, studies have shown that inflammation can be modulated by phagocytosis of micro/nanomaterials, such as liposomes (11), polymer particles (12, 13), and inorganic particles (14). Macrophages play a central role in regulating infarct-induced inflammation because they adopt proinflammatory (M1) phenotypes. In this study, we found that MSNs showed great potential in inhibiting M1 polarization following inflammation both in vitro and in vivo (see details in Results). Therefore, we engineered an MSN/miR-21-5p complex by combining MSN, a potential anti-inflammatory nanomaterial, and miR-21-5p, a proangiogenic therapeutic.

RNA interference (RNAi) is a promising therapeutic approach for various diseases (15). An important aspect in RNAi delivery system design is to ensure precise spatiotemporal release (1621). Uncontrolled delivery of miRNA in the heart could result in sudden arrhythmia, as reported by Gabisonia et al. (22). In addition, studies have also identified that a big challenge for RNAi-based therapeutics is to achieve highly localized RNAi delivery (16, 18, 19, 23). Drug release from conventional hydrogels (24, 25) is controlled by passive diffusion and often results in off-target effects (26). In contrast, MSN/miR-21-5p complexes were conjugated within an injectable hydrogel matrix via pH-responsive bonds to form Gel@MSN/miR-21-5p, which accurately released MSN/miR-21-5p complexes only in the acidic infarct area.

Here, we designed an injectable hydrogel loaded with MSN/miR-21-5p complexes (Gel@MSN/miR-21-5p) to deliver miR-21-5p in a two-stage mechanism: The first stage comprises pH-triggered on-demand delivery of MSN/miR-21-5p complexes from the hydrogel matrix in acidic infarct areas, and the second stage involves intracellular delivery of miR-21-5p from MSN/miR-21-5p complexes. This drug delivery system is designed to harness the synergy of inflammation suppression and angiogenesis enhancement in treating MI, the efficacy of which was evaluated in a clinically relevant MI swine model.

Amino (-NH2) and trimethylamine [-N(CH3)3, TMA] functionalized MSNs (MSN-NH2-TMA) were first synthesized (fig. S1A), which had positive charges for miRNA loading (fig. S1B). The miRNA-loading capacity of the MSN-NH2-TMA complex was quantitatively evaluated by a gel retardation assay and potential measurements (fig. S1C), which showed complete encapsulation of miRNA when the mass ratio between the MSN-NH2-TMA complex and miRNA increased to 10:1. Subsequent studies were all using MSN/miRNA complexes with this ratio. Direct evidence of miRNAs loading in MSNs was also provided by transmission electron microscopy (fig. S1D) and energy-dispersive x-ray spectroscopy (EDS) analysis (fig. S1E), which revealed obvious miRNAs residing in MSN pores and signals corresponding to the element P from loaded miRNAs.

Gel@MSN/miR-21-5p was fabricated by mixing the MSN/miR-21-5p complex aqueous solution (30 wt%) with an aqueous solution of -CD (66.7 mg/ml) and aldehyde-capped polyethylene glycol (PEGCHO; 66.7 mg/ml). The hydrogel matrix had a porous structure with pore sizes of around 10 m in diameter and MSN/miR-21-5p complexes covering the wall surface (fig. S1F). Scanning electron microscope image of the injectable colloidal hydrogel (Gel@MSN) showed plenty of MSNs conjugated in the hydrogel (red arrows). The presence of MSNs was also confirmed by EDS, which showed an obvious elemental signal of Si (fig. S1G). Hydrogel formation resulted from two interactions (fig. S2): (i) hydrophobic interaction between cyclodextrins (CDs) along the PEGCHO chains (27) and (ii) Schiff base between the NH2 group from MSNs and the aldehyde (CHO) group from PEGCHO/CD complexes. The stepwise gelation was confirmed by comparing different gelation processes between the MSN/PEGCHO/CD (group with both Schiff base and hydrophobic interaction) and control groups (PEG/MSN, group without hydrophobic interactions and Schiff bases; PEG/MSN/CD, group only with hydrophobic interactions) (fig. S3A) as well as the different rheological characterization of the resulting hydrogels (fig. S3, B and C). The cross-linking relies on hydrophobic and Schiff interactions, which are relatively weaker than conventional covalent bonds. The liquid-gel transition takes approximately 5 min, after which point the hydrogel is injected into the infarct area. The weak interaction allows the hydrogel to exhibit a shear-thinning property, which permitted it to switch from hydrogel to fluid during injection and subsequently formed a firm hydrogel at the MI area along with the further cross-linking process (fig. S3D). The retention property of the hydrogel in the beating heart was also evaluated. During bench testing, hydrogel (labeled with blue dye) was injected into myocardium tissue, and no detachment or cracks were observed between the hydrogel and tissue after bending, distorting, long-time immersing underwater, or stretching (fig. S3E).

The MSN/miRNA complexes were conjugated onto an injectable hydrogel by Schiff bonds. The Schiff base bond is stable at pH 7.4 but is disrupted in an acidic environment (pH 6.8) (fig. S3, F to H), enabling an on-demand release of MSN/miRNA (step 3 in fig. S2B) (28, 29). The 1H NMR (nuclear magnetic resonance) of 1,6-diaminohexane (HDA) functionalized PEG with Schiff base in between (HDA-PEG-HDA) after incubation in phosphate-buffered saline (PBS) buffer with pH 7.4 (red line) and pH 6.8 (black line) for 24 hours, which presented a clear proton peak of aldehyde only in the pH 6.8 treated group, demonstrating the high stability of Schiff base bonds at pH 7.4 and its gradual cleavage to form an aldehyde group at an acidic environment (fig. S3F). The gel permeation chromatography results of HDA functionalized PEG with Schiff base in between (HDA-PEG-HDA) after incubation in PBS buffer with pH 7.4 (i) and pH 6.8 (ii) for 24 hours, which presented an obvious drop of molecule weight only in the pH 6.8 treated group. Moreover, the molecule weight loss is close to twice the molecule weight of HDA, indicating the separation of HDA with PEG, due to the break of Schiff base (fig. S3H). These data comprehensively demonstrated the high stability of Schiff base bonds at pH 7.4 and its gradual cleavage at the slightly acidic environment.

The on-demand release profile was characterized in PBS buffer with pH 7.4 and pH 6.8 [which respectively simulated the microenvironment of healthy tissue (pH 7.4) and infarcted myocardium (pH 6.8)] (30, 31). There was a sustained release of MSN/miRNA complexes from the hydrogel matrix with ~75% release after 7 days at pH 6.8 (fig. S3I). In contrast, only ~6% MSN/miRNA was released from the hydrogel after 7 days at pH 7.4, which could be attributed to the diffusion of MSN/miR-21-5p at the different hydrogel degradation rates under different pH conditions (fig. S3I). The miRNA release from the MSN/miRNA complexes is presented in fig. S3J, which shows that a further decrease in the pH value to 5 (simulated intracellular endosomes and lysosomes environment) (32) could trigger miRNA release from MSN/miRNA complexes, leading to a cumulative release of miRNA of up to 60% over 48 hours.

Hydrogel degradation in vitro was monitored by measuring dry weight loss as a function of time following incubation in PBS (pH 6.8) at 37C (fig. S3K). As shown in fig. S3K, Gel@MSN/miRNA lost approximately 93% of the initial gel mass within 20 days. For in vivo measurements, the PEG frame of the hydrogel was labeled by fluorescent dye. Following injection, the fluorescence signal in the injected area was detected at the indicated time points. Figure S4 shows that the fluorescence signal decay is down to 67% at day 3 and 16% at day 14. At day 28, we could not detect any fluorescence signal, indicating that the hydrogel was completely degraded.

The retention of MSNs in vivo was monitored by fluorescence in vivo imaging system (IVIS) imaging at the indicated time points. Figure S5 shows that the fluorescence signal decay is down to 54% at day 3, 18% at day 14, and 2% at day 28, indicating that accumulation of MSNs was gradually decreased at tissue. At day 36, no positive signal was observed, indicating that almost no residual MSNs could be detectable at tissue.

To assess the in vitro uptake of the MSN/miR-21-5p complex by endothelial cells, the miR-21-5p was labeled with Cy3 (orange-red), the MSNs were labeled with fluorescein isothiocyanate (FITC) (green), and the cell nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI) (blue). For in vitro uptake analysis, endothelial cells were cocultured with MSN/miR-NC complexes or MSN/miR-21-5p complexes. The confocal images and quantification analysis showed that MSN/miR-21-5p complexes showed high transfection efficiency of miR-21-5p and resulted in an approximately 37-fold enhanced miR-21-5p levels in endothelial cells compared to that of control cells (Fig. 1, A and B). Representative profiles from the flow cytometry analysis revealed that the CD31 expression level was 96.5% in endothelial cells (Fig. 1C). Flow cytometry analysis indicated that more than 70% of endothelial cells internalized the MSN/miR-21-5p complexes (identified by the CD31+Cy3+) (Fig. 1D). The cytokine levels were determined by Western blot and real-time quantitative polymerase chain reaction (PCR) assay. Figure 1E shows that compared to the endothelial cell group and the MSN/miR-NCtreated group, MSN/miR-21-5p significantly promoted the expression of proangiogenic cytokines (VEGFA and PDGF-BB) from endothelial cells. MSN/miR-21-5ptreated endothelial cells also had increased capillary tube network formation (as measured by branch points and total tube length via tube formation assay) (as shown in Fig. 1G). We then simulated serum-free and hypoxic infarct-like conditions in vitro to assess the protective effect of MSN/miR-21-5p on the hypoxia/ischemia-induced cardiomyocyte apoptosis (Fig. 1H). The cardiomyocytes were exposed to a combination of ischemic/hypoxic conditions for 24 hours. Endothelial cells were pretreated with MSN/miR-21-5p or MSN/miR-NC and then cocultured with cardiomyocytes subjected to hypoxia/ischemia. Notably, at 24 hours of coculture, we found that coculture with MSN/miR-21-5ptreated endothelial cells reduced the apoptosis of hypoxia/ischemia-induced cardiomyocytes. This correlated with increased secretion of proangiogenic cytokines (VEGFA and PDGF-BB) from endothelial cells treated with MSN/miR-21-5p (Fig. 1, I and J). Previous studies demonstrated that VEGFA or PDGF-BB inhibits apoptosis (33, 34). These data may suggest that miR-21-5pinduced expression of proangiogenic factors in endothelial cells could prevent cardiomyocytes from undergoing apoptosis under ischemic and hypoxic conditions.

(A) In vitro uptake of the MSN/miR-21-5p complex by adherent endothelial cells (ECs) and macrophages (MCs). (B) In vitro transfection efficiency of miR-21-5p was determined by quantifying the miRNA level using real-time quantitative PCR. (C) Representative flow cytometry analysis of CD31 levels in ECs and F4/80 levels in MCs. (D) In vitro uptake of the MSN/miR-21-5p complex by ECs and MCs was determined by quantifying the double-positive cells (CD31 or F4/80 and Cy3) using flow cytometric analysis. The protein expression levels of VEGFA and PDGF-BB in endothelial cells (E) and tumor necrosis factor- (TNF-), interleukin-1 (IL-1), and IL-6 in macrophages (F) were determined by the real-time quantitative PCR and Western blot analysis. (G) The endothelial cells that formed three-dimensional (3D) capillary-like tubular structures were evaluated at indicated times (8 and 16 hours). (H) Schematic diagram of the experimental setup. TUNEL, terminal deoxynucleotidyl transferasemediated deoxyuridine triphosphate nick end labeling. (I) Apoptosis-positive cardiomyocytes from these treatment groups were further quantified. (J) Protein levels of secreted proangiogenic factors were determined by enzyme-linked immunosorbent assay (ELISA) analysis of cell supernatants from the MSN/miRNA-treated ECs (scale bars, 50 m). *P < 0.05 and ***P < 0.01. All experiments were carried out in triplicate. n = 5 per group. The data are shown as means SD. Photo credit: Yan Li, Shanghai Ninth Peoples Hospital, College of Stomatology, Shanghai Jiao Tong University School of Medicine, Shanghai 200011, China.

To understand the in vitro immunomodulatory effect of MSN/miR-21-5p complexes, MSN/miR-NC complexes or MSN/miR-21-5p complexes were cocultured with isolated macrophages. MSNs were labeled with FITC (green), and miR-21-5p was labeled with Cy3 (red). Representative profiles from the flow cytometry analysis revealed that the F4/80 expression level was 98.2% in isolated macrophages (Fig. 1C). The confocal images and quantification analyses showed that MSN/miR-21-5p complexes had high uptake efficiency in macrophages. Flow cytometry analysis indicated that more than 80% of macrophages took up the MSN/miR-21-5p complexes (identified by the F4/80+Cy3 + staining pattern) (Fig. 1D). We then examined whether the uptake of the MSN/miR-21-5p complexes by macrophages could reduce the inflammatory response. For this purpose, a proinflammatory response was induced by injection of lipopolysaccharide (LPS), a potent inducer of inflammatory response (35), into the peritoneum of mice, and macrophages from the treated mice were collected. Figure 1F shows that the inflammation of the LPS-treated macrophages (LPS-macrophages) was markedly suppressed following uptake of the MSN/miR-21-5p complexes, as indicated by the notable decrease in the expression of tumor necrosis factor- (TNF-), interleukin-1 (IL-1), and IL-6, which are typical cytokines involved in the inflammatory response. These data suggest that the MSN/miR-21-5p complexes released from Gel@MSN/miR-21-5p simultaneously reduced proinflammatory cytokines and increased proangiogenic factors in vitro. The enhanced proangiogenic factors from endothelial cells could effectively prevent cardiomyocytes from apoptosis under ischemic and hypoxic conditions.

To obtain insight into the mechanism by which the MSN/miR-21-5p complex acts on macrophages to modulate the immune response, we performed a proteome analysis of protein alterations in macrophages. We collected three replicates of LPS-induced macrophages (inflammatory stage macrophages) treated with MSN/miR-NC, MSN/miR-21-5p, or pure MSNs. Untreated LPS-macrophages were used as a negative control. We used a label-free quantitative proteomic approach. Hierarchical clustering analysis of the data revealed that the protein expression patterns of the three treatment groups (MSN/miR-NC, MSN/miR-21-5p, or pure MSNs) were obviously different from that of LPS-macrophages without treatment, while the protein expression patterns of the three groups were similar (Fig. 2A). This consistently indicated that the function of immunomodulation originates from the MSNs themselves.

(A) A heatmap of selected proteins representing major altered signaling pathways in three datasets of macrophages treated with MSNs, MSN/miR-NC, or MSN/miR-21-5p complexes. Macrophages with no treatment were used as a negative control. The color bar indicates normalized z score intensity-based absolute quantification. (B) KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis of both up- and down-regulated pathways in macrophages after MSN treatment. The most significant pathways in the phosphoproteome are plotted on the x axis as the log10 of the P value, compared with the proteome. (C) KEGG pathway map of Toll-like signaling pathway. Proteins shown with red backgrounds are down-regulated in macrophages after MSN complex treatments when compared with macrophages with no treatment, as determined by pathway analysis. (D) Real-time quantitative PCR and Western blot analysis of TLR1, TLR2, TLR3, TLR8, P-NFB, TNF-, IL-1, and IL-6 protein content alteration in macrophages after treatment with MSNs, MSN/miR-NC, or MSN/miR-21-5p complexes. (E) Real-time quantitative PCR and Western blot analysis of P-NFB, TNF-, IL-1, and IL-6 protein content alteration in MSN/miR-21-5p complextreated macrophages that overexpress TLR2 with the TLR2 overexpression vector. ***P < 0.01. n = 3 per group. The data are shown as means SD. Photo credit: Yan Li, Shanghai Ninth Peoples Hospital, College of Stomatology, Shanghai Jiao Tong University School of Medicine, Shanghai 200011, China.

Current reports showed that the reduced inflammatory response elicited by MSN resulted from the reduction of transcription factor nuclear B (NFB), caspase-3, and IL-12 (36). The NFB signaling plays a major role in innate immunity and inflammatory responses. It was shown that the NFB signaling pathway plays important roles in MSN-regulated inflammation (37), but the exact mechanism leading to this effect was still obscure.

The present study used the GSEA (gene set enrichment analysis) method to examine the distribution of the functionally related KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway gene sets within the ranked gene list. GSEA showed that there were groups of genes negatively correlated with the immune response after MSN treatments. The majority of genes that were differentially expressed in macrophages after MSN treatments were enriched in several pathways, such as neutrophil degranulation, Toll-like receptor (TLR) signaling pathway, and MyD88 deficiency (Fig. 2B). TLR activation and MyD88 (a downstream adapter of TLR pathways) play important roles in the NFB signaling pathway (37), stimulation of which may lead to activation of NFB signaling and eventually lead to proinflammatory responses and progression to fibrous tissues (38, 39).

We found that abundance of proteins (TLR1, TLR2, TLR4, TLR3, TLR7, TLR8, TLR9, CD14, RAC1, and TAB1) involved in TLR signaling was down-regulated in macrophages after MSN treatments, indicating that TLR signal transduction pathway activity decreased in response to MSN treatment (Fig. 2C). The most significantly down-regulated genes are TLR1, TLR2, TLR3, and TLR8, which had more than threefold change.

To gain further insight into the mechanism by which MSNs modulated the immune response through the TLR signaling pathway, we examined protein alterations of TLR1, TLR2, TLR3, and TLR8 within macrophages after MSN treatment (Fig. 2D). We found that the mRNA and protein expressions of TLR1, TLR2, TLR3, and TLR8 cytokines were substantially lower in all MSN-treated groups. Meanwhile, NFB signaling pathway and downstream proinflammatory cytokines (TNF-, IL-1, and IL-6) were inhibited, which is consistent with previous findings that TLRs act as primary sensors that elicit innate immune responses and activate NFB signaling (Fig. 2D). Among the known TLRs, TLR2 has been characterized extensively as an inducer of proinflammatory cytokines. To determine whether MSNs modulated the immune response by down-regulating TLR2, we first treated macrophages with MSNs and then transfected MSN-treated macrophages with a TLR2 overexpression plasmid vector or empty vectors. We found that the NFB signaling pathway was up-regulated in macrophages transfected with the TLR2 overexpression vector compared to the empty vector control group. Consistently, the amounts of TNF-, IL-1, and IL-6 protein in macrophages were increased by transfection with the TLR2 overexpression vector (Fig. 2E). These comprehensive data suggest that MSNs modulated immune response through down-regulating TLR2, which inhibited the activation of NFB signaling and subsequently decreased the release of proinflammatory cytokines (TNF-, IL-1, and IL-6) (fig. S6).

To obtain insight into the mechanism underlying miR-21-5penhanced angiogenesis, we performed a proteogenomic analysis of protein alterations in endothelial cells after miR-21-5p treatment. We collected three replicates of endothelial cells after treatment with MSN/miR-NC or MSN/miR-21-5p. We applied a label-free quantitative proteomic approach. Hierarchical clustering analysis of the data revealed that the genes could be assigned into two groups based on their protein expression patterns, and the assigned groups matched with the groups by treatment (Fig. 3A). GSEA revealed that there were groups of genes positively correlated with angiogenesis after MSN/miR-21-5p treatment. KEGG analysis suggested that the MSN/miR-21-5p treatment groups were positively associated with key angiogenic signaling pathways (Fig. 3B). Compared to MSN/miR-NCtreated endothelial cells, MSN/miR-21-5ptreated endothelial cells had a larger number of proteins enriched in pathways such as vascular endothelial growth factor (VEGF) signaling pathway and platelet-derived growth factor (PDGF) signaling pathway (Fig. 3B). VEGF is the major mediator in endothelial cells and is considered to be a crucial signal transducer in angiogenesis. The binding of VEGF to the VEGF receptor leads to a cascade of signaling pathways, including ERK-MAPK (extracellular signalregulated kinase/mitogen-activated protein kinase) signaling, which particularly plays a central role in angiogenesis. Therefore, we focused on ERK-MAPK signaling in MSN/miR-21-5ptreated endothelial cells and found that the levels of phospho-Erk1/2, phospho-FAK, phospho-P38, phospho-AKT, VEGFA, and PDGF-BB were up-regulated in the MSN/miR-21-5p treatment group compared to the MSN/miR-NC group, indicating that miR-21-5p could enhance VEGFA expression and subsequently lead to ERK-MAPK signaling activation (Fig. 3C).

(A) A heatmap of selected proteins representing strongly altered signaling pathways in three datasets of endothelial cells treated with MSN/miR-NC or MSN/miR-21-5p complexes. (B) KEGG pathway analysis of both up- and down-regulated pathways in endothelial cells after MSN/miR-21-5p complex treatment. (C) Western blot analysis of changes in SPRY1, P-ERK1/2, P-FAK, P-p38, P-AKT, VEGFA, and PDGF-BB protein content alteration in endothelial cells after treatment with the MSN/miR-21-5p complex. (D) The effect of MSN/miR-21-5p or MSN/miR-NC on SPRY1 mRNA levels (left) and SPRY1 protein levels (right) in endothelial cells. (E) Schematic diagram illustrating the design of luciferase reporters with the WT SPRY1 3 untranslated region (WT 3UTR) or the site-directed mutant SPRY1 3UTR (3UTR-Mut). (F) The effect of MSN/miR-21-5p on luciferase activity in endothelial cells transfected with either the WT SPRY1 3UTR reporter (left) or the mutant SPRY1 3UTR reporter (right). (G) Western blot analysis of P-ERK1/2, P-FAK, P-p38, P-AKT, VEGFA, and PDGF-BB protein level alteration in MSN/miR-21-5p complextreated endothelial cells after overexpressing SPRY1 with the SPRY1 overexpression vector. *P < 0.05 and ***P < 0.01. n = 3 per group. The data are shown as means SD. Photo credit: Yan Li, Shanghai Ninth Peoples Hospital, College of Stomatology, Shanghai Jiao Tong University School of Medicine, Shanghai 200011, China.

To gain further insight into the mechanism by which miR-21-5p promotes angiogenesis, we used a miRNA database to predict the potential target genes of miR-21-5p and found that SPRY1 has a miR-21-5p binding site in its 3 untranslated region (UTR). The amount of SPRY1 protein in endothelial cells was down-regulated by MSN/miR-21-5p treatment but not by MSN/miR-NC treatment, whereas we found no difference in SPRY1 mRNA levels between the two groups (Fig. 3D). To determine whether miR-21-5p functionally targets SPRY1 to promote angiogenesis, we overexpressed SPRY1 in endothelial cells. We found that phospho-Erk1/2, phospho-FAK, phospho-P38, phospho-AKT, VEGFA, and PDGF-BB levels were down-regulated in MSN/miR-21-5ptreated endothelial cells transfected with SPRY1 overexpression plasmid vector, compared to cells transfected with the empty vector (Fig. 3E). To test whether miR-21-5p directly targets SPRY1, we constructed luciferase reporters that had either the wild-type (WT) SPRY1 3UTR or an SPRY1 3UTR containing mutations at the miR-21-5p binding site (Fig. 3F). First, we found that MSN/miR-21-5p, but not MSN/miR-NC, substantially inhibited the luciferase reporter activity of the WT SPRY1 3UTR. Second, the luciferase reporter activity of the SPRY1mRNA with the mutated 3UTR was not suppressed by MSN/miR-21-5p (Fig. 3G). These comprehensive data suggest that delivery of miR-21-5p using MSN/miR-21-5p complexes promotes angiogenesis by targeting SPRY1 and subsequently activating the VEGF-induced ERK-MAPK signaling pathway (fig. S6). Detailed predicted miR-21-5p targets by Venn diagram analysis were revealed in fig. S7.

The in vivo efficacy of Gel@MSN/miR-21-5p was evaluated in an induced MI swine model. Coronary arteries were identified and ligated to induce a uniform and consistent MI, and the morphology and pumping effectiveness of the heart were evaluated ~45 min after the MI induction. The MI animals were then randomly divided into four groups receiving saline (negative control), agomiR-21-5p (a commercially available agent used to up-regulate endogenous miR-21-5p level), Gel@MSN/miR-NC, and Gel@MSN/miR-21-5p injection. Sham-operated animals served as a positive control. Morphological and functional assessments were performed using the modified Simpson method, which can accurately calculate left ventricular ejection fraction (LV EF) to detect any early echocardiographic changes. Changes in the morphology and pumping effectiveness of the heart were assessed through measurements of LV end diastolic volume (LVEDV), LV end systolic volume (LVESV), EF, and LV end diastolic dimension (LVEDd). Representative echocardiography images of short-axis views for each treatment group at baseline (before MI) and 45 min, 14 days, and 28 days after MI are shown in Fig. 4A. MI caused a substantial reduction in LV function 45 min after induction, as indicated by an absolute 20% decline in the EF. The morphological and functional parameters were slightly improved in the agomiR-21-5p and Gel@MSN/miR-NC groups compared with the saline negative control group at 14 and 28 days after MI, indicating that either miR-21-5p or MSNs alone could improve the morphology and pumping effectiveness of the heart but only to a limited degree (~an absolute 4 to 5% increase in EF values at 28 days, as compared to the saline group). More substantial improvement was achieved in the Gel@MSN/miR-21-5p group, with an approximately absolute 10% increase in the LV EF values at 28 days after MI. Time course echocardiography assessment over the 28-day study period is shown in Fig. 4B. These data suggest the importance of the therapeutic itself (miR-21-5p) as well as the delivery system (a two-stage delivery) in mitigating the negative LV remodeling and improving the morphology and pumping effectiveness of the heart after MI.

(A) Representative echocardiography imaging by the modified Simpson method of short-axis views for each treatment group at baseline and 45 min, 14 days, and 28 days after MI. The site of the infarct zone is shown by arrows. Notable chamber dilation and wall thinning occurred at 28 days following MI, consistent with the adverse remodeling process. (B) Time course analysis of the EF, LVEDV, LVESV, and LVPWd. (C) MI caused a gradual decline in the EF over 28 days, which was notably attenuated by Gel@MSN/miR-21-5p. (D) MI caused a gradual increase in the LVEDV at day 14 and day 28. The LVEDV of the Gel@MSN/miR-21-5p treatment group was substantially attenuated compared with those of the other three treatment groups. (E) MI caused progressive thinning of the LVPWd thickness at the diastole, which was attenuated by Gel@MSN/miR-NC and agomiR-21-5p treatment and further attenuated by Gel@MSN/miR-21-5p treatment at day 14 and day 28. *P < 0.05 and ***P < 0.01. Sham, n = 3; MI/saline, n = 5; MI/agomir, n = 5; MI/Gel@MSN/miR-NC, n = 6; and MI/Gel@MSN/miR-21-5p, n = 6. The data are shown as the means SD. Photo credit: Yan Li, Shanghai Ninth Peoples Hospital, College of Stomatology, Shanghai Jiao Tong University School of Medicine, Shanghai 200011, China.

Representative delayed-enhancement computed tomography (CT) images of cross-sectional planes of hearts from two-axis (long axis and short axis) slices at day 28 after MI are shown in Fig. 5. The infarct regions in the LV posterior wall were characterized by wall thinning (identified by red counterstain). Analysis of systolic LV wall thickness showed that the wall thickness in the infarcted zone was retained in the agomiR-21-5p and Gel@MSN/miR-NCtreated groups 28 days after MI to a limited degree (marked with white arrows) compared to that in the saline-treated group. LV wall thickness in the infarcted zone was further persevered with the Gel@MSN/miR-21-5ptreated group. Bulls eye plots (Fig. 5A) display LV wall thickness, wall motion, and regional EFs. Global cardiac functional measures such as LVEDV, LVEDV, and EF are shown in the inserted table.

Representative delayed enhancement CT images of cross-sectional planes of hearts from two-axis (long axis and short axis) slices at day 28 after MI are shown. (A) Bulls eye plots display the LV wall thickness, wall motion, and regional EFs. (B) The infarct zone was characterized by wall thinning (identified by white arrows). (C) Global cardiac functional measures such as cardiac output, stroke volume, and EF are shown in the inserted table. Photo credit: Yan Li, Shanghai Ninth Peoples Hospital, College of Stomatology, Shanghai Jiao Tong University School of Medicine, Shanghai 200011, China.

Infarct size as measured by tetraphenyl tetrazolium chloride (TTC) staining also showed that the Gel@MSN/miR-21-5p group had the smallest infarct size (Fig. 6A; paired multiple slices of an infarcted heart in the same pigs shown in fig. S8). The histological characterization of the LV sections from the infarct region at 28 days after MI showed that the infarcted regions in pigs injected with Gel@MSN/miR-21-5p present preserved distinct and thick muscle layers. However, moderately thickened muscle was observed in the agomiR-21-5p and Gel@MSN/miR-NC groups, and obvious fibrillary layers were observed in the saline group. The muscle layers were verified to be cardiomyocytes by anticardiac troponin-T staining (Fig. 6B). Massons trichrome staining showed approximately two times less fibrous content in the Gel@MSN/miR-21-5p group than in the saline group (Fig. 6D). These observations provided evidence that Gel@MSN/miR-21-5p treatment could effectively attenuate fibrosis and improve cardiac remodeling after MI.

A porcine model of MI was used to investigate the post-MI responsiveness of different groups to treatments. Healing at the infarct zone was analyzed after 28 days after treatment. (A) Representative image of TTC-stained hearts and morphometric measures of the infarct area from each group. White coloring in the TTC-stained sections indicates infarct zone and tissue necrosis. (B) Representative histological analysis of the infarcted myocardium among the treatment groups. H&E (left) staining, Massons trichrome staining (middle), and immunohistochemistry staining for cardiac troponin T (right) 28 days after MI showed a loss of cardiomyocytes and collagen deposition, and interstitial fibrosis was substantially reduced in the infarct zone after the Gel@MSN/miR-21-5p treatment (scale bars, 2000 m in the low-magnification images and 60 m in the high-magnification images). Quantitative analysis showing the percentage of the TTC-negative infarct area (C) and fibrotic area (D). (E) miRNA transfection efficiency was investigated using real-time quantitative PCR at 28 days following MI. *P < 0.05 and ***P < 0.01. Sham, n = 3; MI/Saline, n = 5; MI/Agomir, n = 5; MI/Gel@MSN/miR-NC, n = 6; and MI/Gel@MSN/miR-21-5p, n = 6. The data are shown as the mean SD. Photo credit: Yan Li, Shanghai Ninth Peoples Hospital, College of Stomatology, Shanghai Jiao Tong University School of Medicine; Shanghai, 200011, China.

The in vivo data relating to drug release duration and efficacy of MSNs and miRNA delivery by Gel@MSN/miR-21-5p were characterized. Confocal images and quantification analysis showed that more than 60% of macrophages (identified by the F4/80+ marker) or endothelial cells (identified by the CD31+ marker) took up the MSN/miR-21-5p complexes 1 day after injection (Fig. 7). Furthermore, the high intracellular transfection efficacy was sustained up to ~28 days, as evidenced by an approximately twofold increase in endogenous miR-21-5p levels (Fig. 7), which could contribute to the improved morphology and pumping effectiveness of the heart.

MSNs were prelabeled with FITC (green), and miR-21-5p was prelabeled with Cy3 (red). The hydrogel (FITC-labeled Gel@MSN/miR-21-5p or Cy3-labeled Gel@MSN/miR-21-5p) was injected into the mid-myocardium of each target site in the pigs. The duration and efficiency of MSNs and miRNA delivery upon Gel@MSN/miR-21-5p injection were monitored using time course analysis at 1, 14, and 28 days after injection. (A) Histological sections of the infarct region in the Gel@MSN/miR-21-5p group were immunolabeled with the hematoxylin and eosin (H&E) macrophage marker F4/80. (B) Histological sections of the infarct region in the Gel@MSN/miR-21-5p group were immunolabeled with the endothelial marker CD31. Cell nuclei were counterstained with DAPI (blue). (C) F4/80+FITC+ and CD31+Cy3+ double-positive cells were quantified from at least eight high-resolution images acquired from at least eight different regions of each heart. (D) miR-21-5p levels were detected using real-time quantitative PCR at different time points. The transfection efficiency was determined by quantifying the miRNA level. Scale bars, 100 m. n = 3 per group. The data are shown as means SD. Photo credit: Yan Li, Shanghai Ninth Peoples Hospital, College of Stomatology, Shanghai Jiao Tong University School of Medicine, Shanghai 200011, China.

We further compared the effects of MSN/miR-21-5p complexes without a hydrogel matrix (MSN/miR-21-5p alone) and with a hydrogel matrix (Gel@MSN/miR-21-5p) on treating MI. The morphological and functional parameters of the MSN/miR-21-5p group alone were worse than those of the Gel@MSN/miR-21-5p group at 14 and 28 days, and the parameters did not improve over time. The Gel@MSN/miR-21-5p delivery system provided sustained release of miR-21-5p (fig. S9) and sustained a superior therapeutic benefit compared to that from a bolus shot of MSN/miR-21-5p (fig. S10). Histological examination and the quantification of the total infarct size showed similar results. These data suggest that the hydrogel matrix could maintain a long-term drug release, which is important to achieve a persistent therapeutic effect. The hearts were harvested at 28 days after MI for fluorescent imaging, RNA extraction, and real-time quantitative PCR analysis. The fluorescent images showed that the areas of FITC and Cy3 fluorescence enhancement exactly overlapped with the infarct region (Fig. 8A). The confocal images and quantification of miR-21-5p levels showed that MSN/miRNA complexes were effectively transfected into cells within the infarct region in vivo (Fig. 8B). These data indicate that the hydrogel matrix achieved localized sustained drug release, triggered by the acidic microenvironment in the infarct region.

For examination of on-demand delivery, the hearts were harvested at 28 days after MI for fluorescent imaging, RNA extraction, and real-time quantitative PCR analysis. (A) The fluorescent images showed that there were no transfecting cells detected in the sham group. In contrast, it showed that the area of FITC and Cy3 fluorescence exactly overlapped with the infarct region. (B) Quantification of miR-21-5p levels showed that the MSN/miRNA complex could be highly transfected into cells within the infarct region in vivo. Scale bar, 100 m. ***P < 0.01. The data are shown as means SD. Photo credit: Yan Li, Shanghai Ninth Peoples Hospital, College of Stomatology, Shanghai Jiao Tong University School of Medicine, Shanghai 200011, China.

The three-dimensional (3D) organization of the vascular network within the infarct regions was characterized by micro-CT angiography. The vascular density and volume were significantly improved with Gel@MSN/miR-21-5p (Fig. 9A). CD31 and smooth muscle actin (-SMA) are typical biomarkers of endothelial cells and mural cells in blood vessels. Immunofluorescence characterization showed that expression levels of CD31 and -SMA were significantly enhanced and that more newly formed vessels were observed in the Gel@MSN/miR-21-5p treatment group than in the other groups. These observations provided evidence that Gel@MSN/miR-21-5p treatment enhanced vascularization after MI.

(A) Micro-CT angiography analysis of 3D vascular structures within the infarct zone 28 days after MI indicates that the vascular volume was significantly increased in the Gel@MSN/miR-21-5p treatment group. The vascular volume within the infarct zone was quantitatively analyzed. *P < 0.05 and ***P < 0.01. n = 3 per group. (B) Immunofluorescence staining for CD31 (red) identified the vascular endothelium, and staining for -SMA (green) identified myofibroblasts and pericytes, showing that the cardiac capillary density in histological sections of the healing infarct zone was significantly higher in the Gel@MSN/miR-21-5p treatment group than in the other groups. The CD31 and -SMA staining intensities in the above-described groups were quantitatively analyzed (scale bars, 500 mm). *P < 0.05 and ***P < 0.01. Sham, n = 3; MI/saline, n = 5; MI/agomir, n = 5; MI/Gel@MSN/miR-NC, n = 6; and MI/Gel@MSN/miR-21-5p, n = 6. The data are shown as the means SD. Photo credit: Yan Li, Shanghai Ninth Peoples Hospital, College of Stomatology, Shanghai Jiao Tong University School of Medicine, Shanghai 200011, China.

Immunofluorescence analysis of LV sections taken from the infarct region 1 day after MI showed that Gel@MSN/miR-21-5p effectively protected cardiomyocytes (fig. S11) and inhibited the expression of several key inflammatory mediators (TNF-, IL-1, and IL-6) (Fig. 10). Furthermore, concordant with reduced fibrotic area in the infarcted region in the Gel@MSN/miR-21-5ptreated group at 28 days after MI, the expression of key inflammatory mediators (TNF-, IL-1, and IL-6) was obviously reduced (fig. S12). These results suggested that Gel@MSN/miR-21-5p treatment modulated the immune response after MI by inhibiting the expression of proinflammatory cytokines.

Histological sections of the infarct zone (day 1 after MI) were immunolabeled with antibodies targeting TNF- (A), IL-6 (B), or IL-1 (C) and colabeled with the macrophage marker F4/80 (green). Cell nuclei were counterstained with DAPI (blue). (D) The percentages of cells double positive for F4/80 and TNF-, IL-1, or IL-6 (TNF-, IL-1, or IL-6expressing macrophages, respectively) were quantified. Quantification was performed in at least eight high-resolution images acquired from at least eight different regions of each heart. Scale bars, 100 m. ***P < 0.01. n = 3 per group. The data are shown as the means SD. Photo credit: Yan Li, Shanghai Ninth Peoples Hospital, College of Stomatology, Shanghai Jiao Tong University School of Medicine, Shanghai 200011, China.

The use of large-animal models of MI provides valuable information regarding the safety and efficacy of new therapies. Pig models offer an alternative because of their anatomical and physiological similarities to humans (40, 41). The treatment groups used materials such as PEG derivatives, CD, silica, and miRNA, and an obvious inflammatory response to foreign bodies was not observed in the treated pigs, indicating its potential for clinical transition.

Here, we report the potential for an efficient miRNA delivery system that simultaneously integrates immune modification and angiogenesis enhancement in the field of MI therapy. This study demonstrates the efficacy and feasibility of a delivery system in a clinically relevant porcine MI model, where both the pathophysiology and the administration mimic what would be observed and performed in humans.

Current therapeutic strategies (angiogenic therapy or anti-inflammatory therapy) involving protein delivery or gene therapy for treating MI have limited success in reducing infarct size (42, 43). The results of our study suggest that therapeutic outcome relies on both immunomodulation and angiogenesis. This study demonstrated that MSNs could reduce the inflammatory responses that can modify tissue remodeling and prevent fibrous tissue formation for improved repair after MI. Specifically, the effect of the resultant microenvironment can be further enhanced with sustained miR-21-5p delivery via MSNs and synergistically stimulate angiogenesis as well as changes in the morphology and pumping effectiveness of the heart after MI.

To date, the study to use miRNA for the treatment of ischemic cardiovascular disease in a preclinical pig model was performed by Gabisonia et al. (22). Gabisonia et al. used miR-199a therapy in an attempt to stimulate cardiomyocyte proliferation. The approach enabled the induction of preexisting cardiomyocytes to reenter the cell cycle and rebuild the injured heart (44). Substantial improvements in cardiac function and structure were attributed to this process. However, there are potential limitations of cardiomyocyte proliferation after birth including cardiomegaly or hypertrophy, as well as possible arrhythmias due to the immaturity of myocyte conduction or poor coupling with existing myocardium (45). As reported by Gabisonia, the generation of areas of poorly differentiated cardiomyocytes might cause tachyarrhythmias and eventually determine fatal reentry electric circuits. The adverse effects were also observed in several other studies, that long-term stimulation of cardiomyocyte proliferation might result in impaired cardiac function or arrhythmic events (4648). In the current study, we attempted to use specific miR-21-5pbased therapies to promote angiogenesis in infarct areas, which may further facilitate rescuing resident cardiomyocytes in an injured heart. We focused on myocardial salvage rather than replacement. The proangiogenic effects of miR-21-5p were characterized with multiple in vitro and in vivo experiments and could be attributed to targeting SPRY1. Loss of SPRY1 leads to the expression of proangiogenic cytokines (VEGFA and PDGF-BB) in endothelial cells. While manipulation of proteins in the Hippo pathway (identified as miR-199a targets) promotes adult cardiomyocyte cell cycling, animals subjected to this type of treatment also exhibit cardiac dysfunction and heart failure in the long term (47, 48). Our strategy represents another direction to promote MI repair. Until now, no major case of arrhythmias has been reported to be associated with long-term proangiogenic therapies in either animal studies or clinical trials. In addition, Gabisonia et al. used adeno-associated virus vectors as therapeutic and investigational tools, which have advantages such as high transfection efficiency. However, such virus-based delivery systems could lead to uncontrollable continuous miR-199a expression and unrestrained cardiac growth in the long term, which would eventually result in sudden death due to arrhythmia at weeks 7 to 8 in most of the treated pigs because controlled miRNA delivery was beyond the current capabilities of virus-mediated gene transfer. Therefore, the treatment needs to be carefully dosed, which could be achieved through the delivery of naked, synthetic miRNA mimics. In our study, a local on-demand and controlled delivery system was described. The system provided a controlled miR-21-5p mimic delivery, with ~75% release over 7 days at pH 6.8 in vitro. In addition, considering the limitation of current RNAi-based therapy associated with potential off-target accumulation, multiple works have been done in this area to optimize the RNAi delivery system (16, 18, 19, 23). For example, a hydrogel system used ultraviolet as the external stimulus to achieve on-demand controlled localized release of RNA at designated time points to induce human mesenchymal stem cell (hMSC) osteogenesis (18, 19). In the present study, the hydrogel is designed to be pH stimuli responsive to achieve on-demand miRNA delivery for persistent and accuracy therapeutic effect on MI. The miRNA delivery system (Gel@MSN/miR-21-5p) specifically released MSN/miR-21-5p only at the infarct region without affecting the surrounding healthy tissues, which addresses the safety issue associated with miRNA-based therapy. As shown in fig. S15, two pigs survived out to 11 months after Gel@MSN/miR-21treatment, and electrocardiography (ECG) was performed. ECG analysis of Q wave and T wave showed that ECG signal at 11 months is similar to that at 4 weeks after Gel@MSN/miR-21 treatment, indicating that Gel@MSN/miR-21 was not likely to pose a long-term safety risk.

Acute inflammation caused by MI is a protective response that kills invading pathogens, should be self-limiting, and leads to healing (49). However, excess activation of the acute inflammatory response leads to cardiac myocyte death. Macrophages play a central role in regulating inflammation. Modulation of macrophage activation may contribute to the resolution of cardiac injury after MI. The results of this study indicate that MSNs can be used to inhibit proinflammatory polarization (M1) in an inflammatory microenvironment following ischemic muscle injury in vivo (50). Gulin-Sarfraz et al. (13) also noticed that empty mesoporous silica particles could reduce the number of neutrophils and down-modulate the inflammatory response in a mouse airway inflammation model. In addition, our data showed that MSNs modulated immune response through down-regulating TLR2, which inhibited the activation of NFB signaling and subsequently decreased the release of proinflammatory cytokines (TNF-, IL-1, and IL-6). Our results are similar to the findings of Lee et al. (51), who demonstrated that exposure to MSNs decreased the expression of proinflammatory cytokines such as TNF-, IL-1, and IL-6 in macrophages. Consistent with these results, a more recent study indicated that MSNs inhibit lymphocyte proliferation, suppress the killing activity of natural killer cells, and decrease proinflammatory cytokine and nitric oxide production in macrophage cells (36).

Previous studies have demonstrated that angiogenesis can be promoted by the fine-tuned delivery of multiple growth factors and cells with biomaterials (52, 53). It relies on the precisely controlled sequential release or direct serial delivery, which are unfavorable for clinical use. The present study has provided a relatively simple approach that shows not only equivalent efficacy in promoting angiogenesis but also a modified cardiac inflammatory response in pigs after MI, suggesting that achieving cardiac repair through the stimulation of angiogenesis in the infarct region with a miRNA (miR-21-5p)based strategy is attainable in large mammals. The vascular volume was significantly improved within the infarct region in pigs treated with Gel@MSN/miR-21-5p. The enhanced vessels within the infarct region were associated with the accumulation of endothelial cells (identified by CD31+) and mural cells (identified by -SMA+) 28 days after MI. The mechanism by which miR-21-5p exerts its cardiac proangiogenic effects in the myocardium was also studied. KEGG analysis suggested that treatment with miR-21-5p complex was positively associated with key angiogenic signaling pathways such as VEGF signaling and PDGF signaling. Multiple experiments were further conducted and concluded that the delivery of miR-21-5p promoted angiogenesis by targeting SPRY1 and subsequently activating VEGF-induced ERK-MAPK signaling. Together, these data suggest that endogenous cardiac repair may be facilitated by the miR-21-5pinduced angiogenic network.

Increasing reports have revealed the advantage and importance of biomaterials in cardiac tissue engineering. Despite the enthusiasm, there are relatively few ongoing clinical trials using injected materials for cardiac repair, perhaps due to a lack of evidence in large-animal studies, which are necessary before progressing to human trials. Pig models offer an alternative because of their anatomical and physiological similarities to humans. The use of a pig model of MI may provide valuable information regarding the safety and efficacy of therapeutic strategies for MI in clinic. We performed a large-animal study with a pig model to demonstrate the translational potential. However, because the immediate treatment after MI may not be relevant to clinical situations, whether this approach also works in chronic cases and whether there exists an optimal therapeutic time window require further evaluation. There are also human-specific issues to consider including PEG immunity and species-specific interactions. Thus, understanding the factors that affect PEG immunity is crucial for both researchers and clinicians to ensure the treatment safety in clinic. Optimization of Gel@MSN/miR-21-5p dose and long-term studies are also needed for clinical translation.

In summary, the two-stage gene delivery system Gel@MSN/miR-21-5p developed in this study consists of three key components, pH-responsive hydrogel matrix, MSNs, and miR-21-5p. The responsive hydrogel serves as a matrix to achieve a highly localized drug release triggered by an acidic microenvironment and a 1-week sustained drug release (first stage release); MSN is the gene transfection vector (second stage release) and itself alone also resolves early inflammation by suppressing the TLR/NFB signaling pathway; and miR-21-5p promotes angiogenesis and mature vessel formation by targeting SPRY1 and subsequently activating VEGF-induced ERK-MAPK signaling. The synergy among these three elements demonstrated significance in treating MI in a swine model via a combination of anti-inflammatory and proangiogenic effects. Clinically relevant positive outcomes were observed upon Gel@MSN/miR-21-5p treatment, such as improved cardiac remodeling, reduced fibrosis formation and infarct size, and increased vascularization. The injectable property of Gel@MSN/miR-21-5p makes it potentially translatable to minimally invasive transcatheter-based surgery. In addition, this study is a proof of concept for controlled gene delivery and can serve as a technological platform to better elucidate the dose-dependent response of genes in MI treatment or deliver any other nucleic acids (such as DNAs, mRNAs, siRNAs, and miRNAs) or treat any other disease.

The purpose of this study was to design a controlled on-demand miR-21-5p delivery system (Gel@MSN/miR-21-5p) using MSNs combined with a hydrogel matrix, simultaneously integrating immune modification and angiogenesis enhancement in the field of MI therapy. Gel@MSN/miR-21-5p was fabricated by embedding MSN/miR-21-5p complexes into an injectable hydrogel matrix. We performed studies to determine the mechanical properties, structure, and on-demand release profile of Gel@MSN/miR-21-5p.

For the in vitro experiment, real-time quantitative PCR, Western blot, and enzyme-linked immunosorbent assay (ELISA) were performed to assess the immunomodulatory effect of MSNs. Real-time quantitative PCR, Western blot, ELISA, and tube formation assays were performed to determine the proangiogenic effect of miR-21-5p. The mechanisms underlying MSN-mediated inflammatory effects and miR-21-5pmediated proangiogenic effects were studied by proteogenomic analysis, real-time quantitative PCR, and Western blot.

For the in vivo experiments, pigs were randomly assigned to treatment groups, and, wherever applicable, treatment conditions were kept blinded until statistical analysis. Group sizes of at least five animals were chosen, which indicated that the therapeutic efficacy and safety of the Gel@MSN/miR-21-5p could be robustly identified. MI was characterized using multiple methods including echocardiography, delayed enhancement CT, TTC staining, and histological examination. The potential cardiac-protective effect against apoptosis induced by ischemia was analyzed by immunofluorescence analysis. The duration and efficiency of MSNs and miRNA delivered by Gel@MSN/miR-21-5p injection were monitored using time course analysis.

Animal protocols related to this study were reviewed and approved by the Institutional Animal Care and Use Committee at the School of Medicine of Shanghai Jiao Tong University. All experiments were performed in accordance with the guidelines published by the Institutional Animal Care and Use Committee at the School of Medicine of Shanghai Jiao Tong University, Shanghai. All animals were obtained from the Ninth Peoples Hospital Animal Center (Shanghai, China).

Yucatan mini pigs (male, 45 to 50 kg) were anesthetized with tiletamine hydrochloride and zolazepam hydrochloride (4 mg/kg). To establish the porcine MI model, transthoracic 2D echocardiographic measurement by Simpsons method (S5-1 transducer, PHILIPS Medical Systems) was performed to ensure that the animal was healthy before instrumentation and MI induction. Following baseline echocardiographic measurements, light anesthesia was maintained by continuous intravenous infusion of propofol (30 to 40 g kg1 min1). ECG, heart rate, and arterial pressure were constantly monitored. The pericardium was opened through a left thoracotomy, and the first two obtuse marginal arteries of the circumflex artery (OM1 and OM2) were identified and ligated to induce MI. Past studies demonstrated that this technique creates a uniform and consistent MI (24). The pericardium was left open. Pigs were randomized to receive a total of six distinct injection of saline, agomiR-21-5p, Gel@MSN/miR-NC, or Gel@MSN/miR-21-5p within a targeted 2 2 cm region of mid-myocardium immediately after MI (six injection sites, 100 l per injection). Sham controls were was processed in an identical fashion with the exception of coronary artery ligation. The injection of each target site is shown in fig. S13. For the Gel@MSN/miR-NC and Gel@MSN/miR-21 treatments, the miR-NC or miR-21 was preloaded in the MSN-NH2-TMA with a mass ratio of 1:10 between miRNA/MSNs. Then, the sterilized aqueous solutions (600 l) containing RNA-loaded MSN-NH2-TMA, CHO-PEG-CHO, and -CD with a mass ratio of 1:5:5 were incubated for 5 min to form an injectable hydrogel precursor with weak interaction, which was further drawn into a separate syringe, and injected into the mid-myocardium to form the final hydrogel at the target site immediately following MI induction. Animals were carefully monitored until they fully recovered from anesthesia.

Pigs were sedated at baseline, and 2D echocardiographic measurements by Simpsons method (IE33 digital ultrasonic scanner, PHILIPS Medical Systems, USA) were performed in right lateral recumbency. Echocardiography measurements were taken before surgery (baseline) and at 45 min, 14 days, and 28 days following MI. Transthoracic echocardiography allowed assessment and further calculation of LV dimensions, cardiac chamber size, wall thicknesses, EF, LVEDV, and LVEDd according to the biplane modified Simpsons rule. For these measurements, standard parasternal long-axis and apical chamber views were obtained.

CT examinations were performed at 28 days after MI. Animals were sedated with a cocktail injection of tiletamine hydrochloride (4 mg/kg) and zolazepam hydrochloride (4 mg/kg) injection. Pigs were placed in a right lateral position.

CT images were acquired with a clinical 320-slice scanner (Aquilion One, TOSHIBA Medical Systems). The heart was scanned along two long-axis views (vertical and horizontal) and with one set of short-axis views covering the entire LV from the atrioventricular valve plane to the apex. The following parameters were used: a tube voltage of 100 kV, a tube current of 75 mA, a gantry rotation time of 330 ms, 0.5-mm section thickness, a resolution of 0.5 0.5 mm, and free breathing. The CT contrast medium (Ultravist 370, Schering) was injected at a flow rate of 3.5 ml/s. To identify the scar and quantify the extent of post-infarction fibrosis, delayed contrast-enhanced multidetector CT images were acquired to assess viability 3 to 5 min after the administration of contrast media for LV function.

Multiphase reconstruction was performed with commercially available software (VITAL, TOSHIBA Medical Systems, Japan) by using short-axis slices from the base of the heart to the apex. The end diastole and end systole were defined as the maximal and minimal LV volume, respectively.

The hearts from each group were harvested, and blood vessels within the heart were imaged by angiography, as previously described (54). Briefly, a 50.8-millimeter, 18-gauge catheter (Surflo Teflon IV Catheter, Terumo Medical, USA) was inserted into the left ventricle of the heart and advanced into the ascending aorta. A 0.9% normal saline solution containing heparin sodium (100 U/ml) was perfused through the vasculature. The vasculature was then fixed by perfusion with 10% neutral buffered formalin (NBF) and cleared with saline. Last, 25 ml of polymerizable, lead chromatebased, radiopaque contrast agent (Microfil MV-122, Flow Tech, USA) was injected using a 30-ml syringe. Samples were stored at 4C for 24 hours to allow polymerization of the contrast agent.

Samples were scanned using micro-CT (Y. Cheetah, YXLON, Germany) with the following settings: 90 kV, 50 A source current, exposure time of 907 ms, and two images every 0.5 of a 360 rotation range at a voxel size of 76 m. 3D reconstruction of the micro-CT image was completed and analyzed using the manufacturers evaluation software (VG studiomax 3.0). The reconstruction was performed using binning mode, providing an isotropic voxel size of 76 m.

Since the infarct area is clearly visible in the heart tissue slice, matching the micro-CT image slices with their corresponding tissue slices could identify the infarct zone within the 3D micro-CT reconstructed model. The sectioning planes of the microtomograph and of the tissue samples are parallel. After sectioning, the infarct areas (infarcted myocardium appears pale) of the heart tissue slices were counterstained in red. After obtaining micro-CT images, the infarct areas of the micro-CT images were identified on the basis of the observation of the tissue slices.

The heart tissue was sectioned starting from the base to the apex. After sectioning, slices were immediately immersed in 2% TTC in 0.9% NaCl at 37C for 30 min for vital staining. Infarcted myocardium appeared pale after TTC staining. The MI area (TTC negative, white) is outlined. The infarcted area and the total area of the LV wall were analyzed using ImageJ software. The infarct size was calculated as follows: counts of TTC-negative area/counts of total LV wall area (%) on short-axial middle LV myocardial slices.

The excised hearts were sectioned through four horizontal planes, and each section was then subdivided into subsections for further histological and molecular analyses, as shown in fig. S14. Briefly, each heart was sectioned into four 1-cm-thick slices, starting from the apex toward the base. Then, two regions (indicated by letters) of each slice were chosen for further histological and molecular analyses. In all quantifications, we considered eight sectors of the four heart sections, and the same regions were chosen in animals with different treatments.

Pig hearts were carefully harvested 28 days following infarction. Samples representing the mid-infarct were sliced. These tissue samples were routinely processed for histologic analysis, and sections (5 m thick) were stained with hematoxylin and eosin (H&E) and Massons trichrome, as previously described (55). Capillary densities were examined by counting the number of capillaries stained with anti-CD31 (ab28364, Abcam, USA) and anti-SMA (ab5694, Abcam, USA) antibodies. For hydrogel immunomodulatory investigation, hearts were collected and processed after 1 and 28 days after MI. Immunofluorescence was used as previously described to identify F4/80+ cells (ab6640, Abcam, USA) colabeling with antiTNF- (ab6671, Abcam, USA), antiIL-6 (ab6672, Abcam, USA), or antiIL-1 antibody (NB600-633, Novas, USA); Alexa Fluor 488labeled donkey anti-rat antibody (Jackson ImmunoResearch Laboratories, USA) and the Alexa Fluor 594labeled anti-rabbit antibody (Jackson ImmunoResearch Laboratories, USA) were used for visualization. Slides were counterstained with DAPI (56). Immunohistochemistry was used to verify cardiomyocytes with anticardiac troponin-T antibody (ab10214). ImageJ software was applied to count blue pixels (positive for collagen) within that region in the trichrome images.

Time course analysis of transfection efficiency of Gel@MSN/miR-21-5p was performed in vivo. MSNs were prelabeled with FITC (green), or miR-21-5p was prelabeled with Cy3 (red). The hydrogel (FITC-labeled Gel@MSN/miR-21-5p or Cy3-labeled Gel@MSN/miR-21-5p) was injected into the mid-myocardium of each target site of pigs. The delivery efficiency of miR-21-5p into endothelial cells was examined by identifying CD31+ cells (ab28364, Abcam, USA) colabeling with Cy3-labeled miR-21-5p. The delivery efficiency of MSNs into macrophages was examined by identifying F4/80+ cells (ab6640, Abcam, USA) colabeled with FITC-labeled MSNs.

To assess whether MSNs could protect against apoptosis in cardiomyocytes, a terminal deoxynucleotidyl transferasemediated deoxyuridine triphosphate nick end labeling assay using an In Situ Cell Death Detection Kit (Roche, Switzerland) was performed at an earlier time (1 day) after MI, which labels broken DNA strands that are often associated with apoptosis. Percentages of positively stained cells were determined by counting the numbers of labeled cells and total cells.

The macrophage activation state was evaluated after intraperitoneal injections of LPS (Sigma-Aldrich, St. Louis, MO; 250 g in 0.5 ml of saline) into mice. Primary peritoneal macrophages were obtained from 20 g of female C57BL6J mice, as previously described (57). Briefly, cell lavage was collected by flushing the peritoneum with cold PBS. The peritoneum was centrifuged (800g, 4C, 9 min), and the pellet was incubated with ACK buffer (Fisher Scientific, Chino, USA) for 1 min to lyse erythrocytes. The remaining cells were cultured in RPMI 1640 medium and 10% fetal bovine serum (FBS) (Gibco, Gaithersburg, USA) at 37C in a 5% CO2 atmosphere and plated to select for adherent macrophages.

Primary cardiomyocytes were obtained from adult C57BL6J mice (8 weeks), as previously described (58). Briefly, the animal is euthanized humanely by cervical dislocation, and the heart is excised, taking care to remove the pericardium. Blood is removed from the coronary vessels after adequate perfusion with EDTA. Next, the heart is perfused with enzyme solution for 8 to 14 min. At the end of the enzyme digestion, the enzyme solution is flushed with 100 M Ca solution for 5 min, after which the heart is excised by dissecting the cannula, atria, and aorta. Once the first digestion was completed, the heart was transferred to a sterile petri dish and a second digestion step is carried out. The ventricular tissue is chopped with small scissors. Fresh digestion buffer was added, and the heart was quickly triturated with fine tweezers and forceps. This second digestion was performed at 37C in an incubator with 5% CO2 for 10 min to facilitate the collagenase activity. The reaction was halted by adding stop buffer containing FBS (Gibco), and the sample was filtered through a 100-m mesh. Following this, cardiomyocytes were purified via gravity separation in a falcon tube for 15 min and washed with Ca solution. After purification, cells were counted in a hemocytometer, seeded in laminin-coated culture dishes, and placed in an incubator with 5% CO2 at 37C.

Endothelial cells were purchased from the cell and stem cell bank (GNO 15, Chinese Academy of Sciences, China) and were maintained in culture with Dulbeccos modified Eagles medium (DMEM) (Gibco) supplied with 10% FBS (BioInd, Israel), as detailed by the manufacturer.

The tube formation assay was performed as previously described (59). Briefly, growth factorreduced Matrigel matrix (Life Technology) was plated in a 24-well plate after thawing at 4C overnight. The plate was then incubated at 37C for 30 min to allow the Matrigel to polymerize. MSNs, MSN/miRNA-NC, and MSN/miRNA-21transfected calcein-labeled endothelial cells in endothelial basal medium 2 (EBM2) supplemented with 0.5% FBS and basic fibroblast growth factor (5 ng/ml) (FGF) final were seeded on the Matrigel-coated well. The plate was then incubated at 37C in a 5% CO2 humidified atmosphere. Tube formation was observed at 8 and 16 hours with confocal microscopy. The tube formation ability was determined by measuring the total tube length of endothelial cells with ImageJ software.

For flow cytometric analyses, cells were blocked with 10% FBS for 10 min on ice and subsequently stained with fluorochrome-tagged anti-F4/80 (BM8, BioLegend) or APC-labeled anti-CD31 (eBioscience, 17-0319-42). All stains were performed in 1% bovine serum albumin PBS buffer for 1 hour in the dark at 4C, followed by two washing steps. Samples were analyzed on a FACSCalibur (BD Biosciences, USA). Dead cells were excluded by forward and side scatter, and data analysis was performed using FlowJo software version 7.6.3 (Tree Star Inc., Ashland, USA).

For in vitro uptake analysis, isolated peritoneal macrophages were cocultured with FITC-labeled nanoparticles (100 g/ml). For in vivo uptake analysis, FITC-labeled Gel@MSN/miR-21-5p was injected into the mid-myocardium of the pigs heart. In vitro and in vivo quantitative uptake of the MSNs by macrophages was determined by quantifying the fluorescence intensity of cells that were positive for F4/80 (ab6640, Abcam, USA) and showed colocalization with FITC.

The growth medium of the hypoxic/ischemia group was replaced with serum-free DMEM. Cells were placed in a hypoxic incubator (Sanyo, O2/CO2 incubator MCO-18M) with oxygen adjusted to 1.0% and CO2 adjusted to 5%. Normal culture (regular medium under 21% oxygen and 5% CO2) served as a control.

The hearts of pigs were collected. Total miRNA from the collected cells or the heart was extracted using the RNeasy Mini Kit (Qiagen) according to the manufacturers instructions. For miRNA level detection, reverse transcription was performed using the Reverse Transcription kit (Takara RR037a, USA) with miRNA-specific stem-loop RT primer (ID: miR8001313, RiboBio, China). Reverse transcriptase reactions contained 0.5 g of RNA samples, 0.2 M stem-loop RT primer, 1 RT buffer, 50 pmol of random primers 6, and PrimerScript Reverse Transcriptase (200 Ul1). The 10-l reactions were incubated in a T100 thermal cycler (Bio-Rad, Hercules, USA) for 15 min at 37C, 5 s at 72C, and then held at 4C. One microliter of cDNA was PCR-amplified using Premix Taq (Takara RR902A) with 1 l of forward primer (0.2 M) and 1 l of reverse primer (0.2 M) for miR-21-5p (RiboBio, ID: miR8001314). The 25-l reaction volume consisted of 1 l of cDNA, 12.5 l of Premix Taq, 9.5 l of ddH2O, 1 l of forward primer (0.2 M), and 1 l of reverse primer (0.2 M). The reactions were performed on a T100 thermal cycler.

The cDNAs were diluted 10 times to perform real-time quantitative PCR using TB Green Premix Ex Taq (Takara RR420A) for miR-21-5p level detection. The 25-l reaction volume consisted of 1 l of cDNA, 12.5 l of Green Premix Ex Taq, 9.5 l of ddH2O, 1 l of forward primer (0.4 M), and 1 l of reverse primer (0.4 M) for miR-21-5p (RiboBio, ID: miR8001314).

For miRNA level detection, cDNAs were synthesized using a reverse transcription kit (Takara, RR037a). Reverse transcriptase reactions contained 0.5 g of RNA samples, 25 pmol of Oligo dT Primer, 1 RT buffer, 50 pmol of random six primers, and PrimerScript Reverse Transcriptase (200 Ul1). The 10-l reactions were incubated in a MyCycler thermal cycler (Bio-Rad, Hercules, CA) for 15 min at 37C and 5 s at 72C and then held at 4C. The cDNAs were then diluted 10 times to perform real-time quantitative PCR for expression confirmation and expression pattern analysis.

The primers used are as follows: -actin (5-CAGGATTCCATACCCAAGAAG-3 and 5-AACCCTAAGGCCAACCGTG-3), IL-1 (5-GAAATGCCACCTTTTGACAGTG-3 and 5-TGGATGCTCTCATCAGGACAG-3), TNF- (5-GACGTGGAACTGGCAGAAGAG-3 and 5-TTGGTGGTTTGTGAGTGTGAG-3), IL-6 (5-TCTATACCACTTCACAAGTCGGA-3 and 5-GAATTGCCATTGCACAACTCTTT-3), TLR1 (5-CCGTCCCAAGTTAGCCCATT-3 and 5-TCCCCCATCGCTGTACCTTA-3), TLR2 (5-TGCGGACTGTTTCCTTCTGA-3 and 5-GCGTTTGCTGAAGAGGACTG-3), TLR3 (5-TACAAAGTTGGGAACGGGGG-3 and 5-GGTTCAGTTGGGCGTTGTTC-3), and TLR8 (5-ACAAACGTTTTACCTTCCTTTGTC-3 and 5-ATGCAGTTGACGATGGTTGC-3).

Western blotting was performed as previously described (56). Total protein was extracted using the EpiQuik whole-cell extraction kit (Epigentek, USA). The protein concentration was measured following the manufacturers instructions (Bio-Rad, USA). Protein was applied to and separated on 4 to 15% NuPAGE gels (Bio-Rad) and transferred to polyvinylidene difluoride membranes (Millipore, USA). The membranes were blocked with 5% bovine serum albumin and incubated with specific primary antibodies against the following: TNF- (AF-410-NA, R&D, USA), IL-1 (Novus, AF-401-NA), IL-6 (bs-0782R, Bioss, USA), VEGFA (DF7470, Affinity, USA), PDGF-BB (bs-1316R, Bioss), TLR1 (NB100-56563, Novus), TLR2 (Abcam, ab209217), TLR3 (NBP2-24875, Novus), TLR8 (NBP2-24917, Novus), NFB (CST8242s, Cell Signaling Technology, USA), p-NFB (CST3033s), SPRY1 (Abcam, ab111523), P-ERK1/2 (AF1018, R&D), ERK1/2 (AF1576, R&D), P-AKT (AF887, R&D), AKT (MAB2055, R&D), P-FAK (MAB4528, R&D), FAK (AF4467, R&D), P-P38 (CST4511), P38 (CST8690), and GAPDH (ab181602) at a ratio of 1:1000 overnight.

Horseradish peroxidaseconjugated IgG (1:10,000 dilution) from Santa Cruz Biotechnology (Santa Cruz, USA) was incubated with the membrane for 1 hour, after which the membranes were enhanced with a SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, USA). The relative amounts of the transferred proteins were quantified by scanning the autoradiographic films. Total protein or nuclear protein was normalized to the corresponding -actin.

For VEGFA and PDGF-BB protein secretion analysis, cells were pretreated with MSN complex loaded with 5 nmol of miR-21-5p as described above. After 6 hours of culture, the medium was replaced with fresh growth medium supplemented with 5.0% serum substitute Nu-Serum (NuS, BD, USA). Samples were collected at 48 hours. VEGFA and PDGF-BB protein levels in the medium were determined using an ELISA according to the manufacturers instructions (R&D Corp., USA). Absorbance was measured at 450 nm with a microplate reader (MTP-800Lab, Corona Electric, Japan). A standard curve was plotted to determine the VEGFA and PDGF-BB concentrations. The values are expressed as picograms per milliliter.

To detect the degradation of Gel@MSN/miR-21 in vivo, the PEG frame of the hydrogel was labeled rhodamine B. Sixty microliters of Gel@MSN/miR-21 was injected into the mid-myocardium of rats after induction of MI. To monitor the residual MSNs in vivo, 60 l of hydrogel containing rhodamine Blabeled MSNs was injected into the mid-myocardium of rats after induction of MI. At the indicated time points, rats were euthanized, and the hearts were removed from the animals. The organs were entirely maintained on ice until ex vivo analysis with Xenogen IVIS imaging system (Alameda, USA). Epifluorescence images of the hearts were acquired. Captured images were then analyzed using the Living Image 4.3.1 software (PerkinElmer Inc., USA). All data obtained by Xenogen IVIS were expressed as radiant efficiency, were assumed to be a calibrated measurement of the photon emission from the subject, and were technically defined as fluorescence emission radiance per incident excitation intensity as follows: photons/s/cm2/sr.

All numerical data are presented as the means SD. Statistical analysis was performed using commercially available software (SPSS 26). Data were first checked for normal distribution, and differences among groups were compared by one-way analysis of variance (ANOVA) followed by the Bonferroni post hoc test. Comparisons between two groups were made using the unpaired t test. For all statistical analyses, significance was accepted at P < 0.05.

Acknowledgments: We thank Y. Zhang and X. Wang (Fudan University) for providing primary cardiomyocytes. The research project was carried out in the Shanghai Key Laboratory of Stomatology and Shanghai Research Institute of Stomatology. Funding: We acknowledge financial support from the Innovative Research Unit of Chinese Academy of Medical Sciences (2019-12M-5-037) and the National Natural Science Research Program of China (81970977, 31870969, 81870785, 81801039, 81720108011, and 81601606), the Shanghai Municipal Science and Technology Committee research program (number 18DZ2291100), the National Key Research Program of China (2017YFC0840100 and 2017YFC0840109), the Fundamental Research Funds for the Central Universities (2016qngz02), the National Natural Science Foundation of Shaanxi Province (2017JM5023), the Open Fund of the State Key Laboratory of Military Stomatology (2017KA02), and the Knowledge Innovation Program of Shenzhen (JCYJ20170816100941258). Author contributions: Y.L., L.C., D.Z., H.C., and Y.D. carried out animal studies and tissue analyses. X.C. and R.J. carried out the MSN complex synthesis, polyplex development, and Gel@MSN/miR-21-5p hydrogel fabrication and their characterization. Y.L., B.C., J.J.G., G.B., and S.L. contributed to data analysis and interpretation. C.Y., Z.Z., M.D., and Y.L. were responsible for the overall project design and manuscript organization. Competing interests: The authors declare that they have no competing interests. Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. Additional data related to this paper may be requested from the authors.

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Injectable hydrogel with MSNs/microRNA-21-5p delivery enables both immunomodification and enhanced angiogenesis for myocardial infarction therapy in...

Scientists have Created a ‘Brain in a Dish.’ It Could Potentially Cure Alzheimer, Dementia – News18

Humans are born with a variety of cells. While all of them are absolutely essential for creating us, some cells are more complex and importantlike the nerve cells forming our brain. After all, what is a human body is nothing if not for its brain.

Once the brain cells start to deteriorate, with ageing or injury, humans start to lose cognitive and motor functions. Often seen in cases of Alzheimer's and schizophrenia.

But looking inside a living human brain is impossible; you can only dissect a dead brain that doesnt function. But a group of researchers have overcome this hurdle by building a brain in a dish.

Scientists have been growing living cells in Petri-dishes for a long time. But this research is leaps and bounds ahead as organoids, grown from stem cells, allowed them to conduct extensive genetic analyses. The organoid was allowed to grow for 20 months. They observed it developed in phases, as if on an internal clock, much like the brain of a human infant. This is beyond the former assumption that dish brain could only develop till foetal stage.

Until now, nobody has grown and characterized these organoids for this amount of time, Nor shown they will recapitulate human brain development in a laboratory environment for the most part, said Daniel Geschwind, author of the study. He adds how this will be incredibly useful as models to study the human brain and diseases as the organoids mature and replicate many aspects of normal human development. The study can be found in the journal Nature Neuroscience.

Studying the organoids is helping them understand the physiology and development of diseases like neurological and neurodevelopmental disorders including autism, epilepsy and schizophrenia.

The scientists developed these organoids using pluripotent stem cells. These cells are born one but have the ability to differentiate into multiple specific cells like neurons or cardiac and so on. They induced these cells, derived from skin and blood, to grow into neurons. By manipulating the chemical balance, cell-dish environment and so on, these cells not just developed a rough neural network but self-organised into a structure similar to a 3-D brain.

Excerpt from:
Scientists have Created a 'Brain in a Dish.' It Could Potentially Cure Alzheimer, Dementia - News18

Exosome therapeutic Market Segmentation, Parameters, Prospects 2021 And Forecast Research Report To 2027 KSU | The Sentinel Newspaper – KSU | The…

Exosome therapeutic Market Industry Trends and Forecast to 2028 New Research Report Added to Databridgemarketresearch.com database. The report width of pages: 350 Figures: 60 And Tables: 220 in it. Exosome therapeutic Market describes complete industry Outlook with in-depth analysis. This report also includes the complete analysis of each segment in terms of opportunity, market attractiveness index and growth rate, top players and new comers in industry, competitive landscape, sales, price, revenue, gross margin, market share, market risks, opportunities, market barriers, and challenges. key statistics on the market status. Which give the clear idea about the product differentiation and an understanding of competitive landscape Globally.

Exosome therapeutic Market Research report comprises of a brief summary on the trends and tendency that may help the key market players functioning in the industry to understand the market and strategize for his or her Organization expansion for this reason. This statistical surveying report examines the entire market size, market share, key segments, growth, key drivers, CAGR, historic data, present market trends And End User Demand, environment, technological innovation, upcoming technologies and the technical progress in the industry.

Global Exosome Therapeutic Market By Type (Natural Exosomes, Hybrid Exosomes), Source (Dendritic Cells, Mesenchymal Stem Cells, Blood, Milk, Body Fluids, Saliva, Urine Others), Therapy (Immunotherapy, Gene Therapy, Chemotherapy), Transporting Capacity (Bio Macromolecules, Small Molecules), Application (Oncology, Neurology, Metabolic Disorders, Cardiac Disorders, Blood Disorders, Inflammatory Disorders, Gynecology Disorders, Organ Transplantation, Others), Route of administration (Oral, Parenteral), End User (Hospitals, Diagnostic Centers, Research & Academic Institutes), Geography (North America, Europe, Asia-Pacific and Latin America)

Market Analysis and Insights:Global Exosome Therapeutic Market

Exosome therapeutic market is expected to gain market growth in the forecast period of 2019 to 2026. Data Bridge Market Research analyses that the market is growing with a CAGR of 21.9% in the forecast period of 2019 to 2026 and expected to reach USD 31,691.52 million by 2026 from USD 6,500.00 million in 2018. Increasing prevalence of lyme disease, chronic inflammation, autoimmune disease and other chronic degenerative diseases are the factors for the market growth.

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Exosomes are used to transfer RNA, DNA, and proteins to other cells in the body by making alteration in the function of the target cells. Increasing research activities in exosome therapeutic is augmenting the market growth as demand for exosome therapeutic has increased among healthcare professionals.

Increased number of exosome therapeutics as compared to the past few years will accelerate the market growth. Companies are receiving funding for exosome therapeutic research and clinical trials. For instance, In September 2018, EXOCOBIO has raised USD 27 million in its series B funding. The company has raised USD 46 million as series a funding in April 2017. The series B funding will help the company to set up GMP-compliant exosome industrial facilities to enhance production of exosomes to commercialize in cosmetics and pharmaceutical industry.

Increasing demand for anti-aging therapies will also drive the market. Unmet medical needs such as very few therapeutic are approved by the regulatory authority for the treatment in comparison to the demand in global exosome therapeutics market will hamper the market growth market. Availability of various exosome isolation and purification techniques is further creates new opportunities for exosome therapeutics as they will help company in isolation and purification of exosomes from dendritic cells, mesenchymal stem cells, blood, milk, body fluids, saliva, and urine and from others sources. Such policies support exosome therapeutic market growth in the forecast period to 2019-2026.

This exosome therapeutic market report provides details of market share, new developments, and product pipeline analysis, impact of domestic and localised market players, analyses opportunities in terms of emerging revenue pockets, changes in market regulations, product approvals, strategic decisions, product launches, geographic expansions, and technological innovations in the market. To understand the analysis and the market scenario contact us for anAnalyst Brief, our team will help you create a revenue impact solution to achieve your desired goal.

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Competitive Landscape and Exosome Therapeutic Market Share Analysis

Global exosome therapeutic market competitive landscape provides details by competitor. Details included are company overview, company financials, revenue generated, market potential, investment in research and development, new market initiatives, global presence, production sites and facilities, company strengths and weaknesses, product launch, product trials pipelines, concept cars, product approvals, patents, product width and breadth, application dominance, technology lifeline curve. The above data points provided are only related to the companys focus related to global exosome therapeutic market.

The major players covered in the report are evox THERAPEUTICS, EXOCOBIO, Exopharm, AEGLE Therapeutics, United Therapeutics Corporation, Codiak BioSciences, Jazz Pharmaceuticals, Inc., Boehringer Ingelheim International GmbH, ReNeuron Group plc, Capricor Therapeutics, Avalon Globocare Corp., CREATIVE MEDICAL TECHNOLOGY HOLDINGS INC., Stem Cells Group among other players domestic and global. Exosome therapeutic market share data is available for Global, North America, Europe, Asia-Pacific, and Latin America separately. DBMR analysts understand competitive strengths and provide competitive analysis for each competitor separately.

Many joint ventures and developments are also initiated by the companies worldwide which are also accelerating the global exosome therapeutic market.

For instance,

Partnership, joint ventures and other strategies enhances the company market share with increased coverage and presence. It also provides the benefit for organisation to improve their offering for exosome therapeutics through expanded model range.

Global Exosome Therapeutic Market Scope and Market Size

Global exosome therapeutic market is segmented of the basis of type, source, therapy, transporting capacity, application, route of administration and end user. The growth among segments helps you analyse niche pockets of growth and strategies to approach the market and determine your core application areas and the difference in your target markets.

Based on type, the market is segmented into natural exosomes and hybrid exosomes. Natural exosomes are dominating in the market because natural exosomes are used in various biological and pathological processes as well as natural exosomes has many advantages such as good biocompatibility and reduced clearance rate compare than hybrid exosomes.

Exosome is an extracellular vesicle which is released from cells, particularly from stem cells. Exosome functions as vehicle for particular proteins and genetic information and other cells. Exosome plays a vital role in the rejuvenation and communication of all the cells in our body while not themselves being cells at all. Research has projected that communication between cells is significant in maintenance of healthy cellular terrain. Chronic disease, age, genetic disorders and environmental factors can affect stem cells communication with other cells and can lead to distribution in the healing process. The growth of the global exosome therapeutic market reflects global and country-wide increase in prevalence of autoimmune disease, chronic inflammation, Lyme disease and chronic degenerative diseases, along with increasing demand for anti-aging therapies. Additionally major factors expected to contribute in growth of the global exosome therapeutic market in future are emerging therapeutic value of exosome, availability of various exosome isolation and purification techniques, technological advancements in exosome and rising healthcare infrastructure.

Rising demand of exosome therapeutic across the globe as exosome therapeutic is expected to be one of the most prominent therapies for autoimmune disease, chronic inflammation, Lyme disease and chronic degenerative diseases treatment, according to clinical researches exosomes help to processes regulation within the body during treatment of autoimmune disease, chronic inflammation, Lyme disease and chronic degenerative diseases. This factor has increased the research activities in exosome therapeutic development around the world for exosome therapeutic. Hence, this factor is leading the clinician and researches to shift towards exosome therapeutic. In the current scenario the exosome therapeutic are highly used in treatment of autoimmune disease, chronic inflammation, Lyme disease and chronic degenerative diseases and as anti-aging therapy as it Exosomes has proliferation of fibroblast cells which is significant in maintenance of skin elasticity and strength.

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Exosome therapeutic Market Country Level Analysis

The global exosome therapeutic market is analysed and market size information is provided by country by type, source, therapy, transporting capacity, application, route of administration and end user as referenced above.

The countries covered in the exosome therapeutic market report are U.S. and Mexico in North America, Turkey in Europe, South Korea, Australia, Hong Kong in the Asia-Pacific, Argentina, Colombia, Peru, Chile, Ecuador, Venezuela, Panama, Dominican Republic, El Salvador, Paraguay, Costa Rica, Puerto Rico, Nicaragua, Uruguay as part of Latin America.

Country Level Analysis, By Type

North America dominates the exosome therapeutic market as the U.S. is leader in exosome therapeutic manufacturing as well as research activities required for exosome therapeutics. At present time Stem Cells Group holding shares around 60.00%. In addition global exosomes therapeutics manufacturers like EXOCOBIO, evox THERAPEUTICS and others are intensifying their efforts in China. The Europe region is expected to grow with the highest growth rate in the forecast period of 2019 to 2026 because of increasing research activities in exosome therapeutic by population.

The country section of the report also provides individual market impacting factors and changes in regulation in the market domestically that impacts the current and future trends of the market. Data points such as new sales, replacement sales, country demographics, regulatory acts and import-export tariffs are some of the major pointers used to forecast the market scenario for individual countries. Also, presence and availability of global brands and their challenges faced due to large or scarce competition from local and domestic brands, impact of sales channels are considered while providing forecast analysis of the country data.

Huge Investment by Automakers for Exosome Therapeutics and New Technology Penetration

Global exosome therapeutic market also provides you with detailed market analysis for every country growth in pharma industry with exosome therapeutic sales, impact of technological development in exosome therapeutic and changes in regulatory scenarios with their support for the exosome therapeutic market. The data is available for historic period 2010 to 2017.

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Exosome therapeutic Market Segmentation, Parameters, Prospects 2021 And Forecast Research Report To 2027 KSU | The Sentinel Newspaper - KSU | The...

2021 Updates in Autologous Stem Cell Based Therapies Industry with Global Market Demand Analysis, Industry Chain, Revenue and Forecast 2026 – The…

The Latest Released Autologous Stem Cell Based Therapies market study has evaluated the future growth potential of the Global Autologous Stem Cell Based Therapies Industry and provides information and useful stats on market structure and size. The report is intended to provide market intelligence and strategic insights to help decision-makers take sound investment decisions and identify potential gaps and growth opportunities.

Additionally, the Autologous Stem Cell Based Therapies Market report also identifies and analyses changing dynamics, emerging trends along with essential drivers, challenges, opportunities, and restraints in the Autologous Stem Cell Based Therapies market, which will help the future market to grow with promising CAGR and offers an extensive collection of reports on different markets covering crucial details. The report studies the competitive environment of the Autologous Stem Cell Based Therapies Market is based on company profiles and their efforts on increasing product value and production.

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Keep yourself up to date with the latest market trends and changing dynamics due to COVID Impact and Economic Slowdown globally. Maintain a competitive edge by sizing up with available business opportunities in Autologous Stem Cell Based Therapies Market various segments and emerging territory.

The research offers detailed segmentation of the global Autologous Stem Cell Based Therapies market. Key segments analyzed in the research include Type and Application.

By Type:

By Application:

The report will include a market analysis of Autologous Stem Cell Based Therapies which includes Business to Business (B2B) transactions as well as Autologous Stem Cell Based Therapies aftermarket. The market value has been determined by analyzing the revenue generated by the companies solely. R&D, any third-party channel cost, consulting cost and any other cost except company revenue has been neglected during the analysis of the market. A comprehensive analysis will be provided covering the following points in the report:

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Top Key Players included in Autologous Stem Cell Based Therapies Market:

Region Included are: North America, Europe, Asia Pacific, Oceania, South America, Middle East & AfricaCountry Level Break-Up: United States, Canada, Mexico, Brazil, Argentina, Colombia, Chile, South Africa, Nigeria, Tunisia, Morocco, Germany, United Kingdom (UK), the Netherlands, Spain, Italy, Belgium, Austria, Turkey, Russia, France, Poland, Israel, United Arab Emirates, Qatar, Saudi Arabia, China, Japan, Taiwan, South Korea, Singapore, India, Australia and New Zealand, etc.

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Table of Content For Autologous Stem Cell Based Therapies Market Report

Chapter 1. Research Objective

Chapter 2. Executive Summary

Chapter 3. Strategic Analysis

Chapter 4. Autologous Stem Cell Based Therapies Market Dynamics

Chapter 5. Segmentation & Statistics

Chapter 6. Market Use case studies

Chapter 7. KOL Recommendations

Chapter 8. Investment Landscape

Chapter 9. Competitive Intelligence

Chapter 10. Company Profiles

Chapter 11. Appendix

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2021 Updates in Autologous Stem Cell Based Therapies Industry with Global Market Demand Analysis, Industry Chain, Revenue and Forecast 2026 - The...

[Full text] A Comprehensive Review on Factors Influences Biogenesis, Functions, Th | IJN – Dove Medical Press

Introduction

Extracellular vesicles (EVs) including exosomes, microvesicles, and apoptotic bodies are produced and released by almost all types of cell. EVs vary in size, properties, and secretion pathway depending on the originating cell.1,2 Exosomes are small EVs (sEVs) which are formed by a process of inward budding in early endosomes to form multivesicular bodies (MVBs) with an average size of 100 nm, and released into the extracellular microenvironment to transfer their components.3,4 Microvesicles are composed of lipid components of the plasma membrane and their sizes range from 1001000 nm, whereas apoptotic bodies result from programmed cell death.5 Initially, EVs were considered to maintain cellular waste through release of unwanted proteins and biomolecules; later, these organelles were considered important for intercellular communications through various cargo molecules such as lipids, proteins, DNA, RNA, and microRNAs (miRNAs).6 Previously, it was suggested that EVs play a critical role in normal cells to maintain homeostasis and prevent cancer initiation. Inhibition of EVs secretion causes accumulation of nuclear DNA in the cytoplasm, leading to apoptosis.1 The induction of apoptosis is the principal event of the reactive oxygen species (ROS) dependent DNA damage response.7,8

Several studies reported that exosomes are synthesized by means of two major pathways, the endosomal sorting complexes required for transport (ESCRT)-dependent and ESCRT-independent, and the processes are highly regulated by multiple signal transduction cascades.18 Exosomes released from the cell through normal exocytosis mechanisms are characterized by vesicular docking and fusion with the aid of SNARE complexes. Exosomes are considered to be organelle responsible for garbage disposal agents. However, at a later stage, these secretory bodies play a critical role in maintaining the physiological and pathological conditions of the surrounding cells by transferring information from donor cells to recipient cells. Exosome development begins with endocytosis to form early endosomes, later forming multivesicular endosomes (MVEs), and finally generating late endosomes by inward budding. MVEs merge with the cell membrane and release intraluminal endosomal vesicles that become exosomes into the extracellular space.9,10 Exosome biogenesis is dependent on various critical factors including the site of biogenesis, protein sorting, physicochemical aspects, and transacting mediators.11

Exosomes contain various types of cargo molecules including lipids, proteins, DNAs, mRNAs, and miRNAs. Most of the cargo is involved in the biogenesis and transportation ability of exosomes.12,13 Exosomes are released by fusion of MVBs with the cell membrane via activation of Rab-GTPases and SNAREs. Exosomes are abundant and can be isolated from a wide variety of body fluids and also cell culture medium.14 Exosomes contain tetraspanins that are responsible for cell penetration, invasion, and fusion events. Exosomes are released onto the external surface by the MVB formation proteins Alix and TSG101. Exosome-bound proteins, annexins and Rab protein, govern membrane transport and fusion whereas Alix, flotillin, and TSG101 are involved in exosome biogenesis.15,16 Exosomes contain various types of proteins such as integral exosomal membrane proteins, lipid-anchored outer and inner membrane proteins, peripheral surface and inner membrane proteins, exosomal enzymes, and soluble proteins that play critical roles in exosome functions.11

The functions of exosomes depend on the origin of the cell/tissue, and are involved in the immune response, antigen presentation programmed cell death, angiogenesis, inflammation, coagulation, and morphogen transporters in the creation of polarity during development and differentiation.1720 Ferguson and Nguyen reported that the unique functions of exosomes depend on the availability of unique and specific proteins and also the type of cell.21 All of these categories influence cellular aspects of proteins such as the cell junction, chaperones, the cytoskeleton, membrane trafficking, structure, and transmembrane receptor/regulatory adaptor proteins. The role of exosomes has been explored in different pathophysiological conditions including metabolic diseases. Exosomes are extremely useful in cancer biology for the early detection of cancer, which could increase prognosis and survival. For example, the presence of CD24, EDIL3, and fibronectin proteins on circulating exosomes has been proposed as a marker of early-stage breast cancer.22 Cancer-derived exosomes promoted tumor growth by directly activating the signaling pathways such as P13K/AKT or MAPK/ERK.23 Tumor-derived exosomes are significantly involved in the immune system, particularly stimulating the immune response against cancer and delivering tumor antigens to dendric cells to produce exosomes, which in turn stimulates the T-cell-mediated antitumor immune response.24 Exosomal surface proteins are involved in immunotherapies through the regulation of the tumor immune microenvironment by aberrant cancer signaling.25 A study demonstrated that exosomes have the potential to affect health and pathology of cells through contents of the vesicle.26 Exosomes derived from mesenchymal stem cells exhibit protective effects in stroke models following neural injury resulting from middle cerebral artery occlusion.27 The structural region of the exosome facilitate the release of misfolded and prion proteins, and are also involved in the propagation of neurodegenerative diseases such as Huntington disease, Alzheimers disease (AD), and Parkinsons disease (PD).28,29

Exosomes serve as novel intercellular communicators due to their cell-specific cargo of proteins, lipids, and nucleic acids. In addition, exosomes released from parental cells may interact with target cells, and it can influence cell behavior and phenotype features30 and also it mediate the horizontal transfer of genetic material via interaction of surface adhesion proteins.31 Exosomes are potentially serving as biomarkers due to the wide-spread and cell-specific availability of exosomes in almost all body fluids.13 Therefore, exosomes are exhibited as delivery vehicles for the efficient delivery of biological therapeutics across different biological barriers to target cells.3234

In this review, first, we comprehensively describe the factors involved in exosome biogenesis and the role of exosomes in intercellular signaling and cell-cell communications, immune responses, cellular homeostasis, autophagy, and infectious diseases. In addition, we discuss the role of exosomes as diagnostic markers, and the therapeutic and clinical implications. Finally, we discuss the challenges and outstanding developments in exosome research.

The extracellular vesicles play critical role in inter cellular communication by serving as vehicles for transfer of biomolecules. These vesicles are generally classified into microvesicles, ectosomes, shedding vesicles, or microparticles. MVs bud directly from the plasma membrane, whereas exosomes are represented by small vesicles of different sizes that are formed as the ILV by budding into early endosomes and MVBs and are released by fusion of MVBs with the plasma membrane (Figure 1). Invagination of late endosomal membranes results in the formation of intraluminal vesicles (ILVs) within large MVBs.35 Biogenesis of exosomes occurs in three ways including vesicle budding into discrete endosomes that mature into multivesicular bodies, which release exosomes upon plasma membrane fusion; direct vesicle budding from the plasma membrane; and delayed release by budding at intracellular plasma membrane-connected compartments (IPMCs) followed by deconstruction of IPMC neck(s).11 The mechanisms of biogenesis of exosomes are governed by various types of proteins including the ESCRT proteins Hrs, CHMP4, TSG101, STAM1, VPS4, and other proteins such as the Syndecan-syntenin-ALIX complex, nSMase2, PLD2, and CD9.14,3639 After formation, the MVB can either fuse with the lysosome to degrade its content or fuse with the plasma membrane to release the ILVs as exosomes. The release of exosomes to the extracellular milieu is driven by proteins of the Rab-GTPase family including RAB2B, 5A, 7, 9A, 11, 27, and 35. SNARE family proteins VAMP7 and YKT6 have also been implicated in the release.14,38,4042 Biogenesis of exosomes is influenced by several external factors including cell type, cell confluency, serum conditions, and the presence and absence of cytokines and growth factors. In addition, biogenesis is also regulated by the sites of exosomes, protein sorting, physico-chemical aspects, and trans-acting mediators (Figure 2). For example, THP-1 cells were cultured in RPMI-1640 cell culture medium supplemented with 10% FCS secreted low level of exosomes compared to cells grown on cell culture medium supplemented with 1% FCS (Figure 3). The exogenous factor like serum starvation influences biogenesis and secretion of exosomes.

Figure 1 Biogenesis and cargoes of exosomes.

Figure 2 Effect of various factors on biogenesis of exosomes.

Figure 3 Serum deprivation causes an increase of the number of cellular exosomes in THP-1 cells. Panel (A); 10% FCS. Panel (B); 1% FCS. Panel (C) Quantification of exosomes using DLS and NTA.

Exosome release depends on expression of Rab27 or Ral. For example, exosomes released from the MVB significantly decrease in cells depleted of Rab2741 or Ral.43 The most efficient EV-producing cell types have yet to be determined44 and few reports suggest that immature dendritic cells produce limited amounts of EVs45,46 whereas mesenchymal stem cells secrete vast amounts, relevant for the production of EV therapeutics on a clinical scale.47,48 A few proteins play a critical role in the biogenesis of EVs, such as Rab27a and Rab27b.49 Over expression of Rab27a and Rab27b produce significant amounts of EVs in cancer cells. For example, overexpression of Rab27a and Rab27b in breast cancer cells,50 hepatocellular carcinoma cells,51 glioma cells,52 and pancreas cancer cells53 produces significant levels of EVs. Although all types of cells secrete and release EVs, cancer cells seem to produce higher levels than normal cells.54 Furthermore, the presence of invadopodia that are docking sites for Rab27a-positive MVBs induces secretion of EVs, and also enhances secretion of EVs in cancer cells.55 Thus, inhibition of invadopodia formation greatly reduces exosome secretion into conditioned media. This evidence demonstrates that cancer cells potentially release more EVs than non-cancer cells.

The rate of origin of exosomes from the plasma membrane of stem cells is vigorous, at rates equal to the production of exosomes,56 which is consistent with a report suggesting that stem cells bud ~50100 nm-diameter vesicles directly from the plasma membrane.57 Plasma membrane-derived exosomes contain selectively enriched protein and lipid markers in leukocytes.58 Plasma membrane exosomal budding is also observed for glioblastoma exosomes.59 Conventional transmission electron microscopy revealed that certain cell types contain deep invaginations of the plasma membrane that are indistinguishable from MVBs.6062 Certain cell types secrete exosomes containing cargo proteins, which primarily bud from the plasma membrane, and exosome composition is determined predominantly by intracellular protein trafficking pathways, rather than by the distinct mechanisms of exosome biogenesis.63 Biogenesis of exosomes is regulated by syndecan heparan sulphate proteoglycans and their cytoplasmic adaptor syntenin. Syntenin interacts directly with ALIX through LYPX (n) L motifs.64 Glycosylation is an essential factor in the biogenesis of exosomes and N-linked glycosylation directs glycoprotein sorting into EMVs.65 Collectively, these reports suggest that exosomes are made at both plasma and endosome membranes rather than endosome alone. Oligomerization is a critical factor for exosomal protein sorting and it was found to be sufficient to target plasma membrane proteins to exosomes. High-order oligomeric proteins target them to exosomes.66 Further, plasma membrane anchors support exosomal protein budding. For example, budding of CD63 and CD9 from the plasma membrane is much more efficient than endosome-targeted budding of CD63 and CD9.63 Protein clustering is another factor that induces membrane scission.67

Physico-chemical properties determine budding efficiency and are crucial factors of exosome biogenesis, a fundamental process involving the budding of vesicles that are 30200 nm in size. In particular, lipids are critical players in exosome biogenesis, especially those able to form cone and inverse cone shapes. Generally, exosome membranes contain phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylinositols (PIs), phosphatidic acid (PA), cholesterol, ceramides, sphingomyelin, glycosphingolipids, and a number of lower abundance lipids.68,69 Exosomes have a rich content of PE and PS, which increase budding efficiency and promote exosome genesis and release. PA promotes exosome biogenesis and PLD2 is involved in the budding of certain exosomal cargoes.70 Besides these factors, ceramide is an important lipid molecule regulating exosome biogenesis and facilitating membrane curvature, which is essential for vesicular budding. Inhibition of an enzyme that generates ceramide impairs exosome biogenesis.71

The next critical factor is trans-acting mediators that are involved in the biogenesis of exosomes through regulating plasma membrane homeostasis, intracellular protein trafficking pathways, MVB maturation and trafficking, IPMC biogenesis, vesicle budding, and scission.11 For example, Rab proteins regulate exosome biogenesis via endosomes and the plasma membrane by determining organelle membrane identity, recruiting mechanistic effectors, and mediating organelle dynamics.72 The functions of Rab proteins in the control and biogenesis of exosomes depends on cell type. MVB biogenesis is regulated by Rab27a, Rab27b, their effectors Slp4, Slac2b, and Munc13-4, and also Rab 35 and Rab 11.73 Loss of Rab27 function leads to a ~5075% drop in exosome production, and is also involved in assembling the plasma membrane microdomains involved in plasma membrane vesicle budding, by regulating plasma membrane PIP2 dynamics.74 Overall, Rab27 proteins control exosome biogenesis at both endosomes and plasma membranes. In addition, Rab35 also contributes to exosome biogenesis by regulating PIP2 levels of plasma membrane, and its loss leads to a reduction of exosome release by ~50%.75 Gurunathan et al76 reported that yeast produces two classes of secretory vesicles, low density and high density, and dynamin and clathrin are required for the biogenesis of these two different types of vesicle.

The Ral family is involved in the biogenesis of exosomes, and inhibition of Ral causes an accumulation of MVBs near the plasma membrane and a ~50% decrease in the vesicular secretion of exosomes and exosomal marker proteins.43 Ral GTPases function through various effectors proteins, including Arf6 and the phospholipase PLD2, which are involved in exosomal release of SDCs.37 The ESCRT complex machinery (0 through III) are involved in MVB biogenesis on a major level including membrane deformation, sealing, and repair during a wide array of processes. The major contributions of the ESCRT complex to the biogenesis of vesicles are the recognition and sequestration of ubiquitinated proteins to specific domains of the endosomal membrane via ubiquitin binding subunits of ESCRT-0. After interaction with the ESCRT-I and -II complexes, the total complex will then combine with ESCRT-III, a protein complex that is involved in promoting the budding process. Finally, following cleaving of the buds to form ILVs, the ESCRT-III complex separates from the MVB membrane using energy supplied by the sorting protein Vps4.77 In addition, other proteins such as Alix, which is associated with several ESCRT (TSG101 and CHMP4) proteins, are involved in endosomal membrane budding and abscission, as well as exosomal cargo selection via interaction with syndecan.39 Another important factor, autophagy, is critically involved in exosome secretion. Autophagy related (Atg) proteins coordinate initiation, nucleation, and elongation during autophagosome biogenesis in the presence of ESCRT-III components including CHMP2A and VPS4. For instance, the absence of Atg5 in cancer cells causes a reduction in exosome production.78 Conversely, CRISPR/Cas9-mediated knockout of Atg5 in neuronal cells increases the release of exosomes and exosome-associated prions from neuronal cells.79

Exosomes play a critical role in the physiologic regulation of mammary gland development and are important mediators of breast tumorigenesis.80 Biogenesis of exosomes occurs in all cell types; however, production depends on cell type. For example, breast cancer cells (BCC) produce increased numbers of exosomes compared to normal mammary epithelial cells. Studies revealed that patients with BC have increased numbers of MVs in their blood.81 Kavanagh et al reported that several fold changes were observed from exosomes isolated from triple negative breast cancer (TNBC) chemoresistant therapeutic induced senescent (TIS) cells compared with control EVs.82 TIS cells release significantly more EVs compared with control cells, containing chemotherapy and key proteins involved in cell proliferation, ATP depletion, and apoptosis, and exhibit the senescence-associated secretory phenotype (SASP). Cannabidiol (CBD), inhibits exosome and microvesicle (EMV) release in three different types of cancer cells including prostate cancer (PC3), hepatocellular carcinoma (HEPG2), and breast adenocarcinoma (MDA-MB-231). All three different cell lines show variability in the release of exosomes in a dose-dependent manner. These variabilities are all due to mitochondrial function, including modulation of STAT3 and prohibitin expression. This study suggests that the anticancer agent CBD plays critical role in EMV biogenesis.83 Sulfisoxazole (SFX) inhibits sEV secretion from breast cancer cells through interference with endothelin receptor A (ETA) through the reduced expression of proteins involved in the biogenesis and secretion of sEV, and triggers co-localization of multivesicular endosomes with lysosomes for degradation.84 Secreted EVs from human colorectal cancer cells contain 957 vesicular proteins. The direct protein interactions between cellular proteins play a critical role in protein sorting during EV formation. SRC signaling plays a major role in EV biogenesis, and inhibition of SRC kinase decreases the intracellular biogenesis and cell surface release of EVs.85 Proteomic analysis revealed that the exosomes released from imatinib-sensitive GIST882 cell line exhibit 764 proteins. The authors found that significant amount of proteins belong to protein release function and involved in the classical pathway and overlap to a high degree with proteins of exosomal origin.86 Exosomes secreted by antigen-presenting cells contain high levels of MHC class II proteins and costimulatory proteins, whereas exosomes released from other cell types lack these proteins.1,87

The biogenesis of exosomes depends on a percentage of confluency of approximately 6090%, which influences the yield and functions of EVs.44 Gal et al88 observed a 10-fold decreased level of cholesterol metabolism in confluent cell cultures compared to cells in the preconfluent state. The high level of cholesterol content in confluent cells leads to a decreased level of EVs in prostate cancer.68 The major reason behind for the reduced level of vesicle production is contact inhibition, which triggers confluent cells to enter quiescence and/or alters their characteristics compared to actively dividing cells.89,90 Exogenous stimulation could influence the condition of the cells including the phenotype and efficacy of secretion. Previously, several studies demonstrated that various external factors increase biogenesis of EVs such as Ca2+ ionophores,91 hypoxia,9294 and detachment of cells,95 whereas lipopolysaccharide reduces biogenesis and release of EVs.96 Furthermore, serum, which supports adherence of the cells, plays a critical role in the biogenesis of EVs.97 For example, FCS has noticeable effects on cultured cells; however, the effects depend on cell type and differentiation status.97,98 To avoid the immense amounts of vesicles present in FCS, the use of conditioned media has been suggested. Culture viability and health status of cells are important aspects for producing an adequate amount of vesicles with proper cargo molecules such as protein and RNA.99,100 Exogenous stress, such as starvation, can induce phenotypic alterations and changes in proliferation. These changes cause alterations in the cells metabolism and eventually lead to low yields.101,102

Cellular stresses, such as hypoxia, inflammation, and hyperglycemia, influence the RNA and protein content in exosomes. To examine these factors, the effects of cellular stresses on endothelial cells were studied.99 Endothelial cells were exposed to different types of cellular stress such as hypoxia, tumor necrosis factor- (TNF-)-induced activation, and high glucose and mannose concentrations. The mRNA and protein content of exosomes produced by these cells were compared using microarray analysis and a quantitative proteomics approach. The results indicated that endothelial cell-derived exosomes contain 1354 proteins and 1992 mRNAs. Several proteins and mRNAs showed altered levels after exposure of their producing cells to cellular stress. Interestingly, cells exposed to high sugar concentrations had altered exosome protein composition only to a minor extent, and exosome RNA composition was not affected. Low-intensity ultrasound-induced (LIUS) anti-inflammatory effects have been achieved by upregulation of extracellular vesicle/exosome biogenesis. These exosomes carry anti-inflammatory cytokines and anti-inflammatory microRNAs, which inhibit inflammation of target cells via multiple shared and specific pathways. A study suggested that exosome-mediated anti-inflammatory effects of LIUS are feasible and that these techniques are potential novel therapeutics for cancers, inflammatory disorders, tissue regeneration, and tissue repair.103 Another factor, called manumycin-A (MA), a natural microbial metabolite, was analyzed in exosome biogenesis and secretion in castration-resistant prostate cancer (CRPC) C4-2B, cells. The effect of MA on cell growth was observed, and the results revealed that there was no effect on cell growth. However, MA attenuated the ESCRT-0 proteins Hrs, ALIX, and Rab27a, and exosome biogenesis and secretion by CRPC cells. The inhibitory effect of MA on exosome biogenesis and secretion was primarily mediated via targeted inhibition of Ras/Raf/ERK1/2 signaling. These findings suggest that MA is a potential drug candidate for the suppression of exosome biogenesis and secretion by CRPC cells.104

Methods of isolation of exosomes play critical roles in functions and delivery. Although several methods such as ultracentrifugation, density gradient centrifugation, chromatography, filtration, polymer-based precipitation, and immunoaffinity have been adopted to isolate pure exosomes without contamination, there is still a lack of consistency and agreement.105 Isolation of exosomes along with non-exosomal materials and damaged exosomal membranes creates artifacts and alters the protein and RNA profiles. Since exosomes are obtained from a variety of sources, the composition of proteins/lipids influences the sedimentation properties and isolation. Thus, precise and consistent techniques are warranted for the isolation, purification, and application of exosomes.

Although several functions of exosomes have been explored, the precise function of exosomes remains a mystery. Historically, exosomes have been known to function as cellular garbage bags, recyclers of cell surface proteins, cellular signalers, intercellular signaling and cell-cell communications, immune responses, cellular homeostasis, autophagy, and infectious diseases.106 (Figure 4) ECVs are secreted cell-derived membrane particles involved in intercellular signaling and cell-cell communications, and contain immense bioactive information. Most cell types produce exosomes and release these into the extracellular environment, circulating through different bodily fluids such as urine, blood, and saliva and transferring their cargo to recipient cells. These vesicles play a significant role in various pathological conditions, such as different types of cancer, neurodegenerative diseases, infectious diseases, pregnancy complications, obesity, and autoimmune diseases, as reviewed elsewhere.107 Exosomes play a significant role in intercellular communication between cells by interacting with target cells via endocytosis.108 More specifically, exosomes are involved in cancer development, survival and metastasis of tumors, drug resistance, remodeling of the extracellular matrix, angiogenesis, thrombosis, and proliferation of tumor cells.94,109111 Exosomes contribute significantly to tumor vascularization and hypoxia-mediated inter-tumor communication during cancer progression, and premetastatic niches, which are significant players in cancer.16,94,109,112 Exosomes derived from hepatic epithelial cells increase the expression of enhancer zeste homolog 2 (EZH2) and cyclin-D1, and subsequently promotes G1/S transition.113

Figure 4 Multifunctional aspects biological functions of exosomes.

Conventionally, cells communicate with adjacent cells through direct cell-cell contact through gap junctions and cell surface protein/protein interactions, whereas cells communicating with distant cells do so through secreted soluble factors, such as hormones and cytokines, to facilitate signal propagation.114 Cells also communicate through electrical and chemical signals.115 Several studies have suggested that exosomes play vital roles in intercellular communication by serving as vehicles for transferring various cellular constituents, such as proteins, lipids, and nucleic acids, between cells.6,116118 Exosomes function as exosomal shuttle RNAs in which some exosomal RNAs from donor cells functions in recipient cells,6 a form of genetic exchange. Recently, researchers found that cells communicating with other cells through exosomes carrying cell-specific cargoes of proteins, lipids, and nucleic acids may employ novel intercellular communication mechanisms.30 Exosomes exert influences through various mechanistic approaches, such as direct stimulation of target cells via surface-bound ligands; transfer of activated receptors to recipient cells; and epigenetic reprogramming of recipient cells.119,120 Exosomes play critical roles in immunoregulation, including antigen presentation, immune activation, immune suppression, and immune tolerance via exosome-mediated intercellular communication. Mesenchymal stem cell (MSC)-derived exosomes play significant roles in wound healing processes.121 Exosomes from platelet-rich plasma (PRP) inhibit the release of TNF-. PRP-Exos significantly decreases the apoptotic rate of osteoarthritis (OA) chondrocytes compared with activated PRP (PRP-As).122 Extracellular vesicle (ECV)-modified polyethylenimine (PEI) complexes enhance short interfering RNA (siRNA) delivery by forming non-covalent complexes with small RNA molecules, including siRNAs and anti-miRs, in both conditions, in vitro and in vivo.123 Non-GSC glioma cells were treated with GSC-released exosomes. The results showed that GSC-released exosomes increase proliferation, neurosphere formation, invasive capacities, and tumorigenicity of non-GSC glioma cells through the Notch1 signaling pathway and stemness-related protein expressions.124

Exosomal miR-1910-3p promotes proliferation and migration of breast cancer cells in vitro and in vivo through downregulation of myotubularin-related protein 3 and activation of the nuclear factor-B (NF-B) and wnt/-catenin signaling pathway, and promotes breast cancer progression.125 Human hepatic progenitor cell (CdH)-derived exosomes (EXOhCdHs) play a crucial role in maintaining cell viability and inhibit oxidative stress-induced cell death. Experimental evidence suggests that inhibition of exosome secretion treatment with GW4869 results in the acceleration of reactive oxygen species (ROS) production, thereby causing a decrease in cell viability.126 Tumor-derived EXs (TDEs) are vehicles that enable communication between cells by transferring bioactive molecules, also delivering oncogenic molecules and containing different molecular cargoes compared to EXs delivered from normal cells. They can therefore be used as non-invasive biomarkers for the early diagnosis and prognosis of most cancers, including breast and ovarian cancers.127 Exosomes released by ER-stressed HepG2 cells significantly enhance the expression levels of several cytokines, including IL-6, monocyte chemotactic protein-1, IL-10, and tumor necrosis factor- in macrophages. ER stress-associated exosomes mediate macrophage cytokine secretion in the liver cancer microenvironment, and also indicate the potential of treating liver cancer via an ER stress-exosomal-STAT3 pathway.128 Mesenchymal stem cell-derived exosomal miR-223 protects neuronal cells from apoptosis, enhances cell migration and increases miR-223 by targeting PTEN, thus activating the PI3K/Akt pathway. In addition, exosomes isolated from the serum of AD patients promote cell apoptosis through the PTEN-PI3K/Akt pathway and these studies indicate a potential therapeutic approach for AD.129 A mouse model of diabetes demonstrated that mesenchymal stromal cell-derived exosomes ameliorate peripheral neuropathy through increased nerve conduction velocity. In addition, MSC-derived exosomes substantially suppress proinflammatory cytokines.130

Exosomes derived from activated astrocytes promote microglial M2 phenotype transformation following traumatic brain injury (TBI). miR-873a-5p significantly inhibits LPS-induced microglial M1 phenotype transformation.131 Several studies reported that exosomes are involved in cancer progression and metastasis; however, this depends on the type of cells the exosomes were derived from. For example, human umbilical vein endothelial cells (HUVEC) were treated with exosomes derived from HeLa cells (ExoHeLa), and the expression of tight junctions (TJ) proteins, such as zonula occludens-1 (ZO-1) and Claudin-5, was significantly reduced compared with exosomes from human cervical epithelial cells. Thus, permeability of the endothelial monolayer was increased after the treatment with ExoHeLa. Mice studies have shown that injection of ExoHeLa into mice increased vascular permeability and tumor metastasis. The results from this study demonstrated that HeLa cell-derived exosomes promote metastasis by triggering ER stress in endothelial cells and break down endothelial integrity. Such effects of exosomes are microRNA-independent.132 Exosomes mediate the gene expression of target cells and regulate pathological and physiological processes including promoting angiogenesis, inhibiting ventricular remodeling and improving cardiac function, as well as inhibiting local inflammation and regulating the immune response. Accumulating evidence shows that exosomes possess therapeutic potential through their anti-apoptotic and anti-fibrotic roles.

The functions of exosomes in immune responses are well established and do not cause any severe immune responses. A mouse study demonstrated that administration of a low dose of mouse or human cell-derived exosomes for extended periods of time caused no severe immune reactions.133 The function of exosomes in immune regulation is regulated by the transfer and presentation of antigenic peptides. Exosomes contain antigen-presenting cells (APCs) carrying peptide MHC-II and costimulatory signals and directly present the peptide antigen to specific T cells to induce their activation.134 For example, intradermal injection of APC-derived exosomes with MHC-II loaded with tumor peptide delayed tumor progression and growth.135 Exosome-derived immunogenic peptides activate immature mouse dendritic cells and indirectly activate APCs, and induce specific CD4+ T cell proliferation.136 Exosomes containing IFNa and IFNg, tumor necrosis factor a (TNFa), and IL from macrophages promoted dendritic cell maturation, CD4+ and CD8+ T cell activation, and the regulation of macrophage IL expression.137 The cargo of exosomes, such as DNA and miRNA, regulate the innate and adaptive immune responses. Exosomes are able to regulate the immune response by controlling gene expression and signaling pathways in recipient cells through transfer of miRNAs, and eventually control dendritic cell maturation.138 Exosomes containing miR-212-3p derived from tumors down-regulate the MHC-II transcription factor RFXAP (regulatory factor X associated protein) in dendritic cells, possibly promoting immune evasion by cancer cells.139 Exosomes containing miR-222-3p down regulate expression of SOCS3 (suppressor of cytokine signaling 3) in monocytes, which is involved in STAT3-mediated M2 polarization of macrophages.140 In mice, exosomes stimulate adaptive immune responses, including the activation of dendritic cells, with the uptake of breast cancer cell-derived exosomal genomic DNA and activation of cGAS-STING signaling and antitumor responses.141 The priming of dendritic cells is associated with the uptake of exosomal genomic and mitochondrial DNA (mtDNA) from T cells, inducing type I IFN production by cGAS-STING signaling.142 Inhibition of EGFR leads to increased levels of DNA in the exosomes and induces cGAS-STING signaling in dendritic cells, contributing to the overall suppression of tumor growth.143 Conversely, uptake of tumor-derived exosomal DNA by circulating neutrophils was shown to enhance the production of tissue factor and IL-8, which play a role in promoting tumor inflammation and paraneoplastic events.144 Melanoma-derived exosomes containing PD-L1 (programmed cell death ligand 1) suppress CD8+ T cell antitumor function and cancer cell-derived exosomes block dendritic cell maturation and migration in a PD-L1-dependent manner. Engineered cancer cell-derived exosomes promote dendritic cell maturation, resulting in increased proliferation of T cells and antitumor activity.145147

Inflammation is an important process for maintaining homeostasis in cellular systems. Systemic inflammation is an essential component in the pathogenesis of several diseases.148,149 Exosomes seem to play a crucial role in inflammation processes through cargo molecules, such as miRNA and proteins, which act on nearby as well as distant target tissues. Exosomes play a vital role in intercellular communication between cells via endocytosis and are associated with modulation of inflammation, coagulation, angiogenesis, and apoptosis.20,150153 Exosomes derived from dendritic cells, B lymphocytes, and tumor cells release exosomes that can regulate immunological memory through the surface expression of antigen-presenting MHC I and MHC II molecules, and subsequently elicit T cell activation and maturation.134,137,154156 Exosomes play a crucial role in carrying and presenting functional MHC-peptide complexes to modulate antigen-specific CD8+ and CD4+ responses.157,158 Exosomes containing miR-Let-7d influence the growth of T helper 1 (Th1) cells and inhibit IFN- secretion.159 Exosomes derived from choroid plexus epithelial cells containing miR-146a and miR-155 upregulate the expression of inflammatory cytokines in astrocytes and microglia.160 Exosomes containing miR-181c suppress the expression of Toll-like receptor 4 (TLR-4) and subsequently lower TNF- and IL-1 levels in burn-induced inflammation.161 Exosomal miR-155 from bone marrow cells (BMCs) increases the level of TNF- and subsequently enhances innate immune responses in chronic inflammation.162 Exosomes containing miR-150-5p and miR-142-3p derived from dendritic cells (DCs) increase expression of interleukin 10 (IL-10) and a decrease in IL-6 expression.163 Exosomal miR-138 can protect against inflammation by decreasing the expression level of NF-B, a transcription factor that regulates inflammatory cytokines such as TNF- and IL-18.164 HIF-1-inducing exosomal microRNA-23a expression from tubular epithelial cells mediates the cross talk between tubular epithelial cells and macrophages, promoting macrophage activation and triggering tubulointerstitial inflammation.165 A rat model study demonstrated that bone marrow mesenchymal stem cell (BMSC)-derived exosomes reduced inflammatory responses by modulating microglial polarization and maintaining the balance between M2-related and M1-related cytokines.165 Melatonin-stimulated mesenchymal stem cell (MSC)-derived exosomes improve diabetic wound healing through regulating macrophage M1 and M2 polarization by targeting the PTEN/AKT pathway, and significantly suppressed the pro-inflammatory factors IL-1 and TNF- and reduced the relative gene expression of IL-1, TNF-, and iNOS. Increasing levels of anti-inflammatory factor IL-10 are associated with increasing relative expression of Arg-1.166

Immunomodulators are essential factors for the prevention and treatment of disorders occurring due to an over high-spirited immune response, such as the SARS-CoV-2-triggered cytokine storm leading to lung pathology and mortality seen during the ongoing viral pandemic.167 MSC-secreted extracellular vesicles exhibit immunosuppressive capacity, which facilitates the regulation of the migration, proliferation, activation, and polarization of various immune cells, promoting a tolerogenic immune response while inhibiting inflammatory responses.168 Collagen scaffold umbilical cord-derived mesenchymal stem cell (UC-MSC)-derived exosomes induce collagen remodeling, endometrium regeneration, increasing the expression of the estrogen receptor /progesterone receptor, and restoring fertility. Furthermore, exosomes modulate CD163+ M2 macrophage polarization, reduce inflammation, increase anti-inflammatory responses, facilitate endometrium regeneration, and restore fertility through the immunomodulatory functions of miRNAs.169 Exosomes released into the airways during influenza virus infection trigger pulmonary inflammation and carry viral antigens and it facilitate the induction of a cellular immune response.170 Shenoy et al171 reported that exosomes derived from chronic inflammatory microenvironments contribute to the immune suppression of T cells. These exosomes arrest the activation of T cells stimulated via the T cell checkpoint (TCR). Exosomes secreted by normal retinal pigment epithelial cells (RPE) by rotenone-stimulated ARPE-19 cells induce apoptosis, oxidative injury, and inflammation in ARPE-19 cells. Exosomes secreted under oxidative stress induce retinal function damage in rats and upregulate expression of Apaf1. Overexpression of Apaf1 in exosomes secreted under oxidative stress (OS) can cause the inhibition of cell proliferation, increase in apoptosis, and elicitation of inflammatory responses in ARPE-19 cells. Exosomes derived from ARPE-19 cells under OS regulate Apaf1 expression to increase apoptosis and to induce oxidative injury and inflammatory response through a caspase-9 apoptotic pathway.172 Collectively, these findings highlight the critical role of exosomes in inflammation and suggest the possibility of utilizing exosomes as an inducer to attenuate inflammation and restore impaired immune responses in various diseases including cancer.

The endomembrane system of eukaryotic cells is a complex series of interconnected membranous organelles that play vital roles in protecting cells from adverse conditions, such as stress, and maintaining cell homeostasis during health and disease.173 To preserve cellular homeostasis, higher eukaryotic cells are equipped with various potent self-defense mechanisms, such as cellular senescence, which blocks the abnormal proliferation of cells at risk of neoplastic transformation and is considered to be an important tumor-suppressive mechanism.174,175 Exosomes contribute to reduce intracellular stress and preservation of cellular homeostasis through clearance of damaged or toxic material, including proteins, lipids, and even nucleic acids. Therefore, exosomes serve as quality controller in cells.176 The vesicular transport system plays pivotal roles in the maintenance of cell homeostasis in eukaryote cells, which involves the cytoplasmic trafficking of biomolecules inside and outside of cells. Several types of membrane-bound organelles, such as the Golgi apparatus, endoplasmic reticulum (ER), endosomes and lysosomes, in association with cytoskeleton elements, are involved in the intracellular vesicular system. Molecules are transported through exocytosis and endocytosis to maintain homeostasis through the intracellular vesicular system and regulate cells responses to the internal and external environment. To maintain homeostasis and protect cells from various stress conditions, autophagy is an intracellular vesicular-related process that plays an important role through the endocytosis/lysosomal/exocytosis pathways through degradation and expulsion of damaged molecules out of the cytoplasm.177179 Autophagy, as an intracellular waste elimination system, is a synchronized process that actively participates in cellular homeostasis through clearance and recycling of damaged proteins and organelles from the cytoplasm to autophagosomes, and then to lysosomes.38,180182 Cells maintain homeostasis by autophagosomes, which are vesicles derived from autophagic and endosomal compartments. These processes are involved in adaption to nutrient deprivation, cell death, growth, and tumor progression or suppression. Autophagy flux contributes to maintaining homeostasis in the tumor microenvironment of endothelial cells. To support this concept, a study provided evidence suggesting that depletion of Atg5 in ECs could intensify the abnormal function of tumor vessels.183 Exosome secretion plays a crucial role in maintaining cellular homeostasis in exosome-secreting cells. As a consequence of blocking exosome secretion, nuclear DNA accumulates in the cytoplasm, thereby causing the activation of cytoplasmic DNA sensing machinery. Blocking exosome secretion aggravates the innate immune response, leading to ROS-dependent DNA damage responses and thus inducing senescence-like cell-cycle arrest or apoptosis in normal human cells. Thus, cells remove harmful cytoplasmic DNA, protecting them from adverse effects.182 Salomon and Rice reported that the involvement of exosomes in placental homeostasis and pregnancy disorders. EVs of placental origin are found in a variety of body fluids including urine and blood. Moreover, the number of exosomes throughout gestation is higher in complications of pregnancy, such as preeclampsia and gestational diabetes mellitus, compared to normal pregnancies.184

The endolysosomal system is critically involved in maintaining homeostasis through the highly regulated processes of internalization, sorting, recycling, degradation, and secretion. For example, endocytosis allows the internalization of various receptor proteins into cells, and vesicles formed from the plasma membrane fuse and deliver their membrane and protein content to early endosomes. Similarly, significant amounts of internalized content are recycled back to the plasma membrane via recycling endosomes,76 while the remaining material is sequestered in ILVs in late endosomes, also known as multivesicular bodies.185,186 Tetraspanin proteins, such as CD63 and CD81, are regulators of ILV formation. Once ILVs are formed, MVBs can degrade their cargo by fusing with lysosomes or, alternatively, MVBs can secrete their ILVs by fusing with the plasma membrane and release their content into extracellular milieu.187190 Exosomes play an important role in regulating intracellular RNA homeostasis by promoting the release of misfolded or degraded RNA products, and toxic RNA products. Y RNAs are involved in the degradation of structured and misfolded RNAs. Further studies have demonstrated that proteins involved in RNA processing are abundant in exosomes, and the half-lives of secreted RNAs are almost twice as short as those of intracellular mRNAs. These studies suggest that cells maintain intracellular RNA homeostasis through the release of distinct RNA species in extracellular vesicles.191193 Exosomes reduce cholesterol accumulation in Niemann-Pick type C disease, a lysosomal storage disease in which cells accumulate unesterified cholesterol and sphingolipids within the endosomal and lysosomal compartment.194

Autophagy is the intracellular vesicular-related process that regulates the cell environment against pathological and stress conditions. In order to maintain homeostasis and protect the cells against stress conditions, internal vesicles or secreted vesicles serve as a canal to degrade and expel damaged molecules out of the cytoplasm.38,181,182 Autophagy protects the cell from various stress conditions and maintains cellular homeostasis, regulating cell survival and differentiation through clearance and recycling of damaged proteins and organelles from the cytoplasm to autophagosomes, and then to lysosomes.180 Several studies have demonstrated that proteins are involved in controlling tumor cell function and fate, and mediate crosstalk between exosome biogenesis and autophagy. Coordination between exosome-autophagy networks serves as a tool to conserve cellular homeostasis via the lysosomal degradative pathway and/or secretion of cargo into the extracellular milieu.176,195 Autophagy is a multi-step process that occurs by initiation, membrane nucleation, maturation and finally the fusion of autophagosomes with lysosomes. The autophagy process is not only linked with endocytosis but is also linked with the biogenesis of exosomes. For example, subsets of the autophagy machinery involved in the biogenesis of exosomes and the autophagic process itself appear dispensable.78,196 Crosstalk between exosomal and autophagic pathways has been reported in a growing number of diseases. Proteomic studies were performed to analyze the involvement of key proteins in the interconnection between exosome and autophagy pathways. They found that almost all proteins were identified; however, their involvement differed between them. Among 100 proteins, four proteins were highly ranked including HSPA8 (3/100), HSP90AA1 (8/100), VCP (24/100), and Rab7A (81/100). These data suggest an interconnection between the exosome and autophagy.197,198 Endosomal autophagy plays a significant role in the interconnection between exosomes and autophagy. Stress is a major factor for autophagy. In particular, the starvation of cells is a key inducer of autophagy, and induces enlargement of MVB structures and a co-localization of Rab11 and LC3 in these structures, an indication that autophagy-related processes are associated with the MVB.199 The sorting of autophagy-related cargo into MVBs is dependent on Hsc70 (HSPA8), VPS4, and TSG101, and independent on LAMP-2A, thereby excluding a role for, the lysosome.200 Several proteins are involved in the regulation and biogenesis of secretory autophagy compartments such as GRASPs, LC3, Rab8a, ESCRTs, and SNAREs, along with several Atg proteins.181,201,202 Autophagosomes could fuse with MVBs to form amphisomes and release vesicles to the external environment.203

Autophagy and exosome biogenesis and function are interconnected by microRNA. Over-expression of miR-221/222 inhibits the level of PTEN and activates Akt signaling, and subsequently reduces the expression of hallmarks that positively relate to autophagy including LC3, ATG5 and Beclin1, and increases the expression of SQSTM1/p62.204 MiR-221/222 from human aortic smooth muscle cell (HAoSMC)-derived exosomes inhibit autophagy in HUVECs by modulating the PTEN/Akt signaling pathway. miRNA-223 attenuates hypoxia-induced apoptosis and excessive autophagy in neonatal rat cardiomyocytes and H9C2 cells via the Akt/mTOR pathway, by targeting poly(ADP-ribose) polymerase 1 (PARP-1) through increased autophagy via the AMPK/mTOR and Akt/mTOR pathways205 ATG5 mediates the dissociation of vacuolar proton pumps (V1Vo-ATPase) from MVBs, which prevents acidification of the MVB lumen and allows MVB-PM fusion and exosome release. Accordingly, knockout of ATG5 or ATG16L1 significantly reduces exosome release and attenuates the exosomal enrichment of lipidated LC3B. These findings demonstrate that autophagic mechanisms possibly regulate the fate of MVBs and subsequent exosome biogenesis.78 Bone marrow MSC (BMMSC)-derived exosomes contain a high level of miR-29c, which regulates autophagy under hypoxia/reoxygenation (H/R) conditions.206 Human umbilical cord MSC-derived exosomes (HucMDEs) promote hepatic glycolysis, glycogen storage, and lipolysis, and reduce gluconeogenesis. Additionally, autophagy potentially contributes to the effects of HucMDE treatment and increases formation of autophagosomes and the autophagy marker proteins BECN1, MAP, and 1LC3B. These findings suggest that HucMDEs improve hepatic glucose and lipid metabolism in T2DM rats by activating autophagy via the AMPK pathway.207 Liver fibrosis is a serious disorder caused by prolonged parenchymal cell death, leading to the activation of fibrogenic cells, extracellular matrix accumulation, and eventually liver fibrosis. Exosomes derived from adipose-derived mesenchymal stem cells (ADSCs) have been used to deliver circular RNAs mmu_circ_0000623 to treat liver fibrosis. The findings from this study suggest that Exos from ADSCs containing mmu_circ_0000623 significantly suppress CCl4-induced liver fibrosis by promoting autophagy activation. Autophagy inhibitor treatment significantly reverses the treatment effects of Exos.208 Inhibition of autophagy by PDGF and its downstream molecule SHP2 (Src homology 2-containing protein tyrosine phosphatase 2) increased hepatic stellate cell (HSC)-derived EV release. Disruption of mTOR signaling abolishes PDGF-dependent EV release. Activation of mTOR signaling induces the release of MVB-derived exosomes by inhibiting autophagy, as well as microvesicles, through activation of ROCK1 signaling. Furthermore, deletion of SHP2 attenuates CCl4 or BDL-induced liver fibrosis.209 The therapeutic effects of exosomes containing high concentrations of mmu_circ_0000250 were analyzed in diabetic mice. The findings indicated that a high concentration of mmu_circ_0000250 had a better therapeutic effect on wound healing when compared with wild-type exosomes from ADSCs. The results also showed that exosome treatment with mmu_circ_0000250 increased angiopoiesis in wounded skin and suppressed apoptosis by inducing miR-128-3p/SIRT1-mediated autophagy.210 A study showed that mice treated with differentiated cardiomyocyte (iCM) exosomes exhibited significant cardiac improvement post-myocardial infarction, with significantly reduced apoptosis and fibrosis. Apoptosis was associated with reduced levels of hypoxia and inhibition of exosome biogenesis. iCM-exosome-treated groups showed upregulation of autophagosome production and autophagy flux. Hence, these findings indicate that iCM-Ex can improve post-myocardial infarction cardiac function by regulating autophagy in hypoxic cardiomyocytes.211 Exosomes of hepatocytes play a crucial role in inhibiting hepatocyte apoptosis and promoting hepatocyte regeneration. Mesenchymal stem cell-derived hepatocyte-like cell exosomes (MSC-Heps-Exo) were injected into a mouse hepatic Ischemia/reperfusion (I/R) I/R model through the tail. The results demonstrated that MSC-Heps-Exo effectively relieve hepatic I/R damage, reduce hepatocyte apoptosis, and decrease liver enzyme levels. A possible mechanism of reduced hepatic ischemia/reperfusion injury is the enhancement of autophagy.212

Exosomes play a critical role in viral infections, particularly of retroviruses and retroviruses, and use preexisting pathways for intracellular protein trafficking and formation of infectious particles. Exosomes and viruses share several features including biogenesis, uptake by cells, and the intracellular transfer of RNAs, mRNAs, and cellular proteins. Some features are different, including self-replication after infection of new cells, regulation of viral expression, and complex viral entry mechanisms.213,214 Exosomes secreted from virus-infected cells carry mostly cargo molecules such as viral proteins, genomic RNA, mRNA, miRNA, and genetic regulatory elements.215218 These cargo molecules are involved in the alteration of recipient cell behavior, regulating cellular responses, and enabling infection by various types of viruses such as human T-cell lymphotropic virus (HTLV), hepatitis C virus (HCV), dengue virus, and human immunodeficiency virus (HIV).215 Exosomes communicate with host cells through contact between exosomes and their recipient cells, via different kinds of mechanisms. Initially, the transmembrane proteins of exosomes build a network directly with the signaling receptors of target cells and then join with the plasma membrane of recipient cells to transport their content to the cytosol. Finally, the exosomes are incorporated into the recipient cells.219221 A report suggested that disruption of exosomal lipid rafts leads to the inhibition of internalization of exosomes.95 Exosomes derived from HIV-infected patients contain the trans-activating response element, which is responsible for HIV-1 replication in recipient cells through downregulation of apoptosis.222 While exosomes serving as carrier molecules, exosomes contain miRNAs that induce viral replication and immune responses either by direct targeting of viral transcripts or through indirect modulation of virus-related host pathways. In addition, exosomes have been found to act as nanoscale carriers involved in HIV pathogenesis. For example, exosomes enhance HIV-1 entry into human monocytic and T cell lines through the exosomal tetraspanin proteins CD9 and CD81.223 Influenza virus infection causes accumulation of various types of microRNAs in bronchoalveolar lavage fluid, which are responsible for the potentiation of the innate immune response in mouse type II pneumocytes. Serum of influenza virus-infected mice show significant levels of miR-483-3p, which increases the expression of proinflammatory cytokine genes and inflammatory pathogenesis of H5N1 influenza virus infection in vascular endothelial cells.224 Exosomes are involved in the transmission of inflammatory, apoptotic, and regenerative signals through RNAs. Chen et al investigated the potential functions of exosomal RNAs by RNA sequencing analysis in exosomes derived from clinical specimens of healthy control (HC) individuals and patients with chronic hepatitis B (CHB) and acute-on-chronic liver failure caused by HBV (HBV-ACLF). The results revealed that the samples contained unique and distinct types of RNAs in exosomes.225 Zika virus (ZIKV) infection causes severe neurological malfunctions including microcephaly in neonates and other complications associated with Guillain-Barr syndrome in adults. Interestingly, ZIKV uses exosomes as mediators of viral transmission between neurons and increases production of exosomes from neuronal cells. Exosomes derived from ZIKV-infected cells contained both ZIKV viral RNA and protein(s) which are highly infectious to nave cells. ZIKV uses neutral Sphingomyelinase (nSMase)-2/SMPD3 to regulate production and release of exosomes.226

During infections, viruses replicate in host cells through vesicular trafficking through a sequence of complexes known as ESCRT, and assimilate viral constituents into exosomes. Exosomes encapsulate viral antigens to maximize infectivity by hiding viral genomes, entrapping the immune system, and maximizing viral infection in uncontaminated cells. Exosomes can be used as a source of viral antigens that can be targeted for therapeutic use. A Variety of infectious diseases caused by viruses such as HCV, ZIKV, West Nile virus (WNV), and DENV enter into the host cells using clathrin-mediated or receptor-mediated endocytosis. For example, HCV infects host cells by specific targeting of cells through cellular contact, and hepatocyte-derived exosomes that contain HCV RNA can stimulate innate immune cells.217,227230 Exosomes show structural and molecular similarity to HIV-1 and HIV-2, which are enclosed by a lipid bilayer, and in the vital features of size and density, RNA species, and macro biomolecules including carbohydrates, lipids, and proteins. HIV-infected cells release enriched viral RNAs containing exosomes derived from HIV-infected cells and are enhanced with viral RNAs and Nef protein.6,38,231236 Izquierdo-Useros et al reported that both exosomes and HIV-1 express sialyllactose-containing gangliosides and interact with each other via sialic-acid-binding immunoglobulin-like lectins (Siglecs)-1. Siglecs-1 stimulates mature dendritic cell (mDC) capture and storage of both exosomes and HIV-1 in mDCs.237 Exosomes released from HIV-infected T cells contain transactivation response (TAR) element RNA, which stimulate proliferation, migration, and invasion of oral/oropharyngeal and lung cancer cells.238 Nuclear VP40 from Ebola virus VP40 upregulates cyclin D1 levels, resulting in dysregulated cell cycle and EV biogenesis. Synthesized extracellular vesicles contain cytokines and EBOV proteins from infected cells, which are responsible for the destruction of immune cells during EBOV pathogenesis.239 HIV enters into the host cells through human T-cell immunoglobin mucin (TIM) proteins. TIMs are a group of proteins (TIM-1, TIM-3, and TIM-4) that promote phagocytosis of apoptotic cells.240 TIM-4 is involved in HIV-1 exosome-dependent cellular entry mechanisms. Substantiating this hypothesis, neural stem cell (NSC)-derived exosomes containing TIM-4 protein increase HIV-1 exosome-dependent cellular entry into host cells, and antibody against TIM4 inhibits exosome-mediated entry of HIV in various types of cell.241

Exosomes show immense promise in biomedical applications due to their potential in drug delivery, the carriage of biomolecular markers of many diseases, and cellular protection. In addition, they can be used in non-invasive diagnostics or minimum invasive diagnostics.150 Detection of biomarkers is vital for early diagnosis of cancer and also critical for treatment. Several studies have documented the importance of exosomes in a variety of diseases, although further examination of the biology and functions of exosomes is warranted due to the continuing emergence of new diseases in the present world. The complex cargo of exosomes facilitates the exploration of a variety of diagnostic windows into disease detection, monitoring, and treatment. Exosomes are found in all biological fluids and are secreted by all cells, rendering them attractive for use through minimally invasive liquid biopsies, and they have the potential for use in longitudinal sampling to follow disease progression.242 Exosomes are produced and secreted by almost all body fluids, including blood, urine, saliva, breast milk, cerebrospinal fluid, semen, amniotic fluid, and ascites. These exosomes contain micro RNAs, proteins, and lipids serving as diagnostic markers.120 Exosomes are used in diagnostic applications in various kinds of diseases, such as cardiovascular diseases (CVDs),243 diseases of the central nervous system (CNS),244 cancer,245 and other prominent diseases including in the liver,246 kidney,247 and lung.248 Exosomes are potentially used to detect cancer-associated mutations in serum and also for the transfer of genomic DNA from donor cells to recipient cells.249 Exosomes carrying specific miRNAs or groups of miRNAs can be used as diagnostic markers to detect cancer. For example, exosomes containing oncogenic Kras, which have tumor-suppressor miRNAs-100, seem to have high diagnostic value, which could facilitate the differentiation of the expression pattern between cancer cells and normal cells.250,251 Similarly, miR-21 is considered to be diagnostic marker for various types of cancer including glioblastomas and pancreatic, colorectal, colon, liver, breast, ovarian, and esophageal cancers.252 Tumor suppressor miRNAs, such as miR-146a and miR-34a, function as diagnostic tools to detect liver, breast, colon, pancreatic, and hematologic malignancies.251 Exosomes containing GPC1 (glypican 1) are used as diagnostic markers to detect pancreatic, breast, and colon cancer.253,254

Exosomes play critical roles in various types of disease, and particularly in cancer progression and resistance to therapy. The unique biogenesis of exosomes and their biological features have generated excitement for their potential use as biomarkers for cancer.255 Generally, exosomes are produced and secreted by most cells and contain all the biological components of a cell. Hence, exosomes are found in all biological fluids and provide excellent opportunities for use as biomarkers.242 Surface proteins of exosomes are involved in the regulation of the tumor immune microenvironment and the monitoring of immunotherapies. Hence, exosome proteins play a critical role in cancer signaling.256 Exosomes from patients with metastatic pancreatic cancer show a higher mutant Kras allele frequency than exosomes from patients with local disease. In addition, the exosomes also accumulate a significantly higher level of cancer cell-specific DNA such as cytoplasmic DNA.8,257 Exosomes protect DNA and RNA from enzymatic degradation by encapsulation and stability in exosomes. The enhanced stability and retention of exosomes in liquid biopsies increases the availability and performance of exosomes as cancer biomarkers.258 Cancer cells contain cargo molecules, such as nucleic acid, proteins, metabolites, and lipids that are relatively different from normal cells, which is a contributing factor for their candidacy as cancer biomarkers. Exosomes isolated and purified from patient plasma samples enriched for miR-10b-5p, miR-101-3p, and miR-143-5p have been identified as potential diagnostic markers for gastric cancer with lymph node metastasis, gastric cancer with ovarian metastasis, and gastric cancer with liver metastasis, respectively.259 Kato et al analyzed the expression of CD44 protein and mRNA from cell lysates and exosomes from prostate cancer cells.260 Exosomes from serum containing CD44v8-10 mRNA was used as a diagnostic marker for docetaxel resistance in prostate cancer patients. The study was performed to evaluate plasma exosomal mRNA-125a-5p and miR-141-5p miRNAs as biomarkers for the diagnosis of prostate cancer from 19 healthy individuals and 31 prostate cancer patients. In comparing the miR-125a-5p/miR-141-5p level ratio, prostate cancer patients had significantly higher levels of miR-125a-5p/miR-141-5p. The findings from this study demonstrated that plasma exosomal expression of miR-141-3p and miR-125a-5p are markers of specific tumor traits associated with prostate cancer.261 Serum samples from 81 patients with gastric cancer showed that exosomes contained significant levels of long non-coding RNA (lncRNA) H19, which could be a diagnostic marker for gastric cancer.262 Plasma exosomes are suitable candidates as biomarkers for various diseases. For instance, plasma exosome lncRNA expression profiles were examined in esophageal squamous cell carcinoma (ESCC) patients. The findings suggest that five different types of lncRNAs were at significantly higher levels in exosomes from ESCC patients than in non-cancer controls. These lncRNAs may serve as highly effective, noninvasive biomarkers for ESCC diagnosis.263 Differential expression of lncRNAs, such as LINC00462, HOTAIR, and MALAT1, are significantly upregulated in hepatocellular carcinoma (HCC) tissues. The exosomes of the control group had a larger number of lncRNAs with a high amount of alternative splicing compared to hepatic disease patients.264 To demonstrate exosomes as a non-invasive cancer diagnostic tool, RNA-sequencing analysis was performed between three pairs of non-small-cell lung cancer (NSCLC) patients and controls from Chinese populations. The results show that circ_0047921, circ_0056285, and circ_0007761 were significantly expressed and that these exosomal circRNAs are promising biomarkers for NSCLC diagnosis.265 Exosomes were isolated from the serum of 34 patients with acute myocardial infarction (AMI), 31 patients with unstable angina (UA), and 22 healthy controls. The isolated exosomes exhibited higher levels of miR-126 and miR-21 in the patients with UA and AMI than in the healthy controls.266 Xu et al designed a study to examine tumor-derived exosomes as diagnostic biomarkers. In this study exosome miRNA microarray analysis was performed in the peripheral blood from four lung adenocarcinoma patients, including two with metastasis and two without metastasis. The results found that miR-4436a and miR-4687-5p were upregulated in the metastasis and non-metastasis group, while miR-22-3p, miR-3666, miR-4448, miR-4449, miR-6751-5p, and miR-92a-3p were downregulated. Exosomes containing miR-4448 have served as a diagnostic marker of patients with adenocarcinoma metastasis. Increased understanding of exosome biogenesis, structure, and function would enhance the performance of biomarkers in various kinds of disease diagnosis, prognosis, and surveillance.267

Exosomes have unique features such as ease of handling, molecular composition, and critical immunogenicity, and it is particularly easy to use them to transfer genes and proteins into cells. These unique characteristic features can inhibit angiogenesis and cancer metastasis, which are the two main targets of cancer therapy.268,269 Exosomes have potential therapeutic applications in a variety of diseases due to their potential capacity as vehicles for the delivery of therapeutic agents (Figure 5). Exosomes from colon cancer cells contain the highly immunogenic antigens MelanA/Mart-1 and gp100, serving as an indicator of tumor origin in particular organelles. Animal studies have demonstrated that tumor-derived antigen-containing exosomes induce potent antitumor T-cell responses and tumor regression.270 Exosomes containing tumor antigens are able to stimulate CD4+and CD8+T cells, and antigen-presenting exosomes inhibit tumor growth.135,271,272 MSC-derived exosomes exhibit the immunomodulatory and cytoprotective activities of their parent cells.273,274 Similarly, exosomes derived from bone marrow show protective roles in myocardial ischemia/reperfusion injury,109 hypoxia-induced pulmonary hypertension,275 and brain injury,276,277 and inhibit breast cancer growth via vascular endothelial growth factor down-regulation and miR-16 transfer in mice.278 Mesenchymal cell- and epithelial cell-derived exosomes exhibit tolerance and without any undesired side effects in patients and also act as therapeutic agents themselves.48,279 Exosomes engineered with ligands containing RGD peptide are used to induce signaling in specific cell types, and doxorubicin-loaded exosomes derived from dendritic cells show therapeutic responses in mammary tumor-bearing mice.46 Exosomal microRNAs are able to control other cells, and the delivery of miRNA or siRNA payload promotes anticancer activity in mammary carcinoma and glioma.280,281 Rabies virus glycoprotein (RVG)-modified dendritic cell-derived exosomes suppress the expression of BACE1 in the brain, which indicates the therapeutic potential of exosomes to target AD.282 Furthermore, these exosomes stimulated neurite outgrowth in cultured astrocytes by transferring miR-133b between cells.27 Immunotherapy is able to induce tumor-targeting immunity or an antitumor host immune response. For example, tumor-associated antigen-loaded mature autologous dendritic cells increase survival of metastatic castration-resistant patients.283 Exosome therapy induces upregulation of CD122 molecules in CD4+ T cells, whereas the lymphocyte pool is stable. Multiple vaccinations with exosomes increase circulating CD3-/CD56+ natural killer (NK) cells.284 An in vitro study demonstrated that adipose stem cell-derived exosomes up-regulate the peroxisome proliferator-activated receptor gamma coactivator 1, phosphorylate the cyclic AMP response element binding protein, and ameliorate abnormal apoptotic protein levels.285 Exosomes are used as potential carriers to carry anti-inflammatory drugs. Curcumin-encapsulated exosomes show significant anti-inflammatory activity, and exosomes are also used to deliver anti-inflammatory drugs to the brain through a noninvasive intranasal route.286,287 Turturici et al reported that specific progenitor cell-derived EVs contain biological cargo that promotes angiogenesis and tissue repair, and modulates immune functions.288

Figure 5 Therapeutic potential and versatile clinical implications of exosomes.

Generally, exosomes serve as vehicles for the delivery of drugs and are also actively involved as therapeutic agents. Conversely, injected exosomes enter into other cells and deliver functional cargo molecules very efficiently and rapidly, with minimal immune clearance and are well tolerated.16,21,245,289,290 Intravenous administration of human MSC-derived exosomes supports neuroprotection in a swine model of traumatic brain injury.291 In vitro and in vivo models demonstrate that exosomes from human-induced pluripotent stem cell-derived mesenchymal stromal Cells (hiPSC-MSCs) protect the liver against hepatic ischemia/reperfusion injury through increasing the level of proliferation of primary hepatocytes, activity of sphingosine kinase, and synthesis of sphingosine-1-phosphate (S1P).292 Exosomes derived from macrophages show potential for use in neurological diseases because of their easy entry into the brain by crossing the blood-brain barrier (BBB). Catalase-loaded exosomes displayed a neuroprotective effect in a mouse model of PD and exosomes loaded with dopamine entered into the brain better in comparison to free dopamine.33,293 Treatment of tumor-bearing mice with autologous exosomes loaded with gemcitabine significantly suppressed tumor growth and increase longevity, and caused only minimal damage to normal tissues. The study demonstrated that autologous exosomes are safe and effective vehicles for targeted delivery of GEM against pancreatic cancer.294

Generally, lipid-based nanoparticles such as liposomes or micelles, or synthetic delivery systems have been adopted to transport active molecules. However, the merits of synthetic systems are limited due to various factors including inefficiency, cytotoxicity and/or immunogenicity. Therefore, the development of natural carrier systems is indispensable. One of the most prominent examples of such natural carriers are exosomes, which are used to transport drug and active biomolecules. Exosomes are more compatible with other cells because they carry various targeting molecules from their cells of origin. Exosomes are nano-sized membrane vesicles derived from almost all cell types, which carry a variety of cargo molecules from their parent cells to other cells. Due to their natural biogenesis and unique qualities, including high biocompatibility, enhanced stability, and limited immunogenicity, they have advantages as drug delivery systems (DDSs) compared to traditional synthetic delivery vehicles. For instance, extracellular vesicles, including exosomes, carry and protect a wide array of nucleic acids and can potentially deliver these into recipient cells.6 EVs possess inherent targeting properties due to their lipid composition and protein content enabling them to cross biological barriers, and these salient features exploit endogenous intracellular trafficking mechanisms and trigger a response upon uptake by recipient cells.45,295297 The lipid composition and protein content of exocytic vesicles have specific tropism to specific organs.296 The integrin of exosomes determines the ability to alter the pharmacokinetics of EVs and increase their accumulation in various type of organs including brain, lungs, or liver.117 For example, EVs containing Tspan8 in complex with integrin alpha4 were shown to be preferentially taken up by pancreatic cells.298 Similarly, the lipid composition of EVs influences the cellular uptake of EVs by macrophages.299 EVs derived from dendritic cell achieved targeted knockdown by fusion between expression of Lamp2b and neuron-specific RVG peptide by using siRNA in neuronal cell.45 EVs loaded with Cre recombinase protein were able to deliver functional CreFRB to recipient cells through active and passive mechanisms in the presence of endosomal escape, enhancing the compounds chloroquine and UNC10217832A.300 EVs from cardiosphere-derived cells achieved targeted delivery by fusion of the N-terminus of Lamp2b to a cardiomyocyte-specific peptide (CMP).301 RVG-exosomes were used to deliver anti-alpha-synuclein shRNA minicircle (shRNA-MC) therapy to the alpha-synuclein preformed-fibril-induced mouse model of parkinsonism. This therapy decreased alpha-synuclein aggregation, reduced the loss of dopaminergic neurons, and improved clinical symptoms. RVG exosome-mediated therapy prolonged the effectiveness and was specifically delivered into the brain.302 Zhang et al evaluated the effects of umbilical cord-derived macrophage exosomes loaded with cisplatin on the growth and drug resistance of ovarian cancer cells. High loading efficiency of cisplatin was achieved by membrane disruption of exosomes by sonication.303 Incorporation of cisplatin into umbilical cord blood-derived M1 macrophage exosomes increased cytotoxicity 3.3-fold in drug-resistant A2780/DDP cells and 1.4-fold in drug-sensitive A2780 cells, compared to chemotherapy alone. Loading of cisplatin into M2 exosomes increased cytotoxicity by nearly 1.7-fold in drug-resistant A2780/DDP cells and 1.4-fold in drug-sensitive A2780 cells. The findings suggest that cisplatin-loaded M1 exosomes are potentially powerful tools for the delivery of chemotherapeutics to treat cancers regardless of drug resistance. Shandilya et al developed a chemical-free and non-mechanical method for the encapsulation and intercellular delivery of siRNA using milk-derived exosomes through conjugation between bovine lactoferrin with poly-L-lysine, wherein lactoferrin as a ligand was captured by the GAPDH present in exosomes, loading siRNA in an effortless manner.304 Targeted drug delivery was achieved with low immunogenicity and toxicity using exosomes derived from immature dendritic cells (imDCs) from BALB/c mice by expressing the fusion protein RGD. Recombinant methioninase (rMETase) was loaded into tumor-targeting iRGD-Exos. The findings suggest that the iRGD-Exos-rMETase group exhibited significant antitumor activity compared to the rMETase group.305 Several diseases show high inflammatory responses; therefore, amelioration of inflammatory responses is a critical factor. The inflammatory responses in various disease models can be attenuated through introduction of super-repressor IB (srIB), which is the dominant active form of IB, and can inhibit translocation of nuclear factor B into the nucleus. Intraperitoneal injection of purified srIB-loaded exosomes (Exo-srIBs) showed diminished mortality and systemic inflammation in septic mouse models.306 Systemic administration of macrophage-derived exosomes modified with azide and conjugated with dibenzocyclooctyne-modified antibodies of CD47 and SIRP (aCD47 and aSIRP) through pH-sensitive linkers can actively and specifically target tumors through distinguishing between aCD47 and CD47 on the tumor cell surface.307 SPION-decorated exosomes prepared using fusion proteins of cell-penetrating peptides (CPP) and TNF- (CTNF-)-anchored exosomes coupled with superparamagnetic iron oxide nanoparticles (CTNF--exosome-SPIONs) significantly enhanced tumor cell growth inhibition via induction of the TNFR I-mediated apoptotic pathway. Furthermore, in vivo studies in murine melanoma subcutaneous cancer models showed that TNF--loaded exosome-based vehicle delivery enhanced cancer targeting under an external magnetic field and suppressed tumor growth with mitigating toxicity.308 Yu et al309 developed a formulation of erastin-loaded exosomes labeled with folate (FA) to form FA-vectorized exosomes loaded with erastin (erastin@FA-exo) to target triple-negative breast cancer (TNBC) cells with overexpression of FA receptors. Erastin@FA-exo increased the uptake efficiency of erastin and also significantly inhibited the proliferation and migration of MDA-MB-231 cells compared with erastin@exo and free erastin. Interestingly, erastin@FA-exo promoted ferroptosis with intracellular depletion of glutathione and ROS generation. Plasma exosomes (Exo) loaded with quercetin (Exo-Que) improved the drug bioavailability, enhanced the brain targeting of Que and potently ameliorated cognitive dysfunction in okadaic acid (OA)-induced AD mice compared to free quercetin by inhibiting phosphorylated tau-mediated neurofibrillary tangles.310 Spinal cord injury (SCI) causes paralysis of the limbs. To determine the role of resveratrol in SCI, exosomes derived from resveratrol-treated primary microglia were used as carriers which are able to enhance the solubility of resveratrol and enhance penetration of the drug through the BBB, thereby increasing its concentration in the CNS. The findings demonstrated that Exo + Res are highly effective at crossing the BBB with good stability, suggesting they have potential for enhancing targeted drug delivery and recovering neuronal function in SCI therapy, and is likely associated with the induction of autophagy and inhibition of apoptosis via the PI3K signaling pathway.311 Delivery of miR-204-5p by exosomes inhibits cancer cell proliferation and tumor growth, and induces apoptosis and chemoresistance by specifically suppressing the target genes of miR-204-5p in human cancer cells.312 Engineered exosomes with RVG peptide on the surface for neuron targeting and NGF-loaded exosomes (NGF@ExoRVG) were efficiently delivered into ischemic cortex, with a burst release of encapsulated NGF protein and de novo NGF protein translated from the delivered mRNA. The delivered NGF protein showed high stability and a long retention time, and also reduced inflammation by reshaping microglia polarization, promoted cell survival, and increased the population of double cortin-positive cells, a neuroblast marker.313 Intranasal delivery of mesenchymal stem cell-derived extracellular vesicles exerts immunomodulatory and neuroprotective effects in a 3xTg model of AD by activation of microglia cells and increased dendritic spine density.314 Exosome-encapsulated paclitaxel showed efficacy in the treatment of multi-drug resistant cancer cells and it overcomes MDR in cancer cells.315,316 Saari et al found that the loading of Paclitaxel to autologous prostate cancer cell-derived EVs increased its cytotoxic effect.316 Exosome loaded doxorubicin (exoDOX) avoids undesired and unnecessary heart toxicity by partially limiting the crossing of DOX through the myocardial endothelial cells.317 Studies from in vitro and in vivo demonstrate that exosome loaded doxorubicin showed that exosomes did not decrease the efficacy of DOX and there is no cardiotoxicity in DOX-treated mice.318

The intrinsic properties of exosomes have been exploited to control various types of diseases, including neurodegenerative conditions and cancer, through promoting or restraining the delivery of proteins, metabolites, and nucleic acids into recipient cells effectively, eventually altering their biological response. Furthermore, exosomes can be engineered to deliver diverse therapeutic payloads to the target site, including siRNAs, antisense oligonucleotides, chemotherapeutic agents, and immune modulators. The natural lipid and protein composition of exosomes increases bioavailability and minimizes undesirable side effects to the recipients. Due to the availability of exosomes in biological fluid, they can be easily used as potential biomarkers for diagnosis of diseases. Exosomes are naturally decorated with numerous ligands on the surface that can be beneficial for preferential tumor targeting.282 Due to their unique properties, including superior targeting capabilities and safety profile, exosomes are the subject of clinical trials as cancer therapeutic agents.284 Exosomes derived from DCs loaded with tumor antigens have been used to vaccinate cancer patients with the goal of enhancing anti-tumor immune responses.284,319,320

Due to the potential level of various types of cargoes and salient features, exosomes are involved in intercellular messaging and disease diagnosis. As a result of dedicated studies, exosomes have been identified as natural drug delivery vehicles. However, we still face challenges regarding the purity of exosomes due to the lack of standardized techniques for their isolation and purification, inefficient separation methods, difficulties in characterization, and lack of specific biomarkers.321 The first challenge is the use of conventional methods, which are laborious for isolation and purification, time consuming, and vulnerable to contamination by other impurities, which will affect drug delivery processes. The second challenge is the various cellular origins of exosomes, which could affect specific applications. For example, in the application of exosomes in cancer therapy, we should avoid the use of exosomes derived from cancer cells, due to their oncogenic properties. Finally, exosomes have variable properties due to extraction from different types of cell and different cell culture techniques. Therefore, there is a necessity to address and overcome the challenges. There is also a need for an exosome consortium to develop common protocols for the development of rapid and precise methods of exosome isolation, and to assist the selection of sources that are dependent upon the specific therapeutic application. The most important challenge of exosome biology is the clinical translation of exosome-based research using different cell sources. Further characterization studies based on therapeutic applications are needed. Finally, important steps need to be taken to purify exosomes in a feasible, rapid, cost-effective, and scalable manner, which are free from downstream processing and have minimal processing times, that are specifically targeted to therapeutic applications and clinical settings.

The achievement of exosome therapy is based on success rate of clinical trials. Exosomes with size ranges from 60 to 200nm have been used as an active pharmaceutical ingredient or drug carrier in disease treatment. Exosomes derived from human and plant-derived exosomes are registered in clinical trials, but more complete reports are available for humanderived exosomes.322 There are two major exosomes from DCs and MSCs are frequently used in clinical trials, which potentially induce inflammation response and inflammation treatment. The more crucial aspect of exosomes in clinical trials needs to comply with good manufacturing practice (GMP) including upstream, downstream and quality control. Recently, France and USA conducted clinical trials using EVs containing MHCpeptide complexes derived from dendritic could alter tumor growth in immune competent mice and a Phase I anti-non-small cell lung cancer319,320 and several other clinical trial studies are shown in Table 1. Recent clinical case shows promising results with MSC-EVs derived from unrelated bone marrow donors for the treatment of a steroid-refractory graft-vs-host disease patient.279 Similarly, exosomes were used for the treatment of various types of diseases such as melanoma, non-small-cell-lung cancer, colon cancer and chronic kidney disease.284,319,320,323,324

Table 1 Summary of the Exosome Used in Clinical Trials (Source: clinicaltrials.com)

Exosomes are nano-sized membrane vesicles released by the fusion of an organelle of the endocytic pathway, a multivesicular body, with the plasma membrane. Since the last decade, exosomes have played a critical role in nanomedicine and studies related to exosome biology have increased immensely. Exosomes are secreted by almost all cell types and they are found in almost all types of body fluids. They function as mediators of cell-cell communications and play a significant role in both physiological and pathological processes. Exosomes carry a wide range of cargoes including proteins, lipids, RNAs, and DNA, which mediate signaling to recipient cells or tissues, making them a promising diagnostic biomarker and therapeutic tool for the treatment of cancers and other pathologies. In this review, we summarized what is known to date about the factors involved in exosome biogenesis and the role of exosomes in intercellular signaling and cell-cell communications, immune responses, cellular homeostasis, autophagy, and infectious diseases. Further, we reviewed the role of exosomes as diagnostic markers, and their therapeutic and clinical implications. Furthermore, we highlighted the challenges and outstanding developments in exosome research. The clinical application of exosomes is inevitable and they represent multicomponent biomarkers for several diseases including cancer and neurological diseases, etc. Recently, the mortality rate due to various types of cancers has increased. Therefore, therapies are essential to reduce mortality rates. At this juncture, we need sensitive, rapid, cost-effective, and large-scale production of exosomes to use as cancer biomarkers in diagnosis, prognosis, and surveillance. Furthermore, novel technologies are required for further tailoring exosomes as drug delivery vesicles with high drug pay loads, high specificity and low immunogenicity, and free of toxicity undesired side-effects. In addition, standardized and uniform protocols are necessary to isolate and purify exosomes for clinical applications, and more precise isolation and characterization procedures are required to increase understanding of the heterogeneity of exosomes, their cargo, and functions. There is an urgent need for information regarding the composition and mechanisms of action of the various substances in exosomes and to determine how to obtain highly purified exosomes at the right dosage for their clinical use. Currently, exosomes represent a promising tool in the field of nanomedicine and may provide solutions to a variety of todays medical mysteries.

The future direction of exosome research must focus on addressing the differential responses of communication between normal cells and cancer cells, how normal cells rapidly become cancerous, and how exosomes plays critical role in cancer progression via cell-cell communications. In vivo studies need to urgently address the critical factors such as biogenesis, trafficking, and cellular entry of exosomes originating from unmanipulated exosomes that control regulatory pathological functions. Further studies are required to decipher the mechanism of the cell-specific secretion and transport of exosomes, and the biological controls exerted by target cells. Exosomes represent a clinically significant nanoplatform. To substantiate this idea, numerous systematic in vivo studies are necessary to demonstrate the potency and toxicology of exosomes, which could help bring this novel idea a step closer to clinical reality. The most vital part of the system is to optimize the conditions for the engineering of exosomes that are non-toxic, for use in clinical trials. Furthermore, the translation of exosomes into clinical therapies requires their categorization as active drug components or drug delivery vehicles. Finally, future research should focus on the nanoengineering of exosomes that are tailored specifically for drug delivery and clinical efficacy.

Although we are the authors of this review, we would never have been able to complete it without the great many people who have contributed to the field of exosomes biogenesis, functions, therapeutic and clinical implications of exosomes aspects. We owe our gratitude to all those researchers who have made this review possible. We have cited as many references as permitted and apologize to the authors of those publications that we have not cited due to the limitation of references. We apologize to other authors who have worked on these aspects but whom we have unintentionally overlooked.

This study was supported by the KU-Research Professor Program of Konkuk University.

This work was supported by a grant from the Science Research Center (2015R1A5A1009701) of the National Research Foundation of Korea.

The authors report no conflicts of interest related to this work..

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[Full text] A Comprehensive Review on Factors Influences Biogenesis, Functions, Th | IJN - Dove Medical Press

Energy drinks may damage the heart, researchers warnshould the FDA get involved? – Cardiovascular Business

Drinking certain energy drinks may cause significant damage to the heart, according to new findings published in Food and Chemical Toxicology.

Because the consumption of these beverages is not regulated and they are widely accessible over the counter to all age groups, the potential for adverse health effects of these products is a subject of concern and needed research, lead researcher Ivan Rusyn, MD, PhD, a professor at Texas A&M University in College Station, said in a prepared statement.

Rusyn et al. assessed a total of 17 popular energy drinks, studying their chemical profiles and looking for any associations with potential cardiac complications. Energy drinks sold by Adrenaline, Shoc, Bang Star, C4, CELSIUS, HEAT, EBOOST, Game Fuel, GURU, Kill Cliff, Kickstart, Monster Energy, Red Bull, Reign, Rockstar, RUNA, UPTIME, Venom Energy and Xyience Energy were all part of the teams analysis.

Overall, the authors found that stem cell-derived cardiomyocyteshuman heart cells grown in a laboratoryshowed signs of an increased beat rate after being exposed to some energy drinks. Also, theophylline, adenine and azelate were all ingredients the team associated with potentially contributing to QT prolongation in cardiomyocytes.

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Energy drinks may damage the heart, researchers warnshould the FDA get involved? - Cardiovascular Business

bluebird bio Announces Temporary Suspension on Phase 1/2 and Phase 3 Studies of LentiGlobin Gene Therapy for Sickle Cell Disease (bb1111) – BioSpace

Feb. 16, 2021 12:00 UTC

CAMBRIDGE, Mass.--(BUSINESS WIRE)-- bluebird bio, Inc. (Nasdaq: BLUE) announced today that the company has placed its Phase 1/2 (HGB-206) and Phase 3 (HGB-210) studies of LentiGlobin gene therapy for sickle cell disease (SCD) (bb1111) on a temporary suspension due to a reported Suspected Unexpected Serious Adverse Reaction (SUSAR) of acute myeloid leukemia (AML).

In line with the clinical study protocols for HGB-206 and HGB-210, bluebird bio placed the studies on temporary suspension following a report received last week that a patient who was treated more than five years ago in Group A of HGB-206 was diagnosed with AML. The company is investigating the cause of this patients AML in order to determine if there is any relationship to the use of BB305 lentiviral vector in the manufacture of LentiGlobin gene therapy for SCD. In addition, a second SUSAR of myelodysplastic syndrome (MDS) in a patient from Group C of HGB-206 was reported last week to the company and is currently being investigated.

No cases of hematologic malignancy have been reported in any patient who has received treatment with betibeglogene autotemcel for transfusion-dependent -thalassemia (licensed as ZYNTEGLOTM in the European Union and the United Kingdom), however because it is also manufactured using the same BB305 lentiviral vector used in LentiGlobin gene therapy for SCD, the company has decided to temporarily suspend marketing of ZYNTEGLO while the AML case is assessed.

The safety of every patient who has participated in our studies or is treated with our gene therapies is the utmost priority for us, said Nick Leschly, chief bluebird. We are committed to fully assessing these cases in partnership with the healthcare providers supporting our clinical studies and appropriate regulatory agencies. Our thoughts are with these patients and their families during this time.

The independent safety review board monitoring the companys studies as well as the U.S. Food and Drug Administration (FDA) and European Medicines Agency (EMA) have been advised of these cases and bluebird bio will continue to work with regulatory agencies to complete its investigation.

Investor Conference Call Information

bluebird bio will hold a conference call to discuss this update on Tuesday, February 16 at 8:00 a.m. ET. Investors may listen to the call by dialing (844) 825-4408 from locations in the United States or +1 (315) 625-3227 from outside the United States. Please refer to conference ID number 880-6406.

To access the live webcast of bluebird bios presentation, please visit the Events & Presentations page within the Investors & Media section of the bluebird bio website at http://investor.bluebirdbio.com. A replay of the webcast will be available on the bluebird bio website for 90 days following the event.

About HGB-206 and HGB-210

HGB-206 is an ongoing, Phase 1/2 open-label study designed to evaluate the efficacy and safety of LentiGlobin gene therapy for sickle cell disease (SCD) that includes three treatment cohorts: Groups A, B and C. A refined manufacturing process designed to increase vector copy number (VCN) and further protocol refinements made to improve engraftment potential of gene-modified stem cells were used for Group C. Group C patients also received LentiGlobin for SCD made from HSCs collected from peripheral blood after mobilization with plerixafor, rather than via bone marrow harvest, which was used in Groups A and B of HGB-206.

HGB-210 is an ongoing Phase 3 single-arm open-label study designed to evaluate the efficacy and safety of LentiGlobin gene therapy for SCD in patients between two years and 50 years of age with sickle cell disease.

About LentiGlobin for SCD (bb1111)

LentiGlobin gene therapy for sickle cell disease (bb1111) is an investigational treatment being studied as a potential treatment for SCD. bluebird bios clinical development program for LentiGlobin for SCD includes the completed Phase 1/2 HGB-205 study, the ongoing Phase 1/2 HGB-206 study, and the ongoing Phase 3 HGB-210 study.

The U.S. Food and Drug Administration granted orphan drug designation, fast track designation, regenerative medicine advanced therapy (RMAT) designation and rare pediatric disease designation for LentiGlobin for SCD.

LentiGlobin for SCD received orphan medicinal product designation from the European Commission for the treatment of SCD, and Priority Medicines (PRIME) eligibility by the European Medicines Agency (EMA) in September 2020.

bluebird bio is conducting a long-term safety and efficacy follow-up study (LTF-307) for people who have participated in bluebird bio-sponsored clinical studies of LentiGlobin for SCD. For more information visit: https://www.bluebirdbio.com/our-science/clinical-trials or clinicaltrials.gov and use identifier NCT04628585 for LTF-307.

LentiGlobin for SCD is investigational and has not been approved in any geography.

About ZYNTEGLO (betibeglogene autotemcel)

Betibeglogene autotemcel (beti-cel) is a one-time gene therapy that adds functional copies of a modified form of the -globin gene (A-T87Q-globin gene) into a patients own hematopoietic (blood) stem cells (HSCs). Once a patient has the A-T87Q-globin gene, they have the potential to produce HbAT87Q, which is gene therapy-derived adult Hb, at levels that may eliminate or significantly reduce the need for transfusions. In studies of beti-cel, transfusion independence (TI) is defined as no longer needing red blood cell transfusions for at least 12 months while maintaining a weighted average Hb of at least 9 g/dL.

The European Commission granted conditional marketing authorization (CMA) for beti-cel, marketed as ZYNTEGLO gene therapy, for patients 12 years and older with transfusion-dependent -thalassemia (TDT) who do not have a 0/0 genotype, for whom hematopoietic stem cell (HSC) transplantation is appropriate, but a human leukocyte antigen (HLA)-matched related HSC donor is not available.

Non-serious adverse events (AEs) observed during clinical studies that were attributed to beti-cel included abdominal pain, thrombocytopenia, leukopenia, neutropenia, hot flush, dyspnea, pain in extremity, tachycardia and non-cardiac chest pain. One serious adverse event (SAE) of thrombocytopenia was considered possibly related to beti-cel.

Additional AEs observed in clinical studies were consistent with the known side effects of HSC collection and bone marrow ablation with busulfan, including SAEs of veno-occlusive disease.

For details, please see the Summary of Product Characteristics (SmPC).

On April 28, 2020, the European Medicines Agency (EMA) renewed the CMA for beti-cel. The CMA for beti-cel is valid in the 27 member states of the EU as well as the UK, Iceland, Liechtenstein and Norway.

The U.S. Food and Drug Administration granted beti-cel Orphan Drug status and Breakthrough Therapy designation for the treatment of TDT. Beti-cel is not approved in the U.S. Beti-cel continues to be evaluated in the ongoing Phase 3 Northstar-2 (HGB-207) and Northstar-3 (HGB-212) studies.

bluebird bio is conducting a long-term safety and efficacy follow-up study, LTF-303 for people who have participated in bluebird bio-sponsored clinical studies of ZYNTEGLO.

About bluebird bio, Inc.

bluebird bio is pioneering gene therapy with purpose. From our Cambridge, Mass., headquarters, were developing gene and cell therapies for severe genetic diseases and cancer, with the goal that people facing potentially fatal conditions with limited treatment options can live their lives fully. Beyond our labs, were working to positively disrupt the healthcare system to create access, transparency and education so that gene therapy can become available to all those who can benefit.

bluebird bio is a human company powered by human stories. Were putting our care and expertise to work across a spectrum of disorders: cerebral adrenoleukodystrophy, sickle cell disease, -thalassemia and multiple myeloma, using gene and cell therapy technologies including gene addition, and (megaTAL-enabled) gene editing.

bluebird bio has additional nests in Seattle, Wash.; Durham, N.C.; and Zug, Switzerland. For more information, visit bluebirdbio.com.

Follow bluebird bio on social media: @bluebirdbio, LinkedIn, Instagram and YouTube.

ZYNTEGLO, betibeglogene autotemcel, beti-cel, and bluebird bio are trademarks of bluebird bio, Inc.

Forward-Looking Statements

This release contains forward-looking statements within the meaning of the Private Securities Litigation Reform Act of 1995, including statements regarding the Companys timing and expectations regarding its investigation of the relationship of the AML and MDS events to the use of lentiviral vector BB305 in LentiGlobin gene therapy for SCD, and any myeloablation regimen used in connection with treatment. Any forward-looking statements are based on managements current expectations of future events and are subject to a number of risks and uncertainties that could cause actual results to differ materially and adversely from those set forth in or implied by such forward-looking statements, many of which are beyond the Companys control. These risks and uncertainties include, but are not limited to: the risk that the Company may not be able to definitively determine whether the lentiviral vector BB305 used in LentiGlobin gene therapy for SCD and in betibeglogene autotemcel is related to the patients AML in a timely manner, or at all; the risk that the lentiviral vector BB305 has caused insertional oncogenic events, including AML; the risk that insertional oncogenic events associated with lentiviral vector or additional MDS events associated with myeloablation will be discovered or reported over time; the risk that regulatory authorities may impose a clinical hold, in addition to our temporary clinical hold on the HGB-206 and HGB-210 studies, or on additional programs; the risk that we may not be able to address regulatory authorities concerns quickly or at all; the risk that we may not resume patient treatment with ZYNTEGLO in the commercial context in a timely manner or at all; the risk that our lentiviral vector platform across our severe genetic disease programs may be implicated, affecting the development and potential approval of elivaldogene autotemcel; the risk that we may not be able to execute on our business plans, including our commercialization plans, meeting our expected or planned regulatory milestones, submissions, and timelines, research and clinical development plans, and in bringing our product candidates to market; and the risk that with the impact on the execution and timing of our business plans, we may not successfully execute our previously announced plans to spin off our oncology programs into an independent publicly-traded entity. For a discussion of other risks and uncertainties, and other important factors, any of which could cause our actual results to differ from those contained in the forward-looking statements, see the section entitled Risk Factors in our most recent Form 10-Q, as well as discussions of potential risks, uncertainties, and other important factors in our subsequent filings with the Securities and Exchange Commission. All information in this press release is as of the date of the release, and bluebird bio undertakes no duty to update this information unless required by law.

View source version on businesswire.com: https://www.businesswire.com/news/home/20210216005442/en/

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bluebird bio Announces Temporary Suspension on Phase 1/2 and Phase 3 Studies of LentiGlobin Gene Therapy for Sickle Cell Disease (bb1111) - BioSpace

Beti-Cel Gene Therapy Frees Patients With Beta-Thalassemia From Red Blood Cell Transfusions – OncLive

Betibeglogene autotemcel (beti-cel), a one-time gene therapy, enabled durable transfusion independence in most patients with transfusion-dependent -thalassemia (TDT) who were treated across 4 clinical studies.

Of 60 patients enrolled overall, 17 of 22 (77%) treated in the 2 phase 1/2 studies were able to stop packed red blood cell transfusions. In the 2 phase 3 studies, which used a refined manufacturing process resulting in improved beti-cel characteristics, 89% (n = 31/35) of patients with at least 6 months of follow-up achieved transfusion independence for more than 6 months,1 reported Suradej Hongeng, MD, during the virtual 2021 Transplantation & Cellular Therapy Meetings.

The median follow-up after beti-cel infusion in the 4 studies has been 24.8 months (range, 1.1-71.8).

With up to 6 years of follow-up, 1-time beti-cel gene therapy enabled durable transfusion independence in the majority of patients, said Hongeng, from Ramathibodi Hospital of Mahidol University, in Bangkok, Thailand.

Patients who achieved transfusion independence experienced a 38% median reduction in liver iron concentration (LIC) from baseline to month 48. The median reduction in LIC was 59% in patients with a baseline LIC more than 15 mg/g dw. A total of 21 of 37 (57%) patients who achieved transfusion independence have stopped iron chelation for 6 months or longer, with a median duration of 18.5 months from stopping iron chelation to last follow-up.

Erythropoiesis as determined by soluble transferrin receptor level was also improved in transfusion-independent patients. Bone marrow biopsies showed improvement in the myeloid:erythroid ratio.

Beti-cel adds functional copies of a modified form of the -globin (A-T87Q-globin) gene into a patients own hematopoietic stem cells (HSCs) through transduction of autologous CD34+ cells using a BB305 lentiviral vector. Following single-agent busulfan myeloablative conditioning, beti-cel is infused, after which the transduced HSCs engraft and reconstitute red blood cells containing functional adult hemoglobin derived from the gene therapy.

Of the 60 patients treated, 43 were genotype non-/ and 17 were / . The median age at consent was 20 years in the phase 1/2 trials and 15 years in the phase 3 trials. Median LIC at baseline was 7.1 and 5.5 mg Fe/g dw, respectively, and median cardiac T2 was 34 and 37 msec, respectively. The vector copy number was 0.8 in the phase 1/2 trial and 3.0 in the phase 3 study. Additionally, 32t and 78t CD34+ cells were transduced, respectively.

The phase 1/2 studies showed promising results but lower achievement of transfusion independence in patients with the / genotype, leading to a refinement in the manufacturing process, which resulted in a higher number of transduced cells and a higher number of vector copy number, said Hongeng.

The median time to neutrophil engraftment was 22.5 days and the median time to platelet engraftment was 44 days. Lymphocyte subsets were generally within the normal range after beti-cel infusion, which is different from allogeneic stem cell [transplantation], which is probably around 6 months to a year to get complete recovery of immune reconstitution, he said. The median duration of hospitalization was 42 days.

All patients were alive at the last follow-up (March 3, 2020). Eleven of 60 (18%) of patients experienced at least 1 adverse event (AE) considered related or possibly related to beti-cel, the most common being abdominal pain (8%) and thrombocytopenia (5%). Serious AEs were those expected after myeloablative conditioning: veno-occlusive liver disease (8%), neutropenia (5%), pyrexia (5%), thrombocytopenia (5%), and appendicitis, febrile neutropenia, major depression, and stomatitis (3% each).

Of the 7 patients experiencing veno-occlusive liver disease, 3 were of grade 4 and 2 were of grade 3. Two other patients had grade 2 veno-occlusive disease. There were no cases of insertional oncogenesis.

Persistent vector-positive hematopoietic cells and durable HbaT87Q levels supported stable total hemoglobin over time. In phase 3 trials, the median peripheral blood vector copy number was 1.2 c/dg at month 12 and 2.0 c/dg at month 24, and the median total hemoglobin was 11.5 g/dL at month 12 and 12.9 g/dL at month 24.

The weighted average of hemoglobin during transfusion independence in the phase 1/2 trials was 10.4 g/dL, and patients were transfusion-independent for a median of 51.2 months. In the phase 3 studies, the weighted average of hemoglobin during transfusion independence was 11.9 g/dL, and patients were transfusion-independent for a medium 17.7 months.

Hongeng S, Thompson AA, Kwiatkowski JL, et al. Efficacy and safety of betibeglogene autotemcel (beti-cel; LentiGlobin for -thalassemia) gene therapy in 60 patients with transfusion-dependent -thalassemia (TDT) followed for up to 6 years post-infusion. Presented at: 2021 Transplantation & Cellular Therapy Meetings; February 8-12, 2021; virtual. Abstract 1.

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Beti-Cel Gene Therapy Frees Patients With Beta-Thalassemia From Red Blood Cell Transfusions - OncLive

Worldwide Cell Therapy Industry to 2027 – Profiling Allosource, Medipost and Mesoblast Among Others – PRNewswire

DUBLIN, Feb. 9, 2021 /PRNewswire/ -- The "Cell Therapy Market by Cell Type, Therapy Type, Therapeutic Area, and End User: Global Opportunity Analysis and Industry Forecast, 2020-2027" report has been added to ResearchAndMarkets.com's offering.

The global cell therapy market accounted for $7,754. 89 million in 2019, and is expected to reach $48,115. 40 million by 2027, registering a CAGR of 25. 6% from 2020 to 2027.

Cell therapy involves administration of somatic cell preparations for treatment of diseases or traumatic damages. Cell therapy aims to introduce new, healthy cells into a patient's body to replace diseased or missing ones.

This is attributed to the fact that specialized cells, such as brain cells, are difficult to obtain from human body. In addition, specialized cells typically have a limited ability to multiply, making it difficult to produce sufficient number of cells required for certain cell therapies. Some of these issues can be overcome through the use of stem cells. In addition, cells such as blood and bone marrow cells, mature, immature & solid tissue cells, adult stem cells, and embryonic stem cells are widely used in cell therapy procedures.

Moreover, transplanted cells including induced pluripotent stem cells (iPSCs), embryonic stem cells (ESCs), neural stem cells (NSCs), and mesenchymal stem cells (MSCs) are divided broadly into two main groups including autologous cells and non-autologous cells. Development of precision medicine and advancements in Advanced Therapies Medicinal Products (ATMPS) in context to their efficiency and manufacturing are expected to be the major drivers for the market. Furthermore, automation in adult stem cells and cord blood processing and storage are the key technological advancements that fuel growth of the market for cell therapy.

In addition, growth in aging patient population, The rise in cell therapy transplantations globally, and surge in disease awareness drive growth of the global cell therapy market. Furthermore, The rise in adoption of human cells over animal cells for cell therapeutics research, technological advancements in field of cell therapy, and increase in incidences of diseases such as cancer, cardiac abnormalities, and organ failure are the key factors that drive growth of the global market.

Moreover, implementation of stringent government regulations regarding the use of cell therapy is anticipated to restrict growth of the market. On the contrary, surge in number of regulations to promote stem cell therapy and increase in funds for research in developing countries are expected to offer lucrative opportunities to the market in the future.

The global cell therapy market is categorized on the basis of therapy type, therapeutic area, cell type, end user, and region. On the basis of therapy type, the market is segregated into autologous and allogenic. By therapeutics, it is classified into malignancies, musculoskeletal disorders, autoimmune disorders, dermatology, and others.

The global cell therapy market is categorized on the basis of therapy type, therapeutic, cell type, end user and region. On the basis of therapy type, the market is segregated into autologous and allogenic. By therapeutic area, it is classified into malignancies, musculoskeletal disorders, autoimmune disorders, dermatology, and others. On the basis of cell type, it is segregated into stem cell therapy and non-stem cell type. On the basis of end user, it is segregated into hospital & clinics and academic & research institutes. On the basis of region, the market is studied across North America, Europe, Asia-Pacific, and LAMEA.

Key Benefits

Key Topics Covered:

Chapter 1: Introduction1.1. Report Description1.2. Key Benefits for Stakeholders1.3. Key Market Segments1.4. Research Methodology1.4.1. Secondary Research1.4.2. Primary Research1.4.3. Analyst Tools & Models

Chapter 2: Executive Summary2.1. Key Findings of the Study2.2. Cxo Perspective

Chapter 3: Market Overview3.1. Market Definition and Scope3.2. Key Findings3.2.1. Top Player Positioning3.2.2. Top Investment Pockets3.2.3. Top Winning Strategies3.3. Porter's Five Forces Analysis3.4. Impact Analysis3.4.1. Drivers3.4.1.1. Technological Advancements in the Field of Cell Therapy3.4.1.2. The Rise in Number of Cell Therapy Clinical Studies3.4.1.3. The Rise in Adoption of Regenerative Medicine3.4.2. Restraint3.4.2.1. Developing Stage and Pricing3.4.3. Opportunity3.4.3.1. High Growth Potential in Emerging Markets3.5. Impact of Covid-19 on Cell Therapy Market

Chapter 4: Cell Therapy Market, by Cell Type4.1. Overview4.1.1. Market Size and Forecast4.2. Stem Cell4.2.1. Key Market Trends and Opportunities4.2.2. Market Size and Forecast, by Region4.2.3. Market Size and Forecast, by Type4.2.3.1. Bone Marrow, Market Size and Forecast4.2.3.2. Blood, Market Size and Forecast4.2.3.3. Umbilical Cord-Derived, Market Size and Forecast4.2.3.4. Adipose-Derived Stem Cell, Market Size and Forecast4.2.3.5. Others (Placenta, and Nonspecific Cells), Market Size and Forecast4.3. Non-Stem Cell4.3.1. Key Market Trends and Opportunities4.3.2. Market Size and Forecast, by Region

Chapter 5: Cell Therapy Market, by Therapy Type5.1. Overview5.1.1. Market Size and Forecast5.2. Autologous5.2.1. Key Market Trends and Opportunities5.2.2. Market Size and Forecast, by Region5.2.3. Market Analysis, by Country5.3. Allogeneic5.3.1. Key Market Trends and Opportunities5.3.2. Market Size and Forecast, by Region5.3.3. Market Analysis, by Country

Chapter 6: Cell Therapy Market, by Therapeutic Area6.1. Overview6.1.1. Market Size and Forecast6.2. Malignancies6.2.1. Market Size and Forecast, by Region6.2.2. Market Analysis, by Country6.3. Musculoskeletal Disorders6.3.1. Market Size and Forecast, by Region6.3.2. Market Analysis, by Country6.4. Autoimmune Disorders6.4.1. Market Size and Forecast, by Region6.4.2. Market Analysis, by Country6.5. Dermatology6.5.1. Market Size and Forecast, by Region6.5.2. Market Analysis, by Country6.6. Others6.6.1. Market Size and Forecast, by Region6.6.2. Market Analysis, by Country

Chapter 7: Cell Therapy Market, by End-user7.1. Overview7.1.1. Market Size and Forecast7.2. Hospitals & Clinics7.2.1. Key Market Trends and Opportunities7.2.2. Market Size and Forecast, by Region7.2.3. Market Analysis, by Country7.3. Academic & Research Institutes7.3.1. Key Market Trends and Opportunities7.3.2. Market Size and Forecast, by Region7.3.3. Market Analysis, by Country

Chapter 8: Cell Therapy Market, by Region8.1. Overview8.2. North America8.3. Europe8.4. Asia-Pacific8.5. LAMEA

Chapter 9: Company Profiles9.1. Allosource9.1.1. Company Overview9.1.2. Company Snapshot9.1.3. Operating Business Segments9.1.4. Product Portfolio9.1.5. Key Strategic Moves and Developments9.2. Cells for Cells9.2.1. Company Overview9.2.2. Company Snapshot9.2.3. Operating Business Segments9.2.4. Product Portfolio9.3. Holostem Terapie Avanzate Srl9.3.1. Company Overview9.3.2. Company Snapshot9.3.3. Operating Business Segments9.3.4. Product Portfolio9.4. Jcr Pharmaceuticals Co. Ltd.9.4.1. Company Overview9.4.2. Company Snapshot9.4.3. Operating Business Segments9.4.4. Product Portfolio9.4.5. Business Performance9.4.6. Key Strategic Moves and Developments9.5. Kolon Tissuegene, Inc.9.5.1. Company Overview9.5.2. Company Snapshot9.5.3. Operating Business Segments9.5.4. Product Portfolio9.5.5. Key Strategic Moves and Developments9.6. Medipost Co. Ltd.9.6.1. Company Overview9.6.2. Company Snapshot9.6.3. Operating Business Segments9.6.4. Product Portfolio9.6.5. Business Performance9.7. Mesoblast Ltd9.7.1. Company Overview9.7.2. Company Snapshot9.7.3. Operating Business Segments9.7.4. Product Portfolio9.7.5. Business Performance9.8. Nuvasive, Inc.9.8.1. Company Overview9.8.2. Company Snapshot9.8.3. Operating Business Segments9.8.4. Product Portfolio9.8.5. Business Performance9.9. Osiris Therapeutics, Inc.9.9.1. Company Overview9.9.2. Company Snapshot9.9.3. Operating Business Segments9.9.4. Product Portfolio9.10. Stemedica Cell Technologies, Inc.9.10.1. Company Overview9.10.2. Company Snapshot9.10.3. Operating Business Segments9.10.4. Product Portfolio

For more information about this report visit https://www.researchandmarkets.com/r/shw12n

Media Contact:

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Worldwide Cell Therapy Industry to 2027 - Profiling Allosource, Medipost and Mesoblast Among Others - PRNewswire

Evotec and Medical Center Hamburg-Eppendorf Enter Partnership to Develop iPSC-Based Tissue Therapy for Heart Failure – Yahoo Finance UK

The Daily Beast

Andrew Harnik/APSenate Majority Leader Chuck Schumer (D-NY) stood alongside high-profile progressives in Congress in front of a podium that said #CANCEL STUDENT DEBT, a favorite slogan of the activist class, to push the Biden administration on a key economic issue.The resolution, which Schumer first introduced last fall with Sen. Elizabeth Warren (D-MA), would cancel $50,000 in student loan debt for each borrower through executive action, a sum that goes far beyond what Biden has already pledged to nix while in office.In an outdoor briefing on Thursday, the Democratic leader said he has already had a receptive response from the White House.We have met with the president, we are pushing the president and his people, and we are very hopeful, Schumer said, sharing that he and Warren met with Biden and administration officials privately for 45 minutes to lay out a proposed executive action.Biden has promised to eliminate $10,000 in federal student loan debt for each student.Asked about the renewed push later in the afternoon, White House press secretary Jen Psaki reiterated Bidens support for his original proposal, suggesting that it was unlikely that anything more would be done through executive orders.On day one, the first day of his administration, he directed the Department of Education to extend the existing pause on student loan payments and interest for millions of Americans with federal student loans, Psaki said. That was a step he took through executive action, but he certainly supports efforts by members in Congress to take additional steps, and he would look forward to signing it.Schumer was joined by Warren and Squad Reps. Ayanna Pressley (D-MA) and Ilhan Omar (D-MN)the original co-sponsors of the companion House resolution from last Decemberas well as other House members pressing the issue.America does not suffer from scarcity, we suffer from greed, Omar said, linking burdensome debt to the differing chances of students who come from wealthy families versus those in middle and working class households.Progressives Cant Find Anyone in Biden's Cabinet to Be Mad AboutYetSchumers desire to publicly present a loan forgiveness alternative to what Biden has offered has been perceived by some on the left as a way to help stave off a possible primary challenge in his native New York. The senior Democrat is up for re-election in 2022 and Rep. Alexandria Ocasio-Cortez (D-NY) is thought to be contemplating a primary challenge for this Senate seat.Read more at The Daily Beast.Get our top stories in your inbox every day. Sign up now!Daily Beast Membership: Beast Inside goes deeper on the stories that matter to you. Learn more.

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Evotec and Medical Center Hamburg-Eppendorf Enter Partnership to Develop iPSC-Based Tissue Therapy for Heart Failure - Yahoo Finance UK

Harnessing the Potential of Cell and Gene Therapy – OncLive

Excitement took wing in the scientific community in the early 1990s, when the first gene therapy trial showed significant success, only to crash at the end of the decade with a patients tragic death.

Twenty years later, the excitement is back and greater than before. Although safety remains a concern, investigators are breaking ground in cell and gene therapy, and many believe that ultimately, a string of cured cancers will follow.

In 2017, the excitement over these therapies returned in spades when the FDA signed off on a cell-therapy drug for the first time, approving the chimeric antigen receptor (CAR) T-cell treatment tisagenlecleucel (Kymriah; Novartis) for patients with B-cell precursor acute lymphoblastic leukemia. At last, scientists had devised a way to reprogram a persons own T cells to attack tumor cells.

Were entering a new frontier, said Scott Gottlieb, MD, then-FDA commissioner, in announcing the groundbreaking approval.

Gottlieb was not exaggerating. The growth in CAR T-cell research is exploding. Although only a handful of cell and gene therapies are on the market, the FDA predicted in 2019 that it will receive more than 200 investigational new drug applications per year for cell and gene therapies, and that by 2025, it expects to have accelerated to 10 to 20 cell and gene therapy approvals per year.

We can absolutely cut the number of cancer deaths down so that one day in our lifetimes it can be a rare thing for people to die of cancer, said Patrick Hwu, MD, president and CEO of Moffitt Cancer Center in Florida and among gene therapys pioneers. It still may happen here and there, but itll be kind of like people dying of pneumonia. Its like, He died of pneumonia? Thats kind of weird. I think cancer can be the same way.

Essentially, you can kill any cancer cell that has an antigen that is recognized by the immune cell, Hwu said. The key to curing every single cancer, which is our goal, is to have receptors that can recognize the tumor but dont recognize the normal cells.

Community oncologists will need to be increasingly familiar about the various products, including their immediate and longer-term risks, Bo Wang, MD, and Deepu Madduri, MD, recently wrote in OncologyLive.1 It is key to understand the optimal time for referring these patients to an academic institution, as well as how to manage the requisite post CAR T-cell therapy in the community setting. Madduri is an assistant professor of medicine, hematology and medical oncology, as well as associate director of cellular therapy service, and director of clinical operations with the Center of Excellence for Multiple Myeloma at The Tisch Cancer Institute and the Icahn School of Medicine at Mount Sinai in New York, New York. Wang is a third-year clinical fellow in hematology/oncology at Mount Sinai.

Early referral to academic centers and hospitals equipped to deliver therapies is crucial for patients eligible for therapy. However, as advances continue in the field, community practices may be called upon to administer therapies in their clinic.

The Community Oncology Alliance (COA) envisions a broader role for the settings in which CAR T-cell therapies can be administered. When the Centers for Medicare & Medicaid Services (CMS) was considering coverage for CAR T-cell therapies in 2019, COA officials argued against limiting approvals to hospitals.

It is important to understand that there are state-of-the-art community oncology practices that have significant experience and capabilities in administering highly complex treatments, COA officials wrote in a letter to CMS. For example, stem cell transplants, which are similar in complexity to CAR T therapy, are performed successfully in the community oncology practice setting.2

Broader use of gene therapies depends on several factors, including navigating the logistics of gene therapies, addressing the high costs, and managing toxicities.3

Autologous CAR T-cell therapies involve a manufacturing process that requires coordination between the treating facility and the processing facility. Following leukapheresis, patients may require maintenance therapy to control disease progression during the manufacturing time, which can take 3 to 5 weeks.

In terms of cost, gene and cell therapies can cost from $375,000 to $475,000 per dose and they may face coverage restrictions from payers. Approvals could take weeks to obtain.3,4

Because of cytokine release syndrome and neurotoxicities associated with CAR T-cell therapy, the FDA mandates risk evaluation and mitigation strategy training for centers.

Further, providers may find that real-world experiences with gene therapies are different from those seen in the clinical trial setting, according to Ankit J. Kansagra, MD.

In a presentation at the 2020 American Society of Clinical Oncology Virtual Education Program, Kansagra, an assistant professor of medicine and Eugene P. Frenkel, MD, Scholar in Clinical Medicine at Harold C. Simmons Comprehensive Cancer Center in Dallas, Texas, said that in practice patients may be older and have more aggressive disease, with double- and triple-hit lymphomas.4

Specifically, Kansagra noted that medications such as steroids and/or tocilizumab (Actemra) to prevent or treat cytokine release syndrome or other toxicities were more frequently used in the real-world setting than what had been seen in clinical trials.

As it stands now, only a fraction of eligible patients are receiving CAR T-cell therapies, Kansagra said. Potentially, 9750 patients a year may be eligible for CAR T-cell therapies in approved and upcoming hematologic indications. From 2016 to 2019, a total of 2058 patients received CAR T-cell infusion.4

Next steps for transplanting these novel therapies to clinical practice will require changes in key areas, Kansagra said, such as supply chain management, patient support, and financial systems (Figure).4

Figure. Next Steps for Effective Delivery of Gene and Cell Therapies4

Meanwhile, multiple myeloma experts advise providers to be ready for change. As commercially available myeloma CAR T-cell therapies are approved, it will be even more important for community oncologists to better understand these therapies so they can offer them to their patients, Wang and Madduri wrote.1

Cell therapy involves cultivating or modifying immune cells outside the body before injecting them into the patient. Cells may be autologous (self-provided) or allogeneic (donor-provided); they include hematopoietic stem cells and adult and embryonic stem cells. Gene therapy modifies or manipulates cell expression. There is considerable overlap between the 2 disciplines.

Juliette Hordeaux, PhD, senior director of translational research for the University of Pennsylvanias gene therapy program, is cautious about the FDAs predictions, saying shed be thrilled with 5 cell and/or gene therapy approvals annually.

For monogenic diseases, there are only a certain number of mutations, and then well plateau until we reach a stage where we can go after more common diseases, Hordeaux said.

Safety has been the main brake around adeno-associated virus vector [AAV] gene therapy, added Hordeaux, whose hospitals program has the institutional memory of both Jesse Gelsingers tragic death during a 1999 gene therapy trial as well as breakthroughs by 2015 Giants of Cancer Care winner in immuno-oncology Carl H. June, MD, and others in CAR T-cell therapy. Sometimes there are unexpected toxicity [events] in trials.I think figuring out ways to make gene therapy safer is going to be the next goal for the field before we can even envision many more drugs approved.

In total, 3 CAR T-cell therapies are now on the market, all targeting the CD19 antigen. Tisagenlecleucel was the first. Gilead Sciences received approval in October 2017 for axicabtagene ciloleucel (axi-cel; Yescarta), a CAR T-cell therapy for adults with large B-cell non-Hodgkin lymphoma. Kite Pharma, a subsidiary of Gilead, received an accelerated approval in July 2020 for brexucabtagene autoleucel (Tecartus) for adults with relapsed/ refractory mantle cell lymphoma.

Another CD19-directed therapy under FDA review for relapsed/refractory large B-cell lymphoma, is lisocabtagene maraleucel (liso-cel; JCAR017; Bristol Myers Squibb). Idecabtagene vicleucel (ide-cel; bb2121; Bristol Myers Squibb) is under priority FDA review, with a decision expected by March 31, 2021. The biologics license application for ide-cel seeks approval for the B-cell maturation antigendirected CAR therapy to treat adult patients with multiple myeloma who have received at least 3 prior therapies.5

The number of clinical trials evaluating CAR T-cell therapies has risen sharply since 2015, when investigators counted a total of 78 studies registered on the ClinicalTrials. gov website. In June 2020, the site listed 671 trials, including 357 registered in China, 256 in the United States, and 58 in other countries.6 Natural killer (NK) cells are the research focus of Dean A. Lee, MD, PhD, a physician in the Division of Hematology and Oncology at Nationwide Childrens Hospital in Columbus, Ohio. He developed a method for consistent, robust expansion of highly active clinical-grade NK cells that enables repeated delivery of large cell doses for improved efficacy. This finding led to several first-in-human clinical trials evaluating adoptive immunotherapy with expanded NK cells under an FDA investigational new drug application. Lee is developing both genetic and nongenetic methods to improve tumor targeting and tissue homing of NK cells. His efforts are geared toward pediatric sarcomas.

The biggest emphasis over the past 20 to 25 years has been cell therapy for cancer, talking about trying to transfer a specific part of the immune system for cells, said Lee, who is also director of the Cellular Therapy and Cancer Immunology Program at Nationwide Childrens Hospital, at The Ohio State University Comprehensive Cancer Center Arthur G. James Cancer Hospital, and at the Richard J. Solove Research Institute.

However, Lee said, NKs have wider potential. This is kind of a natural swing back. Now that we know we can grow them, we can reengineer them against infectious disease targets and use them in that [space], he said.

Lee is part of a coronavirus disease 2019 (COVID-19) clinical trial, partnering with Kiadis, for off-the-shelf K-NK cells using Kiadis proprietary platforms. Such treatment would be a postexposure preemptive therapy for treating COVID-19. Lee said the pivot toward treating COVID19 with cell therapy was because some of the very early reports on immune responses to coronavirus, both original [SARS-CoV-2] and the new [mutation], seem to implicate that those who did poorly [overall] had poorly functioning NK cells.

The revolutionary gene editing tool CRISPR is making its initial impact in clinical trials outside the cancer area. Its developers, Jennifer Doudna, PhD, and Emmanuelle Charpentier, PhD, won the Nobel Prize in Chemistry 2020.

For patients with sickle cell disease (SCD), CRISPR was used to reengineer bone marrow cells to produce fetal hemoglobin, with the hope that the protein would turn deformed red blood cells into healthy ones. National Public Radio (NPR) did a story on one patient who, so far, thanks to CRISPR, has been liberated from the attacks of SCD that typically have sent her to the hospital, as well from the need for blood transfusions.7

Its a miracle, you know? the patient, Victoria Gray of Forest, Mississippi, told NPR.

She was among 10 patients with SCD or transfusion-dependent beta-thalassemia treated with promising results, as reported by the New England Journal of Medicine.8

Stephen Gottschalk, MD, chair of the department of bone marrow transplantation and cellular therapy at St Jude Childrens Research Hospital, said, Theres a lot of activity to really explore these therapies with diseases that are much more common than cancer.

Animal models use T cells to reverse cardiac fibrosis, for instance, Gottschalk said. Using T cells to reverse pathologies associated with senescence, such as conditions associated with inflammatory clots, are also being studied.

CAR T, I think, will become part of the standard of care, Gottschalk said. The question is how to best get that accomplished. To address the tribulations of some autologous products, a lot of groups are working with off-the-shelf products to get around some of the manufacturing bottlenecks. I believe those issues will be solved in the long run.

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Harnessing the Potential of Cell and Gene Therapy - OncLive

Stem Cell Study Illuminates the Cause of a Devastating Inherited Heart Disorder – Newswise

Newswise PHILADELPHIAScientists in the Perelman School of Medicine at the University of Pennsylvania have uncovered the molecular causes of a congenital form of dilated cardiomyopathy (DCM), an often-fatal heart disorder.

This inherited form of DCM which affects at least several thousand people in the United States at any one time and often causes sudden death or progressive heart failure is one of multiple congenital disorders known to be caused by inherited mutations in a gene called LMNA. The LMNA gene is active in most cell types, and researchers have not understood why LMNA mutations affect particular organs such as the heart while sparing most other organs and tissues.

In the study, published this week in Cell Stem Cell, the Penn Medicine scientists used stem cell techniques to grow human heart muscle cells containing DCM-causing mutations in LMNA. They found that these mutations severely disrupt the structural organization of DNA in the nucleus of heart muscle cells but not two other cell types studied leading to the abnormal activation of non-heart muscle genes.

Were now beginning to understand why patients with LMNA mutations have tissue-restricted disorders such as DCM even though the gene is expressed in most cell types, said study co-senior author Rajan Jain, MD, an assistant professor of Cardiovascular Medicine and Cell and Developmental Biology at the Perelman School of Medicine.

Further work along these lines should enable us to predict how LMNA mutations will manifest in individual patients, and ultimately we may be able to intervene with drugs to correct the genome disorganization that these mutations cause, said study co-senior author Kiran Musunuru, MD, PhD, a professor of Cardiovascular Medicine and Genetics, and Director of the Genetic and Epigenetic Origins of Disease Program at Penn Medicine.

Inherited LMNA mutations have long puzzled researchers. The LMNA gene encodes proteins that form a lacy structure on the inner wall of the cell nucleus, where chromosomes full of coiled DNA are housed. This lacy structure, known as the nuclear lamina, touches some parts of the genome, and these lamina-genome interactions help regulate gene activity, for example in the process of cell division. The puzzle is that the nuclear lamina is found in most cell types, yet the disruption of this important and near-ubiquitous cellular component by LMNA mutations causes only a handful of relatively specific clinical disorders, including a form of DCM, two forms of muscular dystrophy, and a form of progeria a syndrome that resembles rapid aging.

To better understand how LMNA mutations can cause DCM, Jain, Musunuru, and their colleagues took cells from a healthy human donor, and used the CRISPR gene-editing technique to create known DCM-causing LMNA mutations in each cell. They then used stem cell methods to turn these cells into heart muscle cells cardiomyocytes and, for comparison, liver and fat cells. Their goal was to discover what was happening in the mutation-containing cardiomyocytes that wasnt happening in the other cell types.

The researchers found that in the LMNA-mutant cardiomyocytes but hardly at all in the other two cell types the nuclear lamina had an altered appearance and did not connect to the genome in the usual way. This disruption of lamina-genome interactions led to a failure of normal gene regulation: many genes that should be switched off in heart muscle cells were active. The researchers examined cells taken from DCM patients with LMNA mutations and found similar abnormalities in gene activity.

A distinctive pattern of gene activity essentially defines what biologists call the identity of a cell. Thus the DCM-causing LMNA mutations had begun to alter the identity of cardiomyocytes, giving them features of other cell types.

The LMNA-mutant cardiomyocytes also had another defect seen in patients with LMNA-linked DCM: the heart muscle cells had lost much of the mechanical elasticity that normally allows them to contract and stretch as needed. The same deficiency was not seen in the LMNA-mutant liver and fat cells.

Research is ongoing to understand whether changes in elasticity in the heart cells with LMNA mutations occurs prior to changes in genome organization, or whether the genome interactions at the lamina help ensure proper elasticity. Their experiments did suggest an explanation for the differences between the lamina-genome connections being badly disrupted in LMNA-mutant cardiomyocytes but not so much in LMNA-mutant liver and fat cells: Every cell type uses a distinct pattern of chemical marks on its genome, called epigenetic marks, to program its patterns of gene activity, and this pattern in cardiomyocytes apparently results in lamina-genome interactions that are especially vulnerable to disruption in the presence of certain LMNA mutations.

The findings reveal the likely importance of the nuclear lamina in regulating cell identity and the physical organization of the genome, Jain said. This also opens up new avenues of research that could one day lead to the successful treatment or prevention of LMNA-mutations and related disorders.

Other co-authors of the study were co-first authors Parisha Shah and Wenjian Lv; and Joshua Rhoades, Andrey Poleshko, Deepti Abbey, Matthew Caporizzo, Ricardo Linares-Saldana, Julie Heffler, Nazish Sayed, Dilip Thomas, Qiaohong Wang, Liam Stanton, Kenneth Bedi, Michael Morley, Thomas Cappola, Anjali Owens, Kenneth Margulies, David Frank, Joseph Wu, Daniel Rader, Wenli Yang, and Benjamin Prosser.

Funding was provided by the Burroughs Wellcome Career Award for Medical Scientists, Gilead Research Scholars Award, Pennsylvania Department of Health, American Heart Association/Allen Initiative, the National Institutes of Health (DP2 HL147123, R35 HL145203, R01 HL149891, F31 HL147416, NSF15-48571, R01 GM137425), the Penn Institute of Regenerative Medicine, and the Winkelman Family Fund for Cardiac Innovation.

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Penn Medicineis one of the worlds leading academic medical centers, dedicated to the related missions of medical education, biomedical research, and excellence in patient care. Penn Medicine consists of theRaymond and Ruth Perelman School of Medicine at the University of Pennsylvania (founded in 1765 as the nations first medical school) and theUniversity of Pennsylvania Health System, which together form a $8.6 billion enterprise.

The Perelman School of Medicine has been ranked among the top medical schools in the United States for more than 20 years, according toU.S. News & World Report's survey of research-oriented medical schools. The School is consistently among the nation's top recipients of funding from the National Institutes of Health, with $494 million awarded in the 2019 fiscal year.

The University of Pennsylvania Health Systems patient care facilities include: the Hospital of the University of Pennsylvania and Penn Presbyterian Medical Centerwhich are recognized as one of the nations top Honor Roll hospitals byU.S. News & World ReportChester County Hospital; Lancaster General Health; Penn Medicine Princeton Health; and Pennsylvania Hospital, the nations first hospital, founded in 1751. Additional facilities and enterprises include Good Shepherd Penn Partners, Penn Medicine at Home, Lancaster Behavioral Health Hospital, and Princeton House Behavioral Health, among others.

Penn Medicine is powered by a talented and dedicated workforce of more than 43,900 people. The organization also has alliances with top community health systems across both Southeastern Pennsylvania and Southern New Jersey, creating more options for patients no matter where they live.

Penn Medicine is committed to improving lives and health through a variety of community-based programs and activities. In fiscal year 2019, Penn Medicine provided more than $583 million to benefit our community.

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Stem Cell Study Illuminates the Cause of a Devastating Inherited Heart Disorder - Newswise

Merck Receives Positive EU CHMP Opinion for Expanded Approval of KEYTRUDA (pembrolizumab) in Certain Patients With Relapsed or Refractory Classical…

KENILWORTH, N.J.--(BUSINESS WIRE)--Merck (NYSE: MRK), known as MSD outside the United States and Canada, today announced that the Committee for Medicinal Products for Human Use (CHMP) of the European Medicines Agency (EMA) has adopted a positive opinion recommending approval of an expanded label for KEYTRUDA, Mercks anti-PD-1 therapy. The opinion is recommending KEYTRUDA as monotherapy for the treatment of adult and pediatric patients aged 3 years and older with relapsed or refractory classical Hodgkin lymphoma (cHL) who have failed autologous stem cell transplant (ASCT) or following at least two prior therapies when ASCT is not a treatment option.

This recommendation is based on results from the pivotal Phase 3 KEYNOTE-204 trial, in which KEYTRUDA monotherapy demonstrated a significant improvement in progression-free survival (PFS) compared with brentuximab vedotin (BV), a commonly used treatment. KEYTRUDA reduced the risk of disease progression or death by 35% (HR=0.65 [95% CI, 0.48-0.88]; p=0.00271) and showed a median PFS of 13.2 months versus 8.3 months for patients treated with BV. The recommendation is also based on supportive data from an updated analysis of the KEYNOTE-087 trial, which supported the European Commissions (EC) approval of KEYTRUDA for the treatment of adult patients with relapsed or refractory cHL who have failed ASCT and BV or who are transplant ineligible and have failed BV. The CHMPs recommendation will now be reviewed by the EC for marketing authorization in the European Union (EU), and a final decision is expected in the first quarter of 2021. If approved, this will be the first pediatric indication for KEYTRUDA in the EU.

This positive opinion reinforces the importance of KEYTRUDA for certain adult and pediatric patients with relapsed or refractory classical Hodgkin lymphoma in the European Union, said Dr. Vicki Goodman, vice president, clinical research, Merck Research Laboratories. We look forward to the decision by the European Commission and will continue to expand our clinical development program in blood cancers with KEYTRUDA and our recently acquired investigational therapies to help address the unmet needs of patients.

Merck is studying KEYTRUDA across hematologic malignancies through a broad clinical program, including multiple registrational trials in cHL and primary mediastinal large B-cell lymphoma and more than 60 investigator-initiated studies across 15 tumors. In addition to KEYTRUDA, Merck is evaluating two clinical-stage assets for the treatment of patients with hematologic malignancies: MK-1026 (formerly ARQ 531), a Brutons tyrosine kinase inhibitor, and VLS-101, an antibody-drug conjugate targeting ROR1.

About KEYNOTE-204

KEYNOTE-204 (ClinicalTrials.gov, NCT02684292) is a randomized, open-label, Phase 3 trial evaluating KEYTRUDA monotherapy compared with BV for the treatment of patients with relapsed or refractory cHL. The primary endpoints are PFS and overall survival (OS), and the secondary endpoints include objective response rate (ORR), complete remission rate (CRR) and safety. The study enrolled 304 patients, aged 18 years and older, who were randomized to receive either:

About Hodgkin Lymphoma

Hodgkin lymphoma is a type of lymphoma that develops in the white blood cells called lymphocytes, which are part of the immune system. Hodgkin lymphoma can start almost anywhere most often in lymph nodes in the upper part of the body, with the most common sites being in the chest, neck or under the arms. Worldwide, there were approximately 83,000 new cases of Hodgkin lymphoma diagnosed, and more than 23,000 people died from the disease in 2020. In the EU, there were nearly 20,000 new cases of Hodgkin lymphoma diagnosed, and nearly 4,000 people died from the disease in 2020. Classical Hodgkin lymphoma accounts for more than nine in 10 cases of Hodgkin lymphoma in developed countries.

About KEYTRUDA (pembrolizumab) Injection, 100 mg

KEYTRUDA is an anti-PD-1 therapy that works by increasing the ability of the bodys immune system to help detect and fight tumor cells. KEYTRUDA is a humanized monoclonal antibody that blocks the interaction between PD-1 and its ligands, PD-L1 and PD-L2, thereby activating T lymphocytes which may affect both tumor cells and healthy cells.

Merck has the industrys largest immuno-oncology clinical research program. There are currently more than 1,300 trials studying KEYTRUDA across a wide variety of cancers and treatment settings. The KEYTRUDA clinical program seeks to understand the role of KEYTRUDA across cancers and the factors that may predict a patient's likelihood of benefitting from treatment with KEYTRUDA, including exploring several different biomarkers.

Selected KEYTRUDA (pembrolizumab) Indications in the U.S.

Melanoma

KEYTRUDA is indicated for the treatment of patients with unresectable or metastatic melanoma.

KEYTRUDA is indicated for the adjuvant treatment of patients with melanoma with involvement of lymph node(s) following complete resection.

Non-Small Cell Lung Cancer

KEYTRUDA, in combination with pemetrexed and platinum chemotherapy, is indicated for the first-line treatment of patients with metastatic nonsquamous non-small cell lung cancer (NSCLC), with no EGFR or ALK genomic tumor aberrations.

KEYTRUDA, in combination with carboplatin and either paclitaxel or paclitaxel protein-bound, is indicated for the first-line treatment of patients with metastatic squamous NSCLC.

KEYTRUDA, as a single agent, is indicated for the first-line treatment of patients with NSCLC expressing PD-L1 [tumor proportion score (TPS) 1%] as determined by an FDA-approved test, with no EGFR or ALK genomic tumor aberrations, and is stage III where patients are not candidates for surgical resection or definitive chemoradiation, or metastatic.

KEYTRUDA, as a single agent, is indicated for the treatment of patients with metastatic NSCLC whose tumors express PD-L1 (TPS 1%) as determined by an FDA-approved test, with disease progression on or after platinum-containing chemotherapy. Patients with EGFR or ALK genomic tumor aberrations should have disease progression on FDA-approved therapy for these aberrations prior to receiving KEYTRUDA.

Small Cell Lung Cancer

KEYTRUDA is indicated for the treatment of patients with metastatic small cell lung cancer (SCLC) with disease progression on or after platinum-based chemotherapy and at least 1 other prior line of therapy. This indication is approved under accelerated approval based on tumor response rate and durability of response. Continued approval for this indication may be contingent upon verification and description of clinical benefit in confirmatory trials.

Head and Neck Squamous Cell Cancer

KEYTRUDA, in combination with platinum and fluorouracil (FU), is indicated for the first-line treatment of patients with metastatic or with unresectable, recurrent head and neck squamous cell carcinoma (HNSCC).

KEYTRUDA, as a single agent, is indicated for the first-line treatment of patients with metastatic or with unresectable, recurrent HNSCC whose tumors express PD-L1 [combined positive score (CPS) 1] as determined by an FDA-approved test.

KEYTRUDA, as a single agent, is indicated for the treatment of patients with recurrent or metastatic HNSCC with disease progression on or after platinum-containing chemotherapy.

Classical Hodgkin Lymphoma

KEYTRUDA is indicated for the treatment of adult patients with relapsed or refractory classical Hodgkin lymphoma (cHL).

KEYTRUDA is indicated for the treatment of pediatric patients with refractory cHL, or cHL that has relapsed after 2 or more lines of therapy.

Primary Mediastinal Large B-Cell Lymphoma

KEYTRUDA is indicated for the treatment of adult and pediatric patients with refractory primary mediastinal large B-cell lymphoma (PMBCL), or who have relapsed after 2 or more prior lines of therapy. KEYTRUDA is not recommended for treatment of patients with PMBCL who require urgent cytoreductive therapy.

Urothelial Carcinoma

KEYTRUDA is indicated for the treatment of patients with locally advanced or metastatic urothelial carcinoma (mUC) who are not eligible for cisplatin-containing chemotherapy and whose tumors express PD-L1 (CPS 10), as determined by an FDA-approved test, or in patients who are not eligible for any platinum-containing chemotherapy regardless of PD-L1 status. This indication is approved under accelerated approval based on tumor response rate and duration of response. Continued approval for this indication may be contingent upon verification and description of clinical benefit in confirmatory trials.

KEYTRUDA is indicated for the treatment of patients with locally advanced or metastatic urothelial carcinoma (mUC) who have disease progression during or following platinum-containing chemotherapy or within 12 months of neoadjuvant or adjuvant treatment with platinum-containing chemotherapy.

KEYTRUDA is indicated for the treatment of patients with Bacillus Calmette-Guerin (BCG)-unresponsive, high-risk, non-muscle invasive bladder cancer (NMIBC) with carcinoma in situ (CIS) with or without papillary tumors who are ineligible for or have elected not to undergo cystectomy.

Microsatellite Instability-High or Mismatch Repair Deficient Cancer

KEYTRUDA is indicated for the treatment of adult and pediatric patients with unresectable or metastatic microsatellite instability-high (MSI-H) or mismatch repair deficient (dMMR)

This indication is approved under accelerated approval based on tumor response rate and durability of response. Continued approval for this indication may be contingent upon verification and description of clinical benefit in the confirmatory trials. The safety and effectiveness of KEYTRUDA in pediatric patients with MSI-H central nervous system cancers have not been established.

Microsatellite Instability-High or Mismatch Repair Deficient Colorectal Cancer

KEYTRUDA is indicated for the first-line treatment of patients with unresectable or metastatic MSI-H or dMMR colorectal cancer (CRC).

Gastric Cancer

KEYTRUDA is indicated for the treatment of patients with recurrent locally advanced or metastatic gastric or gastroesophageal junction (GEJ) adenocarcinoma whose tumors express PD-L1 (CPS 1) as determined by an FDA-approved test, with disease progression on or after two or more prior lines of therapy including fluoropyrimidine- and platinum-containing chemotherapy and if appropriate, HER2/neu-targeted therapy. This indication is approved under accelerated approval based on tumor response rate and durability of response. Continued approval for this indication may be contingent upon verification and description of clinical benefit in the confirmatory trials.

Esophageal Cancer

KEYTRUDA is indicated for the treatment of patients with recurrent locally advanced or metastatic squamous cell carcinoma of the esophagus whose tumors express PD-L1 (CPS 10) as determined by an FDA-approved test, with disease progression after one or more prior lines of systemic therapy.

Cervical Cancer

KEYTRUDA is indicated for the treatment of patients with recurrent or metastatic cervical cancer with disease progression on or after chemotherapy whose tumors express PD-L1 (CPS 1) as determined by an FDA-approved test. This indication is approved under accelerated approval based on tumor response rate and durability of response. Continued approval for this indication may be contingent upon verification and description of clinical benefit in the confirmatory trials.

Hepatocellular Carcinoma

KEYTRUDA is indicated for the treatment of patients with hepatocellular carcinoma (HCC) who have been previously treated with sorafenib. This indication is approved under accelerated approval based on tumor response rate and durability of response. Continued approval for this indication may be contingent upon verification and description of clinical benefit in the confirmatory trials.

Merkel Cell Carcinoma

KEYTRUDA is indicated for the treatment of adult and pediatric patients with recurrent locally advanced or metastatic Merkel cell carcinoma (MCC). This indication is approved under accelerated approval based on tumor response rate and durability of response. Continued approval for this indication may be contingent upon verification and description of clinical benefit in the confirmatory trials.

Renal Cell Carcinoma

KEYTRUDA, in combination with axitinib, is indicated for the first-line treatment of patients with advanced renal cell carcinoma (RCC).

Tumor Mutational Burden-High

KEYTRUDA is indicated for the treatment of adult and pediatric patients with unresectable or metastatic tumor mutational burden-high (TMB-H) [10 mutations/megabase] solid tumors, as determined by an FDA-approved test, that have progressed following prior treatment and who have no satisfactory alternative treatment options. This indication is approved under accelerated approval based on tumor response rate and durability of response. Continued approval for this indication may be contingent upon verification and description of clinical benefit in the confirmatory trials. The safety and effectiveness of KEYTRUDA in pediatric patients with TMB-H central nervous system cancers have not been established.

Cutaneous Squamous Cell Carcinoma

KEYTRUDA is indicated for the treatment of patients with recurrent or metastatic cutaneous squamous cell carcinoma (cSCC) that is not curable by surgery or radiation.

Triple-Negative Breast Cancer

KEYTRUDA, in combination with chemotherapy, is indicated for the treatment of patients with locally recurrent unresectable or metastatic triple-negative breast cancer (TNBC) whose tumors express PD-L1 (CPS 10) as determined by an FDA-approved test. This indication is approved under accelerated approval based on progression-free survival. Continued approval for this indication may be contingent upon verification and description of clinical benefit in the confirmatory trials.

Selected Important Safety Information for KEYTRUDA

Severe and Fatal Immune-Mediated Adverse Reactions

KEYTRUDA is a monoclonal antibody that belongs to a class of drugs that bind to either the programmed death receptor-1 (PD-1) or the programmed death ligand 1 (PD-L1), blocking the PD-1/PD-L1 pathway, thereby removing inhibition of the immune response, potentially breaking peripheral tolerance and inducing immune-mediated adverse reactions. Immune-mediated adverse reactions, which may be severe or fatal, can occur in any organ system or tissue, can affect more than one body system simultaneously, and can occur at any time after starting treatment or after discontinuation of treatment. Important immune-mediated adverse reactions listed here may not include all possible severe and fatal immune-mediated adverse reactions.

Monitor patients closely for symptoms and signs that may be clinical manifestations of underlying immune-mediated adverse reactions. Early identification and management are essential to ensure safe use of antiPD-1/PD-L1 treatments. Evaluate liver enzymes, creatinine, and thyroid function at baseline and periodically during treatment. In cases of suspected immune-mediated adverse reactions, initiate appropriate workup to exclude alternative etiologies, including infection. Institute medical management promptly, including specialty consultation as appropriate.

Withhold or permanently discontinue KEYTRUDA depending on severity of the immune-mediated adverse reaction. In general, if KEYTRUDA requires interruption or discontinuation, administer systemic corticosteroid therapy (1 to 2 mg/kg/day prednisone or equivalent) until improvement to Grade 1 or less. Upon improvement to Grade 1 or less, initiate corticosteroid taper and continue to taper over at least 1 month. Consider administration of other systemic immunosuppressants in patients whose adverse reactions are not controlled with corticosteroid therapy.

Immune-Mediated Pneumonitis

KEYTRUDA can cause immune-mediated pneumonitis. The incidence is higher in patients who have received prior thoracic radiation. Immune-mediated pneumonitis occurred in 3.4% (94/2799) of patients receiving KEYTRUDA, including fatal (0.1%), Grade 4 (0.3%), Grade 3 (0.9%), and Grade 2 (1.3%) reactions. Systemic corticosteroids were required in 67% (63/94) of patients. Pneumonitis led to permanent discontinuation of KEYTRUDA in 1.3% (36) and withholding in 0.9% (26) of patients. All patients who were withheld reinitiated KEYTRUDA after symptom improvement; of these, 23% had recurrence. Pneumonitis resolved in 59% of the 94 patients.

Pneumonitis occurred in 8% (31/389) of adult patients with cHL receiving KEYTRUDA as a single agent, including Grades 3-4 in 2.3% of patients. Patients received high-dose corticosteroids for a median duration of 10 days (range: 2 days to 53 months). Pneumonitis rates were similar in patients with and without prior thoracic radiation. Pneumonitis led to discontinuation of KEYTRUDA in 5.4% (21) of patients. Of the patients who developed pneumonitis, 42% of these patients interrupted KEYTRUDA, 68% discontinued KEYTRUDA, and 77% had resolution.

Immune-Mediated Colitis

KEYTRUDA can cause immune-mediated colitis, which may present with diarrhea. Cytomegalovirus infection/reactivation has been reported in patients with corticosteroid-refractory immune-mediated colitis. In cases of corticosteroid-refractory colitis, consider repeating infectious workup to exclude alternative etiologies. Immune-mediated colitis occurred in 1.7% (48/2799) of patients receiving KEYTRUDA, including Grade 4 (<0.1%), Grade 3 (1.1%), and Grade 2 (0.4%) reactions. Systemic corticosteroids were required in 69% (33/48); additional immunosuppressant therapy was required in 4.2% of patients. Colitis led to permanent discontinuation of KEYTRUDA in 0.5% (15) and withholding in 0.5% (13) of patients. All patients who were withheld reinitiated KEYTRUDA after symptom improvement; of these, 23% had recurrence. Colitis resolved in 85% of the 48 patients.

Hepatotoxicity and Immune-Mediated Hepatitis

KEYTRUDA as a Single Agent

KEYTRUDA can cause immune-mediated hepatitis. Immune-mediated hepatitis occurred in 0.7% (19/2799) of patients receiving KEYTRUDA, including Grade 4 (<0.1%), Grade 3 (0.4%), and Grade 2 (0.1%) reactions. Systemic corticosteroids were required in 68% (13/19) of patients; additional immunosuppressant therapy was required in 11% of patients. Hepatitis led to permanent discontinuation of KEYTRUDA in 0.2% (6) and withholding in 0.3% (9) of patients. All patients who were withheld reinitiated KEYTRUDA after symptom improvement; of these, none had recurrence. Hepatitis resolved in 79% of the 19 patients.

KEYTRUDA with Axitinib

KEYTRUDA in combination with axitinib can cause hepatic toxicity. Monitor liver enzymes before initiation of and periodically throughout treatment. Consider monitoring more frequently as compared to when the drugs are administered as single agents. For elevated liver enzymes, interrupt KEYTRUDA and axitinib, and consider administering corticosteroids as needed. With the combination of KEYTRUDA and axitinib, Grades 3 and 4 increased alanine aminotransferase (ALT) (20%) and increased aspartate aminotransferase (AST) (13%) were seen, which was at a higher frequency compared to KEYTRUDA alone. Fifty-nine percent of the patients with increased ALT received systemic corticosteroids. In patients with ALT 3 times upper limit of normal (ULN) (Grades 2-4, n=116), ALT resolved to Grades 0-1 in 94%. Among the 92 patients who were rechallenged with either KEYTRUDA (n=3) or axitinib (n=34) administered as a single agent or with both (n=55), recurrence of ALT 3 times ULN was observed in 1 patient receiving KEYTRUDA, 16 patients receiving axitinib, and 24 patients receiving both. All patients with a recurrence of ALT 3 ULN subsequently recovered from the event.

Immune-Mediated Endocrinopathies

Adrenal Insufficiency

KEYTRUDA can cause primary or secondary adrenal insufficiency. For Grade 2 or higher, initiate symptomatic treatment, including hormone replacement as clinically indicated. Withhold KEYTRUDA depending on severity. Adrenal insufficiency occurred in 0.8% (22/2799) of patients receiving KEYTRUDA, including Grade 4 (<0.1%), Grade 3 (0.3%), and Grade 2 (0.3%) reactions. Systemic corticosteroids were required in 77% (17/22) of patients; of these, the majority remained on systemic corticosteroids. Adrenal insufficiency led to permanent discontinuation of KEYTRUDA in <0.1% (1) and withholding in 0.3% (8) of patients. All patients who were withheld reinitiated KEYTRUDA after symptom improvement.

Hypophysitis

KEYTRUDA can cause immune-mediated hypophysitis. Hypophysitis can present with acute symptoms associated with mass effect such as headache, photophobia, or visual field defects. Hypophysitis can cause hypopituitarism. Initiate hormone replacement as indicated. Withhold or permanently discontinue KEYTRUDA depending on severity. Hypophysitis occurred in 0.6% (17/2799) of patients receiving KEYTRUDA, including Grade 4 (<0.1%), Grade 3 (0.3%), and Grade 2 (0.2%) reactions. Systemic corticosteroids were required in 94% (16/17) of patients; of these, the majority remained on systemic corticosteroids. Hypophysitis led to permanent discontinuation of KEYTRUDA in 0.1% (4) and withholding in 0.3% (7) of patients. All patients who were withheld reinitiated KEYTRUDA after symptom improvement.

Thyroid Disorders

KEYTRUDA can cause immune-mediated thyroid disorders. Thyroiditis can present with or without endocrinopathy. Hypothyroidism can follow hyperthyroidism. Initiate hormone replacement for hypothyroidism or institute medical management of hyperthyroidism as clinically indicated. Withhold or permanently discontinue KEYTRUDA depending on severity. Thyroiditis occurred in 0.6% (16/2799) of patients receiving KEYTRUDA, including Grade 2 (0.3%). None discontinued, but KEYTRUDA was withheld in <0.1% (1) of patients.

Hyperthyroidism occurred in 3.4% (96/2799) of patients receiving KEYTRUDA, including Grade 3 (0.1%) and Grade 2 (0.8%). It led to permanent discontinuation of KEYTRUDA in <0.1% (2) and withholding in 0.3% (7) of patients. All patients who were withheld reinitiated KEYTRUDA after symptom improvement. Hypothyroidism occurred in 8% (237/2799) of patients receiving KEYTRUDA, including Grade 3 (0.1%) and Grade 2 (6.2%). It led to permanent discontinuation of KEYTRUDA in <0.1% (1) and withholding in 0.5% (14) of patients. All patients who were withheld reinitiated KEYTRUDA after symptom improvement. The majority of patients with hypothyroidism required long-term thyroid hormone replacement. The incidence of new or worsening hypothyroidism was higher in 1185 patients with HNSCC, occurring in 16% of patients receiving KEYTRUDA as a single agent or in combination with platinum and FU, including Grade 3 (0.3%) hypothyroidism. The incidence of new or worsening hypothyroidism was higher in 389 adult patients with cHL (17%) receiving KEYTRUDA as a single agent, including Grade 1 (6.2%) and Grade 2 (10.8%) hypothyroidism.

Type 1 Diabetes Mellitus (DM), Which Can Present With Diabetic Ketoacidosis

Monitor patients for hyperglycemia or other signs and symptoms of diabetes. Initiate treatment with insulin as clinically indicated. Withhold KEYTRUDA depending on severity. Type 1 DM occurred in 0.2% (6/2799) of patients receiving KEYTRUDA. It led to permanent discontinuation in <0.1% (1) and withholding of KEYTRUDA in <0.1% (1). All patients who were withheld reinitiated KEYTRUDA after symptom improvement.

Immune-Mediated Nephritis With Renal Dysfunction

KEYTRUDA can cause immune-mediated nephritis. Immune-mediated nephritis occurred in 0.3% (9/2799) of patients receiving KEYTRUDA, including Grade 4 (<0.1%), Grade 3 (0.1%), and Grade 2 (0.1%) reactions. Systemic corticosteroids were required in 89% (8/9) of patients. Nephritis led to permanent discontinuation of KEYTRUDA in 0.1% (3) and withholding in 0.1% (3) of patients. All patients who were withheld reinitiated KEYTRUDA after symptom improvement; of these, none had recurrence. Nephritis resolved in 56% of the 9 patients.

Immune-Mediated Dermatologic Adverse Reactions

KEYTRUDA can cause immune-mediated rash or dermatitis. Exfoliative dermatitis, including Stevens-Johnson syndrome, drug rash with eosinophilia and systemic symptoms, and toxic epidermal necrolysis, has occurred with antiPD-1/PD-L1 treatments. Topical emollients and/or topical corticosteroids may be adequate to treat mild to moderate nonexfoliative rashes. Withhold or permanently discontinue KEYTRUDA depending on severity. Immune-mediated dermatologic adverse reactions occurred in 1.4% (38/2799) of patients receiving KEYTRUDA, including Grade 3 (1%) and Grade 2 (0.1%) reactions. Systemic corticosteroids were required in 40% (15/38) of patients. These reactions led to permanent discontinuation in 0.1% (2) and withholding of KEYTRUDA in 0.6% (16) of patients. All patients who were withheld reinitiated KEYTRUDA after symptom improvement; of these, 6% had recurrence. The reactions resolved in 79% of the 38 patients.

Other Immune-Mediated Adverse Reactions

The following clinically significant immune-mediated adverse reactions occurred at an incidence of <1% (unless otherwise noted) in patients who received KEYTRUDA or were reported with the use of other antiPD-1/PD-L1 treatments. Severe or fatal cases have been reported for some of these adverse reactions. Cardiac/Vascular: Myocarditis, pericarditis, vasculitis; Nervous System: Meningitis, encephalitis, myelitis and demyelination, myasthenic syndrome/myasthenia gravis (including exacerbation), Guillain-Barr syndrome, nerve paresis, autoimmune neuropathy; Ocular: Uveitis, iritis and other ocular inflammatory toxicities can occur. Some cases can be associated with retinal detachment. Various grades of visual impairment, including blindness, can occur. If uveitis occurs in combination with other immune-mediated adverse reactions, consider a Vogt-Koyanagi-Harada-like syndrome, as this may require treatment with systemic steroids to reduce the risk of permanent vision loss; Gastrointestinal: Pancreatitis, to include increases in serum amylase and lipase levels, gastritis, duodenitis; Musculoskeletal and Connective Tissue: Myositis/polymyositis rhabdomyolysis (and associated sequelae, including renal failure), arthritis (1.5%), polymyalgia rheumatica; Endocrine: Hypoparathyroidism; Hematologic/Immune: Hemolytic anemia, aplastic anemia, hemophagocytic lymphohistiocytosis, systemic inflammatory response syndrome, histiocytic necrotizing lymphadenitis (Kikuchi lymphadenitis), sarcoidosis, immune thrombocytopenic purpura, solid organ transplant rejection.

Infusion-Related Reactions

KEYTRUDA can cause severe or life-threatening infusion-related reactions, including hypersensitivity and anaphylaxis, which have been reported in 0.2% of 2799 patients receiving KEYTRUDA. Monitor for signs and symptoms of infusion-related reactions. Interrupt or slow the rate of infusion for Grade 1 or Grade 2 reactions. For Grade 3 or Grade 4 reactions, stop infusion and permanently discontinue KEYTRUDA.

Complications of Allogeneic Hematopoietic Stem Cell Transplantation (HSCT)

Fatal and other serious complications can occur in patients who receive allogeneic HSCT before or after antiPD-1/PD-L1 treatment. Transplant-related complications include hyperacute graft-versus-host disease (GVHD), acute and chronic GVHD, hepatic veno-occlusive disease after reduced intensity conditioning, and steroid-requiring febrile syndrome (without an identified infectious cause). These complications may occur despite intervening therapy between antiPD-1/PD-L1 treatment and allogeneic HSCT. Follow patients closely for evidence of these complications and intervene promptly. Consider the benefit vs risks of using antiPD-1/PD-L1 treatments prior to or after an allogeneic HSCT.

Increased Mortality in Patients With Multiple Myeloma

In trials in patients with multiple myeloma, the addition of KEYTRUDA to a thalidomide analogue plus dexamethasone resulted in increased mortality. Treatment of these patients with an antiPD-1/PD-L1 treatment in this combination is not recommended outside of controlled trials.

Embryofetal Toxicity

Based on its mechanism of action, KEYTRUDA can cause fetal harm when administered to a pregnant woman. Advise women of this potential risk. In females of reproductive potential, verify pregnancy status prior to initiating KEYTRUDA and advise them to use effective contraception during treatment and for 4 months after the last dose.

Adverse Reactions

In KEYNOTE-006, KEYTRUDA was discontinued due to adverse reactions in 9% of 555 patients with advanced melanoma; adverse reactions leading to permanent discontinuation in more than one patient were colitis (1.4%), autoimmune hepatitis (0.7%), allergic reaction (0.4%), polyneuropathy (0.4%), and cardiac failure (0.4%). The most common adverse reactions (20%) with KEYTRUDA were fatigue (28%), diarrhea (26%), rash (24%), and nausea (21%).

In KEYNOTE-054, KEYTRUDA was permanently discontinued due to adverse reactions in 14% of 509 patients; the most common (1%) were pneumonitis (1.4%), colitis (1.2%), and diarrhea (1%). Serious adverse reactions occurred in 25% of patients receiving KEYTRUDA. The most common adverse reaction (20%) with KEYTRUDA was diarrhea (28%).

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Merck Receives Positive EU CHMP Opinion for Expanded Approval of KEYTRUDA (pembrolizumab) in Certain Patients With Relapsed or Refractory Classical...

Worldwide Industry for Biopreservation to 2026 – Key Drivers, Restraints and Opportunities – Yahoo Finance

DUBLIN, Jan. 4, 2021 /PRNewswire/ -- The "Biopreservation Market by Type, Application, End-user, and Geography - Global Forecast to 2026" report has been added to ResearchAndMarkets.com's offering.

Biopreservation is a process that assists in the conservation of biospecimens such as DNA, saliva, and plasma. This process of biopreservation generally increases the durability, shelf life, and purity of the biosamples. The types of equipment in this process include freezers, liquid nitrogen, consumables, and also media & laboratory information management systems.

This process is also used to preserve food and extend its shelf life, specifically by using lactic acid bacteria. Growth in healthcare spending is assumed for better access to quality healthcare and advanced technology products such as biopreservation facilities, thereby widening the growth expectations. Moreover, the bio-banks, hospitals, and gene banks, which are major end-users for this market, are stimulating the key providers to establish technologically advanced biopreservation products to improve patient outcomes. The Biopreservation Market is projected to grow at a rate of 9.2% CAGR by 2026.

The biopreservation market has been analyzed by utilizing the optimum combination of secondary sources and in-house methodology, along with an irreplaceable blend of primary insights. The real-time assessment of the market is an integral part of our market sizing and forecasting methodology. Our industry experts and panel of primary participants have helped in compiling relevant aspects with realistic parametric estimations for a comprehensive study. The participation share of different categories of primary participants is given below:

In the market for biopreservation, the application of biopreservation consists of therapeutic applications, research applications, clinical trials, and other applications. The biopreservation is primarily applied in therapeutics due to the advancements in regenerative medicine & customized medicine, an increase in the shift of cord blood banking, and the rising incidence of chronic diseases.

The end-users of the biopreservation market include biobanks, gene banks, hospitals, and other end users. The biobanks segment is expected to have a major share in the market. The major share of this segment is attributed to the increasing preference for the preservation of stem cells and the rising numbers of sperm and egg banks.

Further, according to the regional market of biopreservation, the North American region is recorded for the colossal share in the market. This is due to the continuous drug developments and the arrival of advanced therapies in the domain of biomedical research. Additionally, the increasing requirement of expensive and improved treatment for patients' chronic diseases is the key factor.

The rising incidence of chronic diseases, including cardiac, renal diseases, diabetes, and obesity, is the crucial factor that will propel the biopreservation market growth in the prevailing period. Government initiatives to encourage stem cell therapies to treat the disease, which will again propel market growth. Conversely, the strict regulations for producing biopreservation products and the evolution of room temperature storage procedures may limit the biopreservation market growth.

Merck KGaA, Avantor, Inc., Bio-Techne Corporation, BioLife Solutions, Inc., Thermo Fisher Scientific Inc, ThermoGenesis Holdings, Inc., Worthington Industries, Inc., Chart Industries, Inc, So-Low Environmental Equipment Co., Inc., Princeton BioCision, LLC, Shanghai Genext Medical Technology Co. Ltd, Exact Sciences Corporation, Helmer Scientific, Inc., CryoTech, Inc., Arctiko, Nippon Genetics Europe, PHC Holdings Corporation, STEMCELL Technologies, Inc., AMS Biotechnology, and OPS Diagnostics. These are the few companies list of the biopreservation market.

Since the rapid increase in the number of research and developments gives the way of potentials for market growth, the biopreservation of biological samples has become a crucial segment. This helps the researchers to access the data of the number of people by the preserved biological samples.

This research presents a thorough analysis of market share, the present trends, and forthcoming evaluations to explain the approaching investment pockets.

This research provides market insights from 2020 to 2026, which is predicted to allow the shareholders to capitalize on the forthcoming opportunities.

This report further offers comprehensive insights into the region, which helps to understand the geographical market and assist in strategic business planning and ascertain future opportunities.

Key Topics Covered:

1. Executive Summary

2. Industry Outlook2.1. Industry Overview2.2. Industry Trends

3. Market Snapshot3.1. Market Definition3.2. Market Outlook3.2.1. PEST Analysis3.2.2. Porter Five Forces3.3. Related Markets

4. Market characteristics4.1. Market Evolution4.2. Market Trends and Impact4.3. Advantages/Disadvantages of Market4.4. Regulatory Impact4.5. Market Offerings4.6. Market Segmentation4.7. Market Dynamics4.7.1. Drivers4.7.2. Restraints4.7.3. Opportunities4.8. DRO - Impact Analysis

5. Type: Market Size & Analysis5.1. Overview5.2. Biopreservation Media5.2.1. Nutrient Media5.2.2. Sera5.2.3. Growth Factors & Supplements5.3. Biospecimen Equipment5.3.1. Temperature Control Systems5.4. Freezers5.5. Cryogenic Storage Systems5.6. Thawing Equipment5.7. Refrigerators5.7.1. Accessories5.7.2. Alarms & Monitoring systems5.7.3. Incubators5.7.4. Centrifuges5.7.5. Other Equipment

6. Application: Market Size & Analysis6.1. Overview6.2. Therapeutic Applications6.3. Research Applications6.4. Clinical Trials6.5. Other Applications

7. End User: Market Size & Analysis7.1. Overview7.2. Biobanks7.3. Gene Banks7.4. Hospitals7.5. Other End Users

8. Geography: Market Size & Analysis8.1. Overview8.2. North America8.3. Europe8.4. Asia Pacific8.5. Rest of the World

9. Competitive Landscape9.1. Competitor Comparison Analysis9.2. Market Developments9.2.1. Mergers and Acquisitions, Legal, Awards, Partnerships9.2.2. Product Launches and execution

10. Vendor Profiles10.1. Merck KGaA10.1.1. Overview10.1.2. Financials10.1.3. Products & Services10.1.4. Recent Developments10.1.5. Business Strategy10.2. Avantor, Inc10.2.1. Overview10.2.2. Financials10.2.3. Products & Services10.2.4. Recent Developments10.2.5. Business Strategy10.3. Bio-Techne Corporation10.3.1. Overview10.3.2. Financials10.3.3. Products & Services10.3.4. Recent Developments10.3.5. Business Strategy10.4. BioLife Solutions, Inc10.4.1. Overview10.4.2. Financials10.4.3. Products & Services10.4.4. Recent Developments10.4.5. Business Strategy10.5. Thermo Fisher Scientific Inc10.5.1. Overview10.5.2. Financials10.5.3. Products & Services10.5.4. Recent Developments10.5.5. Business Strategy10.6. ThermoGenesis Holdings, Inc10.6.1. Overview10.6.2. Financials10.6.3. Products & Services10.6.4. Recent Developments10.6.5. Business Strategy10.7. Worthington Industries, Inc10.7.1. Overview10.7.2. Financials10.7.3. Products & Services10.7.4. Recent Developments10.7.5. Business Strategy10.8. Chart Industries, Inc10.8.1. Overview10.8.2. Financials10.8.3. Products & Services10.8.4. Recent Developments10.8.5. Business Strategy10.9. So-Low Environmental Equipment Co.,Inc10.9.1. Overview10.9.2. Financials10.9.3. Products & Services10.9.4. Recent Developments10.9.5. Business Strategy10.10. Princeton BioCision, LLC10.10.1. Overview10.10.2. Financials10.10.3. Products & Services10.10.4. Recent Developments10.10.5. Business Strategy

11. Companies to Watch11.1. Shanghai Genext Medical Technology Co. Ltd11.1.1. Overview11.1.2. Products & Services11.1.3. Business Strategy11.2. Exact Sciences Corporation11.2.1. Overview11.2.2. Products & Services11.2.3. Business Strategy11.3. Helmer Scientific, Inc11.3.1. Overview11.3.2. Products & Services11.3.3. Business Strategy11.4. CryoTech, Inc11.4.1. Overview11.4.2. Products & Services11.4.3. Business Strategy11.5. Arctiko11.5.1. Overview11.5.2. Products & Services11.5.3. Business Strategy11.6. Nippon Genetics Europe11.6.1. Overview11.6.2. Products & Services11.6.3. Business Strategy11.7. PHC Holdings Corporation11.7.1. Overview11.7.2. Products & Services11.7.3. Business Strategy11.8. STEMCELL Technologies, Inc11.8.1. Overview11.8.2. Products & Services11.8.3. Business Strategy11.9. AMS Biotechnology11.9.1. Overview11.9.2. Products & Services11.9.3. Business Strategy11.10. OPS Diagnostics11.10.1. Overview11.10.2. Products & Services11.10.3. Business Strategy

12. Analyst Opinion

13. Annexure13.1. Report Scope13.2. Market Definitions13.3. Research Methodology13.3.1. Data Collation and In-house Estimation13.3.2. Market Triangulation13.3.3. Forecasting13.4. Report Assumptions13.5. Declarations13.6. Stakeholders13.7. Abbreviations

For more information about this report visit https://www.researchandmarkets.com/r/711zgr

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Worldwide Industry for Biopreservation to 2026 - Key Drivers, Restraints and Opportunities - Yahoo Finance

Vape Flavorings Are Cardiotoxic and Can Damage the Heart – SciTechDaily

The vape flavorings so popular with kids and young adults are cardiotoxic and disrupt the hearts normal electrical activity, a University of South Florida Health preclinical study finds.

The appealing array of fruit and candy flavors that entice millions of young people take up vaping can harm their hearts, a preclinical study by University of South Florida Health (USF Health) researchers found.

Mounting studies indicate that the nicotine and other chemicals delivered by vaping, while generally less toxic than conventional cigarettes, can damage the lungs and heart. But so far there has been no clear understanding about what happens when the vaporized flavoring molecules in flavored vaping products, after being inhaled, enter the bloodstream and reach the heart, said the studys principal investigator Sami Noujaim, PhD, an associate professor of molecular pharmacology and physiology at the USF Health Morsani College of Medicine.

In their study published on November 20, 2020, in the American Journal of Physiology- Heart and Circulatory Physiology, Dr. Noujaim and colleagues report on a series of experiments assessing the toxicity of vape flavorings in cardiac cells and in young mice.

The flavored electronic nicotine delivery systems widely popular among teens and young adults are not harm-free, Dr. Noujaim said. Altogether, our findings in the cells and mice indicate that vaping does interfere with the normal functioning of the heart and can potentially lead to cardiac rhythm disturbances.

Dr. Noujaims laboratory is among the first beginning to investigate the potential cardiotoxic effects of the many flavoring chemicals added to the e-liquids in electronic nicotine delivery systems, or ENDS. He recently received a five-year, $2.2-million grant from the NIHs National Institute of Environmental Health Sciences to carry out this laboratory research. Commonly called e-cigarettes, ENDS include different products such as vape pens, mods, and pods.

Sami Noujaim, PhD, associate professor of molecular pharmacology and physiology at the University of South Florida Health (USF Health) Morsani College of Medicine, has begun investigating preclinically the potential cardiotoxic effects of many flavoring chemicals added to the e-liquids in electronic nicotine delivery systems. Credit: Photo courtesy of USF Health

Vaping involves inhaling an aerosol created by heating an e-liquid containing nicotine, solvents such as propylene glycol and vegetable glycerin, and flavorings. The vaping devices battery-powered heat converts this e-liquid into a smoke-like aerosolized mixture (e-vapor). Manufacturers tout e-cigarettes as a tool to help quit smoking, but evidence of their effectiveness for smoking cessation is limited, and they are not FDA approved for this use. E-cigarettes contain the same highly addictive nicotine found in tobacco products, yet many teens and young adults assume they are safe.

Among the USF Health study key findings:

Whether the mouse findings will translate to people is unknown. Dr. Noujaim emphasizes that more preclinical and human studies are needed to further determine the safety profile of flavored ENDS and their long-term health effects.

A partial government ban on flavored e-cigarettes aimed at stopping young people from vaping focused on enforcement against flavored e-cigarettes with pre-filled cartridges, like those produced by industry leader JUUL. However, teens quickly switched to newer disposable e-cigarettes still sold in a staggering assortment of youth-appealing fruity and dessert-like flavors.

Our research matters because regulation of the vaping industry is a work in progress, Dr. Noujaim said. The FDA needs input from the scientific community about all the possible risks of vaping in order to effectively regulate electronic nicotine delivery systems and protect the publics health. At USF Health, in particular, we will continue to examine how vaping may adversely affect cardiac health.

In 2020, 3.6 million U.S. youths still used e-cigarettes, and among current users, more than eight in 10 reported using flavored varieties, according to the Centers for Disease Control and Prevention.

Reference: In Vitro and In Vivo Cardiac Toxicity of Flavored Electronic Nicotine Delivery Systems by Obada Abou-Assali, Mengmeng Chang, Bojjibabu Chidipi, Jose L. Martinez-de-Juan, Michelle Reiser, Manasa Kanithi, Ravi Soni, Thomas Vincent McDonald, Bengt Herweg, Javier Saiz, Laurent Calcul and Sami F. Noujaim, 20 November 2020, American Journal of Physiology-Heart and Circulatory Physiology.DOI: 10.1152/ajpheart.00283.2020

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Vape Flavorings Are Cardiotoxic and Can Damage the Heart - SciTechDaily

Outlook on the Biopreservation Global Market to 2026 – Profiling Avantor, BioLife Solutions and ThermoGenesis Among Others – GlobeNewswire

Dublin, Dec. 21, 2020 (GLOBE NEWSWIRE) -- The "Biopreservation Market by Type, Application, End-user, and Geography - Global Forecast to 2026" report has been added to ResearchAndMarkets.com's offering.

Biopreservation is a process that assists in the conservation of biospecimens such as DNA, saliva, and plasma. This process of biopreservation generally increases the durability, shelf life, and purity of the biosamples. The types of equipment in this process include freezers, liquid nitrogen, consumables, and also media & laboratory information management systems.

This process is also used to preserve food and extend its shelf life, specifically by using lactic acid bacteria. Growth in healthcare spending is assumed for better access to quality healthcare and advanced technology products such as biopreservation facilities, thereby widening the growth expectations. Moreover, the bio-banks, hospitals, and gene banks, which are major end-users for this market, are stimulating the key providers to establish technologically advanced biopreservation products to improve patient outcomes. The Biopreservation Market is projected to grow at a rate of 9.2% CAGR by 2026.

The biopreservation market has been analyzed by utilizing the optimum combination of secondary sources and in-house methodology, along with an irreplaceable blend of primary insights. The real-time assessment of the market is an integral part of our market sizing and forecasting methodology. Our industry experts and panel of primary participants have helped in compiling relevant aspects with realistic parametric estimations for a comprehensive study. The participation share of different categories of primary participants is given below:

In the market for biopreservation, the application of biopreservation consists of therapeutic applications, research applications, clinical trials, and other applications. The biopreservation is primarily applied in therapeutics due to the advancements in regenerative medicine & customized medicine, an increase in the shift of cord blood banking, and the rising incidence of chronic diseases.

The end-users of the biopreservation market include biobanks, gene banks, hospitals, and other end users. The biobanks segment is expected to have a major share in the market. The major share of this segment is attributed to the increasing preference for the preservation of stem cells and the rising numbers of sperm and egg banks.

Further, according to the regional market of biopreservation, the North American region is recorded for the colossal share in the market. This is due to the continuous drug developments and the arrival of advanced therapies in the domain of biomedical research. Additionally, the increasing requirement of expensive and improved treatment for patients' chronic diseases is the key factor.

The rising incidence of chronic diseases, including cardiac, renal diseases, diabetes, and obesity, is the crucial factor that will propel the biopreservation market growth in the prevailing period. Government initiatives to encourage stem cell therapies to treat the disease, which will again propel market growth. Conversely, the strict regulations for producing biopreservation products and the evolution of room temperature storage procedures may limit the biopreservation market growth.

Merck KGaA, Avantor, Inc., Bio-Techne Corporation, BioLife Solutions, Inc., Thermo Fisher Scientific Inc, ThermoGenesis Holdings, Inc., Worthington Industries, Inc., Chart Industries, Inc, So-Low Environmental Equipment Co., Inc., Princeton BioCision, LLC, Shanghai Genext Medical Technology Co. Ltd, Exact Sciences Corporation, Helmer Scientific, Inc., CryoTech, Inc., Arctiko, Nippon Genetics Europe, PHC Holdings Corporation, STEMCELL Technologies, Inc., AMS Biotechnology, and OPS Diagnostics. These are the few companies list of the biopreservation market.

Since the rapid increase in the number of research and developments gives the way of potentials for market growth, the biopreservation of biological samples has become a crucial segment. This helps the researchers to access the data of the number of people by the preserved biological samples.

This research presents a thorough analysis of market share, the present trends, and forthcoming evaluations to explain the approaching investment pockets.

This research provides market insights from 2020 to 2026, which is predicted to allow the shareholders to capitalize on the forthcoming opportunities.

This report further offers comprehensive insights into the region, which helps to understand the geographical market and assist in strategic business planning and ascertain future opportunities.

Key Topics Covered:

1. Executive Summary

2. Industry Outlook2.1. Industry Overview2.2. Industry Trends

3. Market Snapshot3.1. Market Definition3.2. Market Outlook3.2.1. PEST Analysis3.2.2. Porter Five Forces3.3. Related Markets

4. Market characteristics4.1. Market Evolution4.2. Market Trends and Impact4.3. Advantages/Disadvantages of Market4.4. Regulatory Impact4.5. Market Offerings4.6. Market Segmentation4.7. Market Dynamics4.7.1. Drivers4.7.2. Restraints4.7.3. Opportunities4.8. DRO - Impact Analysis

5. Type: Market Size & Analysis5.1. Overview5.2. Biopreservation Media5.2.1. Nutrient Media5.2.2. Sera5.2.3. Growth Factors & Supplements5.3. Biospecimen Equipment5.3.1. Temperature Control Systems5.4. Freezers5.5. Cryogenic Storage Systems5.6. Thawing Equipment5.7. Refrigerators5.7.1. Accessories5.7.2. Alarms & Monitoring systems5.7.3. Incubators5.7.4. Centrifuges5.7.5. Other Equipment

6. Application: Market Size & Analysis6.1. Overview6.2. Therapeutic Applications6.3. Research Applications6.4. Clinical Trials6.5. Other Applications

7. End User: Market Size & Analysis7.1. Overview7.2. Biobanks7.3. Gene Banks7.4. Hospitals7.5. Other End Users

8. Geography: Market Size & Analysis8.1. Overview8.2. North America8.3. Europe8.4. Asia Pacific8.5. Rest of the World

9. Competitive Landscape9.1. Competitor Comparison Analysis9.2. Market Developments9.2.1. Mergers and Acquisitions, Legal, Awards, Partnerships9.2.2. Product Launches and execution

10. Vendor Profiles10.1. Merck KGaA10.1.1. Overview10.1.2. Financials10.1.3. Products & Services10.1.4. Recent Developments10.1.5. Business Strategy10.2. Avantor, Inc10.2.1. Overview10.2.2. Financials10.2.3. Products & Services10.2.4. Recent Developments10.2.5. Business Strategy10.3. Bio-Techne Corporation10.3.1. Overview10.3.2. Financials10.3.3. Products & Services10.3.4. Recent Developments10.3.5. Business Strategy10.4. BioLife Solutions, Inc10.4.1. Overview10.4.2. Financials10.4.3. Products & Services10.4.4. Recent Developments10.4.5. Business Strategy10.5. Thermo Fisher Scientific Inc10.5.1. Overview10.5.2. Financials10.5.3. Products & Services10.5.4. Recent Developments10.5.5. Business Strategy10.6. ThermoGenesis Holdings, Inc10.6.1. Overview10.6.2. Financials10.6.3. Products & Services10.6.4. Recent Developments10.6.5. Business Strategy10.7. Worthington Industries, Inc10.7.1. Overview10.7.2. Financials10.7.3. Products & Services10.7.4. Recent Developments10.7.5. Business Strategy10.8. Chart Industries, Inc10.8.1. Overview10.8.2. Financials10.8.3. Products & Services10.8.4. Recent Developments10.8.5. Business Strategy10.9. So-Low Environmental Equipment Co.,Inc10.9.1. Overview10.9.2. Financials10.9.3. Products & Services10.9.4. Recent Developments10.9.5. Business Strategy10.10. Princeton BioCision, LLC10.10.1. Overview10.10.2. Financials10.10.3. Products & Services10.10.4. Recent Developments10.10.5. Business Strategy

11. Companies to Watch11.1. Shanghai Genext Medical Technology Co. Ltd11.1.1. Overview11.1.2. Products & Services11.1.3. Business Strategy11.2. Exact Sciences Corporation11.2.1. Overview11.2.2. Products & Services11.2.3. Business Strategy11.3. Helmer Scientific, Inc11.3.1. Overview11.3.2. Products & Services11.3.3. Business Strategy11.4. CryoTech, Inc11.4.1. Overview11.4.2. Products & Services11.4.3. Business Strategy11.5. Arctiko11.5.1. Overview11.5.2. Products & Services11.5.3. Business Strategy11.6. Nippon Genetics Europe11.6.1. Overview11.6.2. Products & Services11.6.3. Business Strategy11.7. PHC Holdings Corporation11.7.1. Overview11.7.2. Products & Services11.7.3. Business Strategy11.8. STEMCELL Technologies, Inc11.8.1. Overview11.8.2. Products & Services11.8.3. Business Strategy11.9. AMS Biotechnology11.9.1. Overview11.9.2. Products & Services11.9.3. Business Strategy11.10. OPS Diagnostics11.10.1. Overview11.10.2. Products & Services11.10.3. Business Strategy

12. Analyst Opinion

13. Annexure13.1. Report Scope13.2. Market Definitions13.3. Research Methodology13.3.1. Data Collation and In-house Estimation13.3.2. Market Triangulation13.3.3. Forecasting13.4. Report Assumptions13.5. Declarations13.6. Stakeholders13.7. Abbreviations

For more information about this report visit https://www.researchandmarkets.com/r/pl06wm

Research and Markets also offers Custom Research services providing focused, comprehensive and tailored research.

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Outlook on the Biopreservation Global Market to 2026 - Profiling Avantor, BioLife Solutions and ThermoGenesis Among Others - GlobeNewswire

UC Davis researchers find a way to help stem cells work …

Blocking an enzyme linked with inflammation makes it possible for stem cells to repair damaged heart tissue, new research from UC Davis Health scientists shows.

Researchers Phung Thai (left) and Padmini Sirish were part of a research team seeking stem cell solutions to heart failure care.

The enzyme soluble epoxide hydrolase, or sEH is a known factor in lung and joint disease. Now, it is a focus of heart-disease researchers as well.

The authors expect their work will lead to a new and powerful class of compounds that overcome the cell death and muscle thickening associated with heart failure a common outcome of a heart attack or long-term cardiovascular disease.

The study, conducted in mice, is published in Stem Cells Translational Medicine. The work was led by cardiologist Nipavan Chiamvimonvat.

The science of using stem cell treatments for heart disease has been full of promise but little progress, Chiamvimonvat said. The inflammation that accompanies heart disease is simply not conducive to stem cell survival.

Prior studies show that stem cells transplanted to the heart experience significant attrition in a very short period of time.

We think weve found a way to quiet that inflammatory environment, giving stem cells a chance to survive and do the healing work we know they can do, said lead author and cardiovascular medicine researcher Padmini Sirish.

Heart failure occurs when the heart no longer pumps blood efficiently, reducing oxygen throughout the body. Survival is around 45-60% five years after diagnosis. It affects approximately 5.7 million people in the U.S., with annual costs of nearly $30 billion. By 2030, it could affect as many as 9 million people at a cost of nearly $80 billion.

Chiamvimonvat often treats patients with heart failure and has been frustrated by the lack of effective medications for the disease, especially when it progresses to later stages. The best current therapies for end-stage heart failure are surgical heart transplants or mechanical heart pumps.

This research was led by cardiologist Nipavan Chiamvimonvat.

She expects her outcome will lead to a two-part treatment for end-stage heart failure that combines an sEH-blocking compound with stem cell transplantation.

Chiamvimonvat and her team tested that theory in mice using cardiac muscle cells known as cardiomyocytes, which were derived from human-induced pluripotent stem cells (hiPSCs). A hiPSC is a cell taken from any human tissue (usually skin or blood) and genetically modified to behave like an embryonic stem cell. They have the ability to form all cell types.

The specific sEH inhibitor used in the study TPPU was selected based on the work of co-author and cancer researcher Bruce Hammock, whose lab has provided detailed studies of nearly a dozen of the enzyme inhibitors.

The researchers studied six groups of mice with induced heart attacks. A group treated with a combination of the inhibitor and hiPSCs had the best outcomes in terms of increased engraftment and survival of transplanted stem cells. That group also had less heart muscle thickening and improved cardiac function.

Taken together, our data suggests that conditioning hiPSC cardiomyocytes with sEH inhibitors may help the cells to better survive the harsh conditions in the muscle damaged by a heart attack, Hammock said.

Chiamvimonvat and her team will next test the process in a larger research animal model to provide more insights into the beneficial role of TPPU. She also wants to test the process with additional heart diseases, including atrial fibrillation. Her ultimate goal, in collaboration with Hammock, is to launch human clinical trials to test the safety of the treatment.

It is my dream as a clinician and scientist to take the problems I see in the clinic to the lab for solutions that benefit our patients, Chiamvimonvat said. It is only possible because of the incredible strength of our team and the extraordinarily collaborative nature of research at UC Davis.

Additional co-authors were Phung Thai, Jun Yang, Xiao-Dong Zhang, Lu Ren, Ning Li, Valeriy Timofeyev, Kin Sing Lee, Carol Nader, Douglas Rowland, Sergey Yechikov, Svetlana Ganaga, J. Nilas Young and Deborah Lieu, all from UC Davis.

Their work was funded by the American Heart Association, Harold S. Geneen Charitable Trust. Rosenfeld Heart Foundation, U.S. Department of Veterans Affairs and the National Institutes of Health (grants T32HL86350, F32HL149288, K99R00ES024806, R35ES030443, P42ES04699, IR35 ES0443-1, P01AG051443, R01DC015135, R56HL138392, R01HL085727, R01HL085844, R01HL137228 and S10RR033106).

The study, titled Suppression of Inflammation and Fibrosis using Soluble Epoxide Hydrolase Inhibitors Enhances Cardiac Stem Cell-Based Therapy, is available online.

More information about UC Davis Health, including its cardiovascular medicine and stem cell programs, is at health.ucdavis.edu.

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I Peace, Inc. and Avery Therapeutics announce collaboration to bring iPSC derived cell therapy for heart failure to the clinic – PRNewswire

Avery Therapeutics is projected to be one of the first companies in the US to seek approval for a clinical trial using iPSC-derived technology for heart failure. The goal of this collaboration is to develop a new off-the-shelf treatment to improve the quality of life of patients suffering from heart failure, a debilitating disease that affects tens of millions of people worldwide.

The iPSCs are manufactured at I Peace's state-of-the-art GMP facility in Kyoto, Japan, under comprehensive validation programs of the facility, equipment, and processes including donor recruiting, screening, blood draw, iPSC generation, storage, and distribution. I Peace has obtained a US-based independent institutional review board (IRB) approval for its process of donor sourcing for commercial-use iPSCs. The facility is designed to be PMDA and USFDA compliant.

As Avery Therapeutics expects to expand the application of its regenerative medicine technology to various types of heart diseases and beyond, iPSCs are the key enabling technology for quality and future scalability. This agreement provides a solid foundation to improve the welfare of those suffering from diseases through advancement of tissue-engineered therapeutics.

"We are thrilled to announce this collaboration with I Peace. It is a big step forward in the development of novel cell-based therapeutics for unmet medical needs. Through this collaboration, I Peace brings deep iPSC development and manufacturing expertise to enable Avery's proprietary MyCardia cell delivery platform technology. Together we hope to positively impact millions of patients worldwide in the near future," Said Jordan Lancaster, PhD, Avery Therapeutics' CEO.

This agreement reflects an innovative collaboration involving multiple locations internationally and marks a significant milestone for both I Peace, Inc. and Avery Therapeutics to pursue one of the first US clinical trials using iPSC technology in the area of heart diseases. Koji Tanabe, PhD, founder and CEO of I Peace stated: "By combining I Peace's proprietary clinical grade iPSC technology and Avery's tissue engineering technology, we can bring the regenerative medicine dream closer to reality. We are very excited by Avery's technology and look forward to continue working together."

About I Peace, Inc

I Peace, Inc. is a global supplier of clinical and research grade iPSCs. It was founded in 2015 in Palo Alto, California, USA by Dr. Tanabe, who earned his doctorate at Kyoto University under Nobel laureate Dr. Shinya Yamanaka. I Peace's mission is to alleviate the suffering of diseased patients and help healthy people maintain a high quality of life by making cell therapy accessible to all. I Peace's state-of-the-art GMP facility and proprietary manufacturing platform enables the fully-automated mass production of discrete iPSCs from multiple donors in a single room. Increasing the available number of clinical-grade iPSC lines allows I Peace customers to take differentiation propensity into account to select the most appropriate iPSC line for their clinical research at significantly reduced cost. I Peace aims to create iPSCs for every individual that become their stem cell for life.

Founder, CEO: Koji TanabeSince: 2015Head Quarter: Palo Alto, CaliforniaJapan subsidiary: I Peace, Ltd. (Kyoto, Japan)Cell Manufacturing Facility: Kyoto, JapanWeb: https://www.ipeace.com

About Avery Therapeutics

Avery Therapeutics is a company developing advanced therapies for patients suffering from cardiovascular diseases. Avery's lead candidate is an allogeneic tissue engineered cardiac graft, MyCardia in development for treatment of chronic heart failure. Using Avery's proprietary manufacturing process MyCardia can be manufactured at scale, cryopreserved, and shipped ready to use. Avery is leveraging its proprietary tissue platform to pursue other cardiovascular indications. For more information visit: AveryThera.com. Follow Avery Therapeutics on LinkedInand Twitter.Since: 2016Headquarter: Tucson, AZWebsite: https://www.AveryThera.com

SOURCE I Peace, Inc.

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I Peace, Inc. and Avery Therapeutics announce collaboration to bring iPSC derived cell therapy for heart failure to the clinic - PRNewswire

Flavors added to vaping devices damage the heart, vanilla custard the most toxic of all – Study Finds

TAMPA, Fla. While health officials and lawmakers continue trying to steer young people away from vaping, the wide variety of enticing flavors added to these products make that a tough task. Although most of the worry over vaping comes from the risk of addiction, lung damage, and threat of switching to conventional cigarettes, a new study finds the flavoring chemicals these products use may be just as harmful as anything else. Researchers from the University of South Florida Health say vaporized flavoring molecules are toxic to the heart and damage the organs ability to beat correctly.

While other studies find that vaping is generally less harmful than smoking traditional tobacco products, the nicotine and other chemicals in e-cigarettes still damages the heart and lungs. Until now however, researchers say the impact of flavoring additives inhaled into the bloodstream remained unclear.

The flavored electronic nicotine delivery systems widely popular among teens and young adults are not harm-free, says principal investigator Dr. Sami Noujaim in a university release. Altogether, our findings in the cells and mice indicate that vaping does interfere with the normal functioning of the heart and can potentially lead to cardiac rhythm disturbances.

Dr. Noujaims study is one of the first to investigate the cardiotoxic effects of flavoring chemicals added to the e-liquids in electronic nicotine delivery systems (ENDS). ENDS include a variety of different vaping products like vape pens, mods, and pods.

Researchers define vaping as inhaling aerosols (tiny droplets) which e-cigarettes create by heating liquid nicotine and solvents like propylene glycol and vegetable glycerin. A vaping devices battery-powered heater converts this liquid into a smoke-like mix, or vapor.

The study tested how three popular e-liquid flavors fruit, cinnamon, and vanilla custard affect cardiac muscle cells (HL-1) of mice. After being exposed to e-vapor in a lab dish, the results reveal all three flavors are toxic to HL-1 cells.

The USF team also examined what happens to cardiac cells grown from human stem cells that are exposed to three types of e-vapors. The first substance containing only solvents interfered with the cells electrical activity and beating rate. The second substance, containing both nicotine and solvents, proved to be even more toxic to the heart cells.

The third substance however, containing nicotine, solvents, and vanilla custard flavoring, caused the most damage to the heart and its ability to spontaneously beat correctly. Researchers also determined that vanilla custard flavoring is the most toxic of the varieties tested.

This experiment told us that the flavoring chemicals added to vaping devices can increase harm beyond what the nicotine alone can do, Dr. Noujaim says.

The study also tested flavored vapings impact on live mice. Researchers implanted each subject with a tiny electrocardiogram device before exposing them to 60 puffs of vanilla-flavored e-vapor five days a week for 10 weeks.

Study authors looked at how this exposure impacted heart rate variability (HRV), which is the change in time intervals between successive heartbeats. The results show that HRV decreased in vaping mice compared to those only exposed to puffs of clean air.

The USF team finds vaping interferes with normal HRV by disrupting the autonomic nervous system and its control over heart rate. Mice exposed to flavored vaping are also more prone to a dangerous heart rhythm problem called ventricular tachycardia.

Researchers say they still have to confirm these results in humans. Dr. Noujaim urges policymakers to continue looking at the growing evidence that vaping is not a particularly safer alternative to smoking.

Our research matters because regulation of the vaping industry is a work in progress, Dr. Noujaim explains. The FDA needs input from the scientific community about all the possible risks of vaping in order to effectively regulate electronic nicotine delivery systems and protect the publics health. At USF Health, in particular, we will continue to examine how vaping may adversely affect cardiac health.

The study appears in the American Journal of Physiology- Heart and Circulatory Physiology.

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Flavors added to vaping devices damage the heart, vanilla custard the most toxic of all - Study Finds

Covid-19 can have impact on heart too, say experts – Hindustan Times

The Covid-19 can damage the heart both directly and indirectly, and lead to complications ranging from inflammation of the heart (myocarditis), injury to heart cells (necrosis), heart rhythm disorders (arrhythmias), heart attack, and muscle dysfunction that can lead to acute or protracted heart failure, experts said.

Covid-19 is a vascular disease that injures heart cells and muscle. It also leads to the formation of blood clots, both in the microvasculature and large vessels, which can block blood supply to the heart, brain and lungs and lead to stroke, heart attack and respiratory failure, said Dr Ravi R Kasliwal, chairman of clinical and preventive cardiology department at Medanta -The Medicity Hospital.

Also Read: Few Covid-19 deaths in Indias old-age homes, survey finds

A US study using MRI found cardiac abnormalities in 78 of 100 patients who had recently recovered from Covid-19, including 12 of 18 asymptomatic patients. Sixty patients had ongoing myocardial inflammation consistent with myocarditis, found the study, which was published in the Journal of American Medical Association Cardiology in July.

Even people with mild disease or no symptoms can develop life-threatening cardiovascular complications. Whats worrying is that this holds true for healthy adults with no pre-existing risk factors, which raise their risk of complications, said Dr Kasliwal, who recommends that everyone who has recovered from Covid-19 be screened for heart damage

Cardiac trouble

Extensive cardiac involvement is what differentiates Sars-CoV-2, the virus that causes Covid-19, from the six other coronaviruses that cause infection in humans, writes cardiologist Dr Eric J Topol, founder, director and professor of molecular medicine at the Scripps Research Translational Institute in La Jolla, California, in the journal Science.

The four human coronaviruses that cause cold-like symptoms have not been associated with heart abnormalities, though there have been isolated reports linking the Middle East Respiratory Syndrome (MERS) caused by MERS-CoV) with myocarditis, and cardiac disease with the Severe Acute Respiratory Syndrome (SARS) caused by Sars-CoV.

Also Read| Extraordinary uncertainties: Harvard prof on Covid-19, impact on mental health

Sars-CoV-2 is structurally different from Sars-CoV. The virus targets the angiotensin-converting enzyme 2 (Ace2) receptor throughout the body, facilitating cell entry by way of its spike protein, along with the cooperation of proteases. The heart is one of the many organs with high expression of Ace2. The affinity of Sars-CoV-2 to Ace2 is significantly greater than that of SARS, according to Dr Topol.

Topol notes the ease with which Sars-CoV-2 infects heart cells derived from induced pluripotent stem cells (iPSCs) in vitro, leading to a distinctive pattern of heart muscle cell fragmentation evident in autopsy reports. Besides directly infecting heart muscle cells, Sars-CoV-2 also enters and infects the endothelial cells that line the blood vessels to the heart and multiple vascular beds, leading to a secondary immune response. This causes blood pressure dysregulation, and activation of a proinflammatory response leading to a cytokine storm, which is a potentially fatal systemic inflammatory syndrome associated with Covid-19.

Persisting problems

Studies have found that injury to heart cells reflected in blood concentrations of a cardiac muscle-specific enzyme called troponin affects at least one in five hospitalised patients and more than half of those with pre-existing heart conditions, which raises the risk of death. Patients with higher troponin amounts also have high markers of inflammation (including C-reactive protein, interleukin-6, ferritin, lactate dehydrogenase), high neutrophil count, and heart dysfunction, all of which heighten immune response.

The heightened systemic inflammatory responses and diminished blood supply because of clotting, endotheliitis (blood vessel inflammation), sepsis, or hypoxemia (oxygen deprivation) because of acute lung infection leads to indirect cardiac damage, said Dr Kasliwal.

The cardiovascular damage associated with Sars-CoV-2 infection can persist beyond recovery. Since the virus affects the heart as much as the respiratory tract, further research is needed to understand why some people are more vulnerable to heart damage than others.

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Network of Genes Involved in Congenital Heart Disease Identified – Technology Networks

Over two million babies, children, and adults in the United States are living with congenital heart disease--a range of birth defects affecting the heart's structure or function. Now, researchers at Gladstone Institutes and UC San Francisco (UCSF) have made inroads into understanding how a broad network of genes and proteins go awry in a subset of congenital heart diseases.

"We now have a better understanding of what genes are improperly deployed in some cases of congenital heart disease," says Benoit Bruneau, PhD, director of the Gladstone Institute of Cardiovascular Disease and a senior author of the new study. "Eventually, this might help us get a handle on how to modulate genetic networks to prevent or treat the disease."

Congenital heart disease encompasses a wide variety of heart defects, ranging from mild structural problems that cause no symptoms to severe malformations that disrupt or block the normal flow of blood through the heart. A handful of genetic mutations have been implicated in contributing to congenital heart disease; the first to be identified was in a gene known as TBX5. The TBX5 protein is a transcription factor--it controls the expression of dozens of others genes, giving it far-reaching effects.

Bruneau has spent the last 20 years studying the effect of TBX5 mutations on developing heart cells, mostly conducting research in mice. In the new study published inDevelopmental Cell, he and his colleagues turned instead to human cells, using novel approaches to follow what happens in individual cells when TBX5 is mutated.

"This is really the first time we've been able to study this genetic mutation in a human context," says Bruneau, who is also a professor in the Department of Pediatrics at UCSF. "The mouse heart is a good proxy for the human heart, but it's not exactly the same, so it's important to be able to carry out these experiments in human cells."

The scientists began with human induced pluripotent stem cells (iPS cells), which have been reprogrammed to an embryonic-like state, giving them--like embryonic stem cells--the ability to become nearly every cell type in the body.

Then, Bruneau's group used CRISPR-Cas9 gene-editing technology to mutate TBX5 in the cells and began coaxing the iPS cells to become heart cells. As the cells became more like heart cells, the researchers used a method called single-cell RNA sequencing to track how the TBX5 mutation changed which genes were switched on and off in tens of thousands of individual cells.

The experiment revealed many genes that were expressed at higher or lower levels in cells with mutated TBX5. Importantly, not all cells responded to the TBX5 mutation in the same way; some had drastic changes in gene expression while other were less affected. This diversity, the researchers say, reflects the fact that the heart is composed of many different cell types.

"It makes sense that some are more affected than others, but this is the first experimental data in human cells to show that diversity," says Bruneau.

Bruneau's team then collaborated with computational researchers to analyze how the impacted genes and proteins were related to each other. The new data let them sketch out a complex and interconnected network of molecules that work together during heart development.

"We've not only provided a list of genes that are implicated in congenital heart disease, but we've offered context in terms of how those genes are connected," says Irfan Kathiriya, MD, PhD, a pediatric cardiac anesthesiologist at UCSF Benioff Children's Hospital, an associate professor in the Department of Anesthesia and Perioperative Care at UCSF, a visiting scientist at Gladstone, and the first author of the study.

Several genes fell into known pathways already associated with heart development or congenital heart disease. Some genes were among those directly regulated by TBX5's function as a transcription factor, while others were affected in a less direct way, the study revealed. In addition, many of the altered genes were relevant to heart function in patients with congenital heart disease as they control the rhythm and relaxation of the heart, and defects in these genes are often found together with the structural defects.

The new paper doesn't point toward any individual drug target that can reverse a congenital heart disease after birth, but a better understanding of the network involved in healthy heart formation, as well as congenital heart disease may lead to ways to prevent the defects, the researchers say. In the same way that folate taken by pregnant women is known to help prevent neural tube defects, there may be a compound that can help ensure that the network of genes and proteins related to congenital heart disease stays balanced during embryonic development.

"Our new data reveal that the genes are really all part of one network--complex but singular--which needs to stay balanced during heart development," says Bruneau. "That means if we can figure out a balancing factor that keeps this network functioning, we might be able to help prevent congenital heart defects."

Reference: Kathiriya IS, Rao KS, Iacono G, et al. Modeling Human TBX5 Haploinsufficiency Predicts Regulatory Networks for Congenital Heart Disease. Developmental Cell. 2020. doi:10.1016/j.devcel.2020.11.020.

This article has been republished from the following materials. Note: material may have been edited for length and content. For further information, please contact the cited source.

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Industry News: Hamamatsu Photonics UK Ltd and the Medical Technologies Innovation Facility enter into a partnership agreement – SelectScience

The agreement will accelerate the development and availability of new medical and pharmaceutical therapies to improve patients lives

Hamamatsu Photonics UK Ltd and Medical Technologies Innovation Facility (MTIF) are pleased to announce they have entered into a partnership agreement enabling customers the ability to view and utilize Hamamatsus Functional Drug Screening System (FDSS) CELL. This is the first FDSS/CELL to be made available in the UK in this way.

This new collaboration aims to leverage the photonics expertise, novel proprietary technology and applications of Hamamatsu, with the significant medical technology research and development capabilities of MTIF.

This is a high-end specialist piece of equipment utilised in the development of innovative medicines around the world. We are very excited to be able to provide customers with this capability, that complements our own research using this technically superb equipment. Says Professor John Hunt, Head of Strategic Research at MTIF and within Nottingham Trent University.

This partnership provides companies with a unique opportunity to use cutting edge high through-put technology to screen compounds for pharmacological activity. These capabilities are usually unavailable to all but the largest organisations. This collaboration allows organisations of every size the opportunity to accelerate their drug discovery programme. Says Professor Mike Hannay, Managing Director of the Medical Technologies Innovation Facility (MTIF) .

Hamamatsu has a long history in developing cutting edge scientific equipment for the life science market; our FDSS/CELL enables scientists, such as those working at MTIF, to make breakthroughs in the field of drug discovery and compound research. We are really excited about this new partnership between Hamamatsu and the team at MTIF helping to make such advanced instrumentation available to hundreds of potential users throughout the UK research community. Tim Stokes, Managing Director of Hamamatsu Photonics UK Ltd.

The FDSS/CELL is a compact, easy to use screening system that enables monitoring of GPCRs and ion channels for drug discovery and life science research. Screening various compounds at high throughput (96 / 384 well assays) is enabled by fluorescence or luminescence measurements using a highly sensitive Hamamatsu camera, which captures cell dynamics under the same conditions with no time lag between wells. It is also capable of recording changes in electrical potential in iPSC-derived neuronal and cardiac stem cells to gain a better understanding of toxic compound effects.

Through this new technical collaboration, HPUK and MTIF will organically integrate their respective advanced technologies and development capabilities to showcase this novel laboratory screening technology onsite at MTIF in Nottingham, UK.

Hamamatsu Photonics and MTIF aim to benefit the UK life science sector by accelerating the availability of new medical and pharmaceutical therapies. By aligning capabilities and ambitions, the parties will deliver benefit to clients by helping them to successfully navigate the complexities of discovering drug and cell therapy candidates.

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Osteoporosis treatments could be on the way after scientists identify aging gene – iNews

Hopes for new treatments for osteoporosis and cartilage degeneration have been raised after scientists identified a gene that plays a key role in the ageing of bone, tendon, ligament and cartilage.

The researchers hope that they can use their findings to slow down treat age-related diseases connected to the skeletal system by creating treatments that slow down the ageing process behind them.

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Our findings are novel and significant in finding a critical answer to how skeletal tissues lose their capability to maintain their properties and functions when we age, said Wan-Ju Li, of the University of Wisconsin-Madison.

We can also develop new pharmacological therapies to treat age-associated diseases based on our findings [although] it will take a few years before we can see the application happens, he said.

The study is published in the journal Stem Cells. The journals editor-in-chief, Jan Nolta, of the University of California at Davis, said the discovery is a very important accomplishment.

Researchers said it is possible that the same mechanism that has been identified for the skeletal system may also be present in neural stem cells and cardic stem cells, where it may play a role in causing diseases associated with those areas of the body.

We dont know if the molecule and mechanism we have identified in the paper also play the same role in other stem cells, such as neural stem cells and cardiac stem cells, in causing Parkinsons disease and heart diseases, respectively, since we havent tested it with these cells, Dr Lin said.

But I am sure that other scientists in the fields of aging and brain and heart will follow our study to answer these questions in the future, Dr Lin said.

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Osteoporosis treatments could be on the way after scientists identify aging gene - iNews

Global Myocardial Infarction Drug Market to have sustainable growth over the forecast period 2020-2028| Leading Players BioCardia, Inc., Laboratoires…

Myocardial Infarction Drug used to treat Heart Attack. Medicines and chemical substances that can cause myocardial infarction. Treatment ranges from lifestyle changes and cardiac rehabilitation to medication, stents, and bypass surgery.

Myocardial Infarction Drug Market is anticipated to grow at a CAGR of +6% during the forecast period 2020-2028.

A Global Myocardial Infarction Drug Market analysis and forecast is released based on a wide study of the market. Statistics about the approaching market trends as well as the current scenario of the market is a vital implement for existence and development in the constantly developing industry.

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Due to the pandemic, we have included a special section on the Impact of COVID 19 on the Myocardial Infarction Drug Market which would mention How the Covid-19 is affecting the Myocardial Infarction Drug Industry, Market Trends and Potential Opportunities in the COVID-19 Landscape, Covid-19 Impact on Key Regions and Proposal for Myocardial Infarction Drug Players to Combat Covid-19 Impact.

The Top Key Players of the global Myocardial Infarction Drug Market:

BioCardia, Inc., Laboratoires Pierre Fabre SA, Human Stem Cells Institute, CSL Limited, Capricor Therapeutics, Inc., Hemostemix Ltd, Compugen Ltd., Celyad SA, FibroGen, Inc., Lees Pharmaceutical Holdings Limited, Juventas Therapeutics, Inc., Cynata Therapeutics Limited, CellProthera, Biscayne Pharmaceuticals, Inc., HUYA Bioscience International, LLC, LegoChem Biosciences, Inc, Immune Pharmaceuticals Inc.

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The Global Myocardial Infarction Drug Market has demonstrated an increasing need to alter the policies that are being currently used by the players so as to exhibit commercial capacities of the manufacturers, distributors, and vendors. This helps the key players in developing a firm strategy that is flexible enough to keep up with future events in the market space.

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