Archive for the ‘Cardiac Stem Cells’ Category

New research shows that the novel coronavirus can essentially dice the muscle fibers of the human heart into pieces, sparking concerns about the potential for heart failure among Covid-19 survivors, Elizabeth Cooney reports for STAT News.

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For the study, which was published preprint on bioRxiv and has not yet been peer reviewed, researchers added the new coronavirus, SARS-CoV-2, to three types of human heart cellscardiomyocytes, cardiac fibroblasts, and endothelial cellsthat were grown in lab dishes from stem cells.

Only the cardiomyocytes, which are muscle cells, showed indication of viral infection that spread to other muscle cells, the researchers said. However, what they found in the infected cells was remarkable: The sarcomeres, which are the long muscle fibers that keep the heart beating, had been sliced into small bits. According to the researchers, the fibers looked as if they had been surgically sliced.

The researchers also found black holes where DNA was supposed to be in the nucleus of the infected cells. The researchers said they found similar, but not identical, changes when they observed autopsy specimens from patients with Covid-19, the disease caused by the novel coronavirus.

It's unclear whether the heart is able to reassemble the sarcomeres after they're severed, but that might be possible after the coronavirus infection clears, the researchers said. However, the researchers said they felt an urgency to share their results as quickly as possible, because their findings may help to further scientists' understanding of how the coronavirus causes heart damagesand possibly how to prevent or treat the injuries.

"When we saw this disruption in those microfibers that was when we made the decision to pull the trigger and put out this preprint," Todd McDevitt, a senior investigator at Gladstone Institutes and a co-author of the study, said. "I'm not a scientist who likes to stoke these things [but] I did not sleep, honestly, while we were finishing this paper and putting it out there."

Bruce Conklin, also a senior investigator at Gladstone and a co-author of the study, said the virus caused "carnage in the human cells" unlike anything seen with other diseases. "Nothing that we see in the published literature is like this in terms of this exact cutting and precise dicing," he explained.

Conklin said the findings should alter the way providers and scientists think about the novel coronavirus and Covid-19. "We should think about this as not only a pulmonary disease, but also potentially a cardiac one."

Gregg Fonarow, interim chief of the UCLA Division of Cardiology and director of the Ahmanson-UCLA Cardiomyopathy Center, said the study is "really important and elegant work, helping to define the potential mechanisms by which SARS-CoV-2 is leading to the observed heart damage and clinical manifestations."

Sahil Parikh, an interventional cardiologist at Columbia University Irving Medical Center, called findings "provocative," but added, "[t]he challenge here is that this paper has not been peer-reviewed by people who are experts in cardiology, who have not had a chance to tear it apart." She said, "I am reluctant to make a lot out of a pre-publication manuscript, no matter how provocative the finding."

The researchers who worked on the study agreed that their work should be reviewed, and they've submitted the study to a leading scientific journal (Cooney, STAT News, 9/4).

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How the coronavirus causes 'carnage' in the heart - The Daily Briefing

Concerning heart failure (HF), the current COVID-19 pandemic is having a dramatic effect on the daily life of each individual, ranging from social distancing measures applied in most countries to getting severely diseased due to the virus. Cardiovascular Disease (CVD) is among the most common conditions in people that die of the infection. The burden of CVD accounts for over 60 million people in the EU alone, therefore, it is the leading cause of death in the world.

Although COVID-19 shows us the direct impact of a potential treatment for peoples health, CVD is a stealthy pandemic killer. HF is a chronic disease condition in which the heart is not able to fill properly or efficiently pump blood throughout your body, caused by different stress conditions including myocardial infarction, atherosclerosis, diabetes and high blood pressure. Several measures are commonly used to treat heart disease, such as lifestyle changes and medications like beta-blockers and ACE inhibitors, yet these typically only slow down the progression of the disease.

Biomedical research is exploring new avenues by combining scientific insights with new technologies to overcome chronic diseases like HF. Among the most appealing and promising technologies are the use of cardiac tissue engineering and extracellular vesicles-mediated repair strategies.

Upon an initial cell loss post-infarction, it is appealing to replace this massive loss in contractile cells for new cells and thereby not treating patients symptoms, but repairing the cause of the disease. Cardiac cell therapy has been pursued for many years with variable results in small initial trials upon injection into patients. Different cell types have been used to help the myocardium in need, but the most promising approaches aim to use induced pluripotent cells (iPS) from reprogrammed cells from the patient themselves that can be directed towards contractile myocardial cells. These cells in combination with natural materials, in which the cells are embedded in the heart, can be used for tissue engineering strategies (1). Together with different international partners, Sluijters team are trying to develop strategies to use these iPS-derived contractile cells for myocardial repair via direct myocardial injection (H2020-Technobeat-66724) or to make a scaffold that can be used as a personalised biological ventricular assist device (H2020-BRAV-874827). A combination of engineering and biology to mimic the native myocardium aims to replace the chronically ill tissue for healthy and well-coupled heart tissue that can enhance the contractile performance of the heart.

Recently, a Dutch national programme started, called RegMedXB, in which the reparative treatment of the heart is aimed to be performed outside the patients body. During the time the heart is outside the body; the patient is connected to the heart-lung machine, and after restoring function, it will be re-implanted. The so-called Cardiovascular Moonshot aims to create a therapy that best suits the individual patient, by having their heart beating in a bioreactor, outside the body. Although it sounds very futuristic, many small lessons will be learned to feet novel therapeutic insights.

The initial injection of stem cells did result in a nice improvement of myocardial performance. We have now learned that rather than these delivered cells helping the heart themselves, the release of small lipid carriers called extracellular vesicles (EVs) (2) from these cells occur. These EVs carry different biological molecules, including nucleotides, proteins and lipids, and are considered to be the bodies nanosized messengers for communication. The use of stem cell-derived EVs are now being explored as a powerful means to change the course of the disease. Via these small messengers, natural biologics are delivered to diseased cells and thereby help them to overcome the stressful circumstances. EVs carry reparative signals that can be transferred to the diseased heart and thereby change the course of heart disease in some patients.

Within the EVICARE program (3) (H2020-ERC-725229), Sluijters team are using stem cell-derived EVs to change the response of the heart to injury. Also, to understand which heart cells and processes are being affected, they use materials to facilitate a slow release of biomaterials over an extended period rather than a single dose, which is probably essential for a chronic disease like HF. For now, improved blood flow is the main aim but the team have seen other effects as well, such as cardiovascular cell proliferation (4) by which the heart cells themselves start to repair the organ.

The use of EVs basically aims to enhance the endogenous repair mechanisms of the heart. These natural carriers can be mimicked with synthetic materials, or used as a hybrid of the two, thereby creating an engineered nanoparticle, that is superior in the intracellular delivery of genetic materials. The possibility of loading different biological materials allows a further tuning of its effectiveness and use in different disease conditions, creating a new off-the-shelf delivery system for nanomedicine to treat cancer and CVD (H2020-Expert-825828).

As is true of the current COVID-19 pandemic, HF is also a growing chronic disease that affects millions of people worldwide. The chronic damaged myocardium needs reparative strategies in the future to lower the social burden for patients, but also to keep the economic consequences affordable. New scientific insights with cutting edge technological developments will help to address these needs of CVD patients and their families.

References

(1) Madonna R, Van Laake LW, Botker HE, Davidson SM, De Caterina R, Engel FB, Eschenhagen T, Fernandez-Aviles F, Hausenloy DJ, Hulot JS, Lecour S, Leor J, Menasch P, Pesce M, Perrino C, Prunier F, Van Linthout S, Ytrehus K, Zimmermann WH, Ferdinandy P, Sluijter JPG. ESC Working Group on Cellular Biology of the Heart: position paper for Cardiovascular Research: tissue engineering strategies combined with cell therapies for cardiac repair in ischaemic heart disease and heart failure. Cardiovasc Res. 2019 Mar 1;115(3):488-500.

(2) Sluijter JPG, Davidson SM, Boulanger, CM, Buzs EI, de Kleijn DPV, Engel FB, Giricz Z, Hausenloy DJ, Kishore R, Lecour S, Leor J, Madonna R, Perrino C, Prunier F, Sahoo S, Schiffelers RM, Schulz R, Van Laake LW, Ytrehus K, Ferdinandy P. Extracellular vesicles in diagnostics and therapy of the ischaemic heart: Position Paper from the Working Group on Cellular Biology of the Heart of the European Society of Cardiology. Cardiovasc Res. 2018 Jan 1;114(1):19-34.

(3) https://www.sluijterlab.com/extracellular-vesicle-inspired-ther

(4) Maring JA, Lodder K, Mol E, Verhage V, Wiesmeijer KC, Dingenouts CKE, Moerkamp AT, Deddens JC, Vader P, Smits, AM, Sluijter JPG, Goumans MJ. Cardiac Progenitor Cell-Derived Extracellular Vesicles Reduce Infarct Size and Associate with Increased Cardiovascular Cell Proliferation. J Cardiovasc Transl Res. 2019 Feb;12(1):5-17.

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Innovative treatments for heart failure - Open Access Government

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Some of the major players operating in the globalautologous stem cell and non-stem cell based therapies marketareAntria (Cro), Bioheart, Brainstorm Cell Therapeutics, Cytori, Dendreon Corporation, Fibrocell, Genesis Biopharma, Georgia Health Sciences University, Neostem, Opexa Therapeutics, Orgenesis, Regenexx, Regeneus, Tengion, Tigenix, Virxsys and many more.

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Market Definition:Global Autologous Stem Cell and Non-Stem Cell Based Therapies Market

In autologous stem-cell transplantation persons own undifferentiated cells or stem cells are collected and transplanted back to the person after intensive therapy. These therapies are performed by means of hematopoietic stem cells, in some of the cases cardiac cells are used to fix the damages caused due to heart attacks. The autologous stem cell and non-stem cell based therapies are used in the treatment of various diseases such as neurodegenerative diseases, cardiovascular diseases, cancer and autoimmune diseases, infectious disease.

According to World Health Organization (WHO), cardiovascular disease (CVD) causes more than half of all deaths across the European Region. The disease leads to death or frequently it is caused by AIDS, tuberculosis and malaria combined in Europe. With the prevalence of cancer and diabetes in all age groups globally the need of steam cell based therapies is increasing, according to article published by the US National Library of Medicine National Institutes of Health, it was reported that around 382 million people had diabetes in 2013 and the number is growing at alarming rate which has increased the need to improve treatment and therapies regarding the diseases.

Market Segmentation:Global Autologous Stem Cell and Non-Stem Cell Based Therapies Market

Major Autologous Stem Cell and Non-Stem Cell Based Therapies Market Drivers and Restraints:

Introduction of novel autologous stem cell based therapies in regenerative medicine

Reduction in transplant associated risks

Prevalence of cancer and diabetes in all age groups

High cost of autologous cellular therapies

Lack of skilled professionals

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Autologous Stem Cell and Non-Stem Cell Based Therapies Market Scope And Price Analysis 2020 | Major Giants Fibrocell, Genesis Biopharma, Georgia...

Abstract

Fibrosis, characterized by aberrant tissue scarring from activated myofibroblasts, is often untreatable. Although the extracellular matrix becomes increasingly stiff and fibrous during disease progression, how these physical cues affect myofibroblast differentiation in 3D is poorly understood. Here, we describe a multicomponent hydrogel that recapitulates the 3D fibrous structure of interstitial tissue regions where idiopathic pulmonary fibrosis (IPF) initiates. In contrast to findings on 2D hydrogels, myofibroblast differentiation in 3D was inversely correlated with hydrogel stiffness but positively correlated with matrix fibers. Using a multistep bioinformatics analysis of IPF patient transcriptomes and in vitro pharmacologic screening, we identify matrix metalloproteinase activity to be essential for 3D but not 2D myofibroblast differentiation. Given our observation that compliant degradable 3D matrices amply support fibrogenesis, these studies demonstrate a departure from the established relationship between stiffness and myofibroblast differentiation in 2D, and provide a new 3D model for studying fibrosis and identifying antifibrotic therapeutics.

Fibrosis is implicated in nearly 45% of all deaths in the developed world and plays a role in numerous pathologies, including pulmonary fibrosis, cardiac disease, atherosclerosis, and cancer (1). In particular, interstitial lung diseases, such as idiopathic pulmonary fibrosis (IPF), are fatal and incurable with a median survival of only 2 to 5 years (2). Often described as dysregulated or incessant wound healing, fibrosis involves persistent cycles of tissue injury and deposition of extracellular matrix (ECM) by myofibroblasts (MFs). These critical cellular mediators of fibrogenesis are primarily derived from tissue-resident fibroblasts (1). MFs drive eventual organ failure through excessive fibrous ECM deposition, force generation and tissue contraction, and eventual disruption of parenchymal tissue function (1). As organ transplantation remains the only curative option for late-stage disease, effective antifibrotic therapeutics that slow MF expansion or even reverse fibrosed tissue remain a major unmet clinical need. Undoubtedly, the limited efficacy of antifibrotic drugs at present underscores limitations of existing models for identifying therapeutics, the complexity of the disease, and an incomplete understanding of MF biology.

A strong correlation between lung tissue stiffening and worse patient outcomes suggests an important role for matrix mechanosensing in fibrotic disease progression (3). Preclinical models of fibrosis in mice have supported the link between tissue stiffening and disease progression. However, a precise understanding of how physical cues from the microenvironment influence MF differentiation in vivo is confounded by concurrent structural (e.g., collagen density and laminin/elastin degradation) and biochemical (e.g., matrix composition and inflammatory) changes to the microenvironment (4). Consequently, natural and synthetic in vitro tissue models have provided great utility for the study of MF mechanobiology. Seminal studies using natural type I collagen gels have elucidated the role of profibrotic soluble cues [e.g., transforming growth factor1 (TGF-1)] in promoting cell contractility, ECM compaction, and MF differentiation, and more recently, precision-cut lung slices, have emerged as a powerful tool to study the complexity of the pulmonary microenvironment in IPF (4, 5). However, their utility in identifying physical microenvironmental determinants of MF differentiation suffers from an intrinsic coupling of multiple biochemical and mechanical material properties (6). Rapid degradation kinetics (1 to 3 days) and resulting issues with material stability (1 to 2 weeks) further impede the use of natural materials for studying fibrogenic events and drug responses, which occur over weeks to months in in vivo models or years in patients (7, 8).

Synthetic hydrogels that are more resistant to cell-mediated degradation have provided a better controlled setting for long-term studies of disease-related processes (9). For example, synthetic hydrogel-based cell culture substrates with tunable stiffness have helped establish a paradigm for mechanosensing during MF differentiation in two-dimensions (2D), where compliant matrices maintain fibroblast quiescence in contrast to stiffer matrices that promote MF differentiation (10, 11). Extensive findings in 2D suggest a causal role for matrix mechanics (e.g., stiffness) during MF differentiation in vitro and potentially in human disease, but these models lack the 3D nature of interstitial spaces where fibrosis originates (12). The interstitium surrounding alveoli is structurally composed of two key components: networks of fibrous ECM proteins (namely, type I collagen fibers) and interpenetrating ground substance, an amorphous hydrogel network rich in glycosaminoglycans such as heparan sulfate proteoglycan. Mechanical cues from fibrotic ECM that promote MF differentiation may arise from changes to the collagen fiber architecture or the gel-like ground substance; whether matrix stiffness is a prerequisite for MF differentiation in 3D fibrous interstitial spaces remains unclear (13). Furthermore, the limited efficacy of antifibrotic therapies identified in preclinical and in vitro models of IPF motivates the development of 3D tissue-engineered systems with improved structural and mechanical biomimicry, relevant pharmacokinetics, and the potential to incorporate patient cells (9). Furthermore, recapitulating key features of the fibrotic progression in an in vitro setting that better approximates interstitial tissues could (i) improve our current understanding of MF mechanobiology and (ii) serve as a more suitable test bed for potential antifibrotic therapeutics.

Accordingly, here, we describe a microengineered pulmonary interstitial matrix that recapitulates mechanical and structural features of fibrotic tissue as well as key biological events observed during IPF progression. Design parameters of these engineered microenvironments were informed by mechanical and structural characterization of fibrotic lung tissue from a bleomycin mouse model. We then investigated the influence of dimensionality, matrix cross-linking/stiffness, and fiber density on TGF-1induced MF differentiation in our pulmonary interstitial matrices. Increased hydrogel cross-linking/stiffness substantially hindered MF differentiation in 3D in contrast to findings in 2D, while fibrotic matrix architecture (i.e., high fiber density) potently promoted fibroblast proliferation and differentiation into MFs. Long-term (21 days) culture of hydrogels with a fibrotic architecture engendered tissue stiffening, collagen deposition, and secretion of profibrotic cytokines, implicating fiber density as a potent fibrogenic cue in 3D microenvironments. Pharmacologic screening in fibrotic pulmonary interstitial matrices revealed matrix metalloproteinase (MMP) activity and hydrogel remodeling as a key step during 3D fibrogenesis, but not in traditional 2D settings. To explore the clinical relevance of our findings, we leveraged a multistep bioinformatics analysis of transcriptional profiles from 231 patients, highlighting increased MMP gene expression and enriched signaling domains associated with matrix degradation in patients with IPF. Together, these results highlight the utility of studying fibrogenesis in a physiologically relevant 3D hydrogel model, underscore the requirement of matrix remodeling in IPF, and establish a new platform for screening antifibrotic therapies.

To inform key design criteria for our pulmonary interstitial matrices, we began by characterizing mechanical properties of fibrotic interstitial tissue in a bleomycin-induced lung injury model in mouse. Nave C57BL/6 mice were intratracheally challenged with bleomycin to induce lung injury and subsequent fibro-proliferative repair, with saline-treated animals maintained as a control group. After 2 weeks, animals were sacrificed and lung tissue was dissected out, sectioned and stained, and then mechanically tested by atomic force microscopy (AFM) nanoindentation to map the stiffness of interstitial tissue surrounding alveoli. While single-dose bleomycin administration does not recapitulate human IPF, the fibro-proliferative response is well characterized and leads to MF differentiation, collagen deposition, and lung stiffening events that are reminiscent of what occurs in human disease over longer time scales. As previously documented (14), bleomycin treatment corresponded to an increase in the thickness of interstitial tissue regions surrounding alveoli, a structural change that occurred alongside matrix stiffening (Fig. 1, A and B); bleomycin-treated lungs had elastic moduli nearly fivefold greater than healthy control tissues. To generate synthetic hydrogels with elastic moduli tunable over this range, we functionalized a biocompatible and protein-resistant polysaccharide, dextran, with pendant vinyl sulfone groups amenable to peptide conjugation (termed DexVS; Fig. 1C). To permit cell-mediated proteolytic hydrogel degradation and thus spreading of encapsulated cells, we cross-linked DexVS with a bifunctional peptide (GCVPMSMRGGCG, abbreviated VPMS) primarily sensitive to MMP9 and MMP14, two MMPs implicated in fibrosis-associated matrix remodeling (15, 16). Tuning input VPMS cross-linker concentration yielded stable hydrogels spanning the full range of elastic moduli we measured by AFM nanoindentation of lung tissue (Fig. 1D). Additional functionalization with cell-adhesive moieties (CGRGDS, abbreviated RGD) facilitated adhesion of primary normal human lung fibroblasts (NHLFs) (Fig. 1E).

(A) Histological preparations of healthy control and bleomycin-treated murine lung tissue (n = 3 mice per group) stained for collagen by picrosirius red (scale bar, 100 m). (B) Youngs modulus of mouse lung tissue as measured by AFM nanoindentation, with data fit to the Hertz contact model to determine Youngs modulus (n = 3 mice per group, n = 50 indentations per group on n = 9 tissue sections). (C) Schematic of proteolytically sensitive, cell-adhesive DexVS-VPMS bulk hydrogels. (D) Youngs modulus determined by AFM nanoindentation of DexVS-VPMS hydrogels formed with different concentrations of VPMS cross-linker (n = 4 samples per group, n = 20 total indentations per group). (E and F) Representative images of F-actin (cyan), nuclei (yellow), and -SMA (magenta); image-based quantification of -SMA expression (left axis, magenta bars, day 9) and nuclear Ki67 (right axis, gray bars, day 5) in 2D and 3D (n = 4 samples per group, n = 10 fields of view per group, n > 50 cells per field of view; scale bars, 200 m). All data presented are means SDs with superimposed data points; asterisk denotes significance with P < 0.05 determined by one-way analysis of variance (ANOVA). AU, arbitrary units.

To confirm the role of matrix mechanics on cell proliferation and MF differentiation, we seeded patient-derived NHLFs on 2D DexVS protease-sensitive hydrogel surfaces varying in VPMS cross-linker density and resulting stiffness and stimulated cultures with TGF-1 to promote MF differentiation. In accordance with previous literature, we observed a stiffness-dependent stepwise increase in cell proliferation (day 5) and MF differentiation (day 9) as measured by Ki67 and -smooth muscle actin (-SMA) immunofluorescence, respectively (Fig. 1E) (11). As the influence of matrix elasticity on MF differentiation in 3D synthetic matrices has not previously been documented, we also encapsulated NHLFs in 3D within identical DexVS hydrogels. The opposing trend with respect to stiffness was noted for cells encapsulated in 3D; compliant (E = 560 Pa) hydrogels that limited -SMA expression in 2D plated cells instead exhibited the highest levels of MF differentiation in 3D (Fig. 1F). Decreasing proliferation and cell-cell contact formation as a function of increasing hydrogel stiffness were also noted in 3D matrices and may be one reason why rigid hydrogels limit differentiation in 3D. Similar findings have been reported for mesenchymal stem cells encapsulated in hyaluronic acid matrices, where compliant gels promoted stem cell proliferation and yes-associated protein (YAP) activity in 3D, yet inhibited YAP activity and proliferation in 2D (17). These results suggest that while stiff, cross-linked 2D surfaces promote cell spreading, proliferation, and MF differentiation, an equivalent relationship does not directly translate to 3D settings. High cross-linking and stiffness (E = 6.1 kPa) in 3D matrices sterically hinder cell spreading, proliferation, and the formation of cell-cell contacts, all well-established promoters of MF differentiation (18).

Cell-degradable synthetic hydrogels with elastic moduli approximating that of fibrotic tissue proved nonpermissive to MF differentiation in 3D. Although matrix cross-linking and densification of ground substance has previously been implicated in fibrotic tissue stiffening, remodeled collagenous architecture can also engender changes in tissue mechanics and may modulate MF development in IPF independently. To characterize the fibrous matrix architecture within healthy and fibrotic lung interstitium, we used second-harmonic generation (SHG) microscopy to visualize collagen microstructure in saline- and bleomycin-treated lungs, respectively. Per previous literature, saline-treated lungs contained limited numbers of micrometer-scale (~1-m-diameter) collagen fibers, primarily localized to the interstitial spaces supporting the alveoli (Fig. 2A) (19). In contrast, bleomycin-treated lungs had, on average, fourfold higher overall SHG intensity, with collagen fibers localized to both an expanded interstitial region and in disrupted alveolar networks. While no difference in fiber diameter was noted with bleomycin treatment, we did observe thick (~2- to 5-m) collagen bundles containing numerous individual fibers in fibrotic lungs, potentially arising from physical remodeling by resident fibroblasts (Fig. 2A and fig. S1). Given that typical synthetic hydrogels amenable to cell encapsulation (as in Fig. 1) lack fibrous architecture, we leveraged a previously established methodology for generating fiber-reinforced hydrogel composites (20). Electrospun DexVS fibers approximating the diameter of collagen fibers characterized by SHG imaging (fig. S1) were co-encapsulated alongside NHLFs in DexVS-VPMS hydrogel matrices, yielding a 3D interpenetrating network of DexVS fibers ensconced within proteolytically cleavable DexVS hydrogel (Fig. 2B). To recapitulate the adhesive nature of collagen and fibronectin fibers within interstitial tissues, we functionalized DexVS fibers with RGD to support integrin engagement and 3D cell spreading. While increasing the weight % of type I collagen matrices increases collagen fiber density and simultaneously increases hydrogel stiffness (fig. S2), our synthetic matrix platform enables changes to fiber density (0.0 to 5.0%) without altering mechanical properties assessed by AFM nanoindentation (Fig. 2C), likely due to the constant weight percentage of DexVS and VPMS cross-linker within the bulk hydrogel.

(A) SHG imaging of collagen microstructure within healthy and bleomycin-treated lungs on day 14, with quantification of average signal intensity (arrows indicate interstitial tissue regions adjacent to alveoli; n = 3 mice per group, n = 10 fields of view per group; scale bar, 100 m). (B) Schematic depicting polymer cross-linking and functionalization for generating fibrous DexVS hydrogel composites to model changes in fiber density within lung interstitial tissue ECM. (C) Images and intensity quantification of fluorophore-labeled fibers within composites varying in fiber density (n = 4 samples per group, n = 10 fields of view per group; scale bar, 100 m). Youngs modulus determined by AFM nanoindentation of fibrous composites formed with different concentrations of VPMS cross-linker (n = 4 samples per group, n = 20 measurements per group). (D) Representative high-resolution images of NHLFs on day 1 in fibrous composites formed with bulk hydrogels (12.5 mM VPMS) functionalized with integrin ligand arginylglycylaspartic acid (RGD) or heparin-binding peptide (HBP) [F-actin (cyan), nuclei (yellow), and DexVS fibers (magenta); scale bar, 50 m]. Quantification of fiber recruitment as measured by contact between cells and DexVS fibers (n = 10 fields of view per group, n > 25 cells analyzed). (E) Representative high-resolution images of NHLF on day 1 fibrous composites formed with bulk hydrogels functionalized with integrin ligand RGD or HBP [F-actin (cyan), fibronectin (yellow), and DexVS fibers (magenta); scale bar, 5 m]. Quantification of fibronectin deposition into tshe hydrogel matrix as measured by immunostain intensity (n = 10 fields of view per group, n > 25 cells analyzed). All data presented are means SDs with superimposed data points; asterisk denotes significance with P < 0.05 determined by one-way ANOVA or Students t test, where appropriate; NS denotes nonsignificant comparison.

Beyond recapitulating the multiphase structural composition of interstitial ECM, we also sought to mimic the adhesive ligand presentation and protein sequestration functions of native interstitial tissue. More specifically, the gel-like ground substance within fibrotic tissue intrinsically lacks integrin-binding moieties and is increasingly rich in heparan sulfate proteoglycans, primarily serving as a local reservoir for nascent ECM proteins, growth factors, and profibrotic cytokines. In contrast, synthetic hydrogels are often intentionally designed to have minimal interactions with secreted proteins and require uniform functionalization with a cell-adhesive ligand to support cell attachment and mechanosensing. We hypothesized that RGD-presenting fibers alone would support cell spreading (20), enabling the use of a nonadhesive bulk DexVS hydrogel functionalized with heparin-binding peptide (HBP; CGFAKLAARLYRKAG) (21). While both RGD- and HBP-functionalized bulk DexVS gels supported cell spreading upon incorporation of RGD-presenting fibers, HBP-functionalized hydrogels encouraged matrix remodeling in the form of cell-mediated fiber recruitment (Fig. 2D) and enhanced the deposition of fibronectin fibrils into the adjacent matrix (Fig. 2E). Given the multiphase structure of lung interstitium, changes in collagen fiber density noted with fibrotic progression, and the importance of physical and biochemical matrix remodeling to fibrogenesis, we used HBP-tethered 560-Pa DexVS-VPMS bulk hydrogels with tunable density of RGD-presenting fibers in all subsequent studies.

We next investigated whether changes in fiber density reflecting fibrosis-associated alterations to matrix architecture could influence MF differentiation in our 3D model. NHLFs were encapsulated in compliant DexVS-VPMS hydrogels ranging in fiber density (E = 560 Pa, 0.0 to 5.0 volume % fibers). Examining cell morphology after 3 days of culture, we noted increased cell spreading (Fig. 3, A and B) and evident F-actin stress fibers (fig. S3) in fibrous conditions compared to nonfibrous controls. Increased frequency of direct cell-cell interactions was also observed as a function of fiber density, as evidenced by higher area:perimeter ratios and the number of fibroblasts per contiguous multicellular cluster (Fig. 3A and fig. S3). As evidenced by changes in the ratio of nuclear to cytosolic YAP localization, we detected changes in mechanosensing as a function of fiber density, with the highest nuclear ratio measured in samples containing the highest fiber density examined. Given that nuclear YAP activity (a transcriptional coactivator required for downstream mechanotransduction) has been implicated as a promoter of MF differentiation (22), we also assayed other markers associated with fibroblast activation. With increases in fiber density, we found significant increases in cell proliferation and local fibronectin deposition (Fig. 3, A and B). Luminex quantification of cytokine secretion at this time point revealed elevated secretion of inflammatory and profibrotic cytokines (Fig. 3C), suggesting that matrix fibers may modulate the soluble milieu known to regulate the response to tissue damage and repair in vivo (2325). While no -SMA expression or collagen deposition was observed at this early time point, F-actin stress fibers, YAP activity, and fibronectin expression have been previously established as proto-MF markers in vivo (26), suggesting that physical interactions with matrix fibers prime fibroblasts for activation into MFs. Supplying the profibrotic soluble factor TGF-1 prompted increases in the expression of various profibrotic YAP-target genes (ACTA2, COL1A1, FN1, CD11, and CTGF) relative to nonfibrous (FD 0.0%) controls at day 5 (Fig. 3D). Together, these data suggest that heightened fiber density promotes a fibrotic phenotype (Fig. 3, A to C) and gene expression (Fig. 3D), despite the absence of a stiff surrounding hydrogel.

(A) Immunofluorescence images of NHLFs in hydrogel composites over a range of fiber densities after 3 days of culture [F-actin (cyan), fibronectin (FN, yellow), YAP (magenta), Ki67 (white), and nuclei (blue); scale bars, 100 m (F-actin), 20 m (FN), 20 m (YAP), and 100 m (Ki67/nuclei)]. (B) Corresponding image-based quantification of cell area, deposited FN, YAP nuclear to cytosolic ratio, and % of proliferating cells (n = 4 samples per group; for cell spread area analysis, n > 50 cells per group; for FN, YAP, and Ki67 analyses, n = 10 fields of view per group and n > 25 cells per field of view). (C) Cytokine secretion into culture medium on day 3 (all data were normalized to background levels in control medium, n = 4 samples per condition). (D) Expression of MF-related genes in NHLFs stimulated with TGF-1 on day 3, in either highly fibrous (FD 5.0%) or nonfibrous (FD 0.0%) hydrogels (data presented are GAPDH-normalized fold changes relative to NHLFs within an FD 0% hydrogel lacking TGF-1 supplementation). All data presented are means SDs with superimposed data points; asterisk denotes significance with P < 0.05 determined by one-way ANOVA or Students t test where appropriate.

To explore whether fibrotic matrix cues in the form of heightened fiber density could promote 3D MF differentiation over longer-term culture, NHLFs were encapsulated within hydrogels varying in fiber density and maintained in medium supplemented with TGF-1 beginning on day 1. Immunofluorescent imaging and cytokine quantification were performed on days 3, 5, 7, and 9 to capture dynamic changes in cellular phenotype and secretion, respectively. No -SMApositive stress fibers or changes in total cytokine secretion were observed on day 3 or 5. On day 7, we noted the sparse appearance of -SMApositive cells alongside increased total cytokine secretion (Fig. 4D) in FD 5.0% conditions containing TGF-1, indicating the beginning of a potential phenotypic shift. Extensive MF differentiation (designated by -SMApositive cells) and a sixfold increase in total cytokine secretion occurred rapidly between days 7 and 9 (Fig. 4, B, D, and E) in the highest fiber density (FD 5.0%) condition. Despite the high proliferation within high fiber density hydrogels (Fig. 4C), -SMApositive cells were not evident in samples lacking exogenous TGF-1 supplementation. Moreover, -SMApositive cells were also absent in TGF-1 supplemented conditions that lacked fibrous architecture, indicating a requirement for both soluble and physical fibrogenic cues in 3D. Furthermore, inhibiting integrin engagement by incorporating fibers lacking RGD also abrogated MF differentiation and proliferation despite the presence of TGF-1 (Fig. 4, A and B), suggesting that a fibrotic matrix architecture drives -SMA expression primarily through integrin engagement and downstream mechanosensing pathways. These results were replicated with primary human dermal fibroblasts and mammary fibroblasts, where similar trends with -SMA expression as a function of fiber density were observed (fig. S4). While high fiber density promoted proliferation in dermal fibroblasts, mammary fibroblasts underwent MF differentiation in the absence of higher proliferation rates, demonstrating intrinsic differences between cell populations originating from different tissues. Nevertheless, these results suggest that fibrotic matrix architecture may be promoting MF differentiation in other pathologies, namely, dermal scarring in systemic sclerosis and desmoplasia in breast cancer.

(A) Representative immunofluorescence images of NHLFs in microenvironmental conditions leading to low (top row) or high (bottom row) MF differentiation after 9 days in culture [-SMA (magenta) and nuclei (cyan); n = 4 samples per group, n = 10 fields of view per group, and n > 50 cells per field of view; scale bar, 200 m], with corresponding image-based quantification in (B) and (C). Insets depict representative fiber densities. (D) Measurement of total cytokine secretion over time as a function of fiber density (n = 4 samples per condition; * indicates significant differences between FD 5.0% and all other groups at a given time point; NS denotes nonsignificant comparison). (E) Secretion of specific cytokines and chemoattractants as a function of fiber density on day 9 (n = 4 samples per condition). (F) Representative images and quantification of tissue contraction within day 14 fibroblast-laden hydrogels of varying fiber density (n = 4 samples per group, dashed line indicates initial diameter of 5 mm). Photo credit: Daniel Matera, University of Michigan. (G) AFM measurements of day 14 fibroblast-laden hydrogels of varying fiber density (n = 20 measurements from n = 4 samples per group). Dashed line indicates original hydrogel stiffness. (H) SHG images of fibrous collagen within fibroblast-laden hydrogels after 21 days of culture in medium supplemented with ascorbic acid (scale bar, 100 m). (I) Measurement of total collagen content within digested DexVS hydrogels at day 21 as measured by biochemical assay (n = 4 samples per group). All data presented are means SDs with superimposed data points; asterisk denotes significance with P < 0.05 determined by one-way ANOVA; NS denotes nonsignificant comparison.

While proliferation and -SMA expression are accepted markers of activated fibroblasts, fibrotic lesions contribute to patient mortality through airway inflammation, collagen secretion, tissue contraction, and lung stiffeningpathogenic events that hinder the physical process of respiration (27). Luminex screening of 41 cytokines and chemokines within hydrogel supernatant revealed elevated total cytokine secretion as a function of fiber density over time (Fig. 4D), many of which were soluble mediators known to regulate airway inflammation (Fig. 4E) (23). Numerous other cytokines were additionally secreted at day 9 but did not change as a function of fiber density despite differences in cell number at this time point (fig. S5), suggesting that cell number alone cannot account for the increased cytokine secretion in high fiber density conditions. By generating free-floating hydrogels that allow contraction over time, we also examined macroscale changes in tissue geometry. Consistent with the influence of fiber density on -SMA expression, hydrogels containing high fiber densities underwent greater hydrogel contraction compared to nonfibrous or low fiber density conditions (Fig. 4F). Day 14 fibrotic hydrogels (FD 5.0%) were also fourfold stiffer (2.0 versus 0.5 kPa) as measured by AFM nanoindentation (Fig. 4G) compared to conditions that yielded low rates of MF differentiation in shorter-term studies (i.e., FD 0.0 or FD 0.5% in Fig. 4, A and B). When medium was supplemented with ascorbic acid to permit procollagen hydroxylation, collagen deposition into the surrounding matrix was evident by SHG microscopy by day 21 in high fiber density hydrogels (Fig. 4H) as compared to nonfibrous controls. Further biochemical analysis of hydrogel collagen content confirmed a stepwise increase in collagen production as a function of fiber density (Fig. 4I). Together, these findings demonstrate a clear influence of fiber density on MF differentiation and phenotype in 3D and furthermore suggest that this in vitro model recapitulates key pathogenic events associated with the progression of fibrosis in vivo.

Having established microenvironmental cues that promote robust 3D MF differentiation, we next evaluated the potential of our fibrous hydrogel model for use as an antifibrotic drug screening platform. Nintedanib, a broad-spectrum receptor tyrosine kinase inhibitor, and pirfenidone, an inhibitor of the mitogen-activated protein kinase (MAPK)/nuclear factor B (NF-B) pathway, were selected due to their recent Food and Drug Administration approval for use in patients with IPF (28). We also included dimethyl fumarate, an inhibitor of the YAP/TAZ pathway clinically approved for treatment of systemic sclerosis, and marimastat, a broad-spectrum MMP inhibitor that has shown efficacy in murine preclinical models of fibrosis (29, 30). We generated fibrotic matrices (560-Pa DexVS-VPMS-HBP bulk hydrogels containing 5.0 volume % DexVS-RGD fibers) that elicited the highest levels of MF differentiation, matrix contraction, and collagen secretion in our previous studies (Fig. 4). As a comparison to the current standard for high-throughput compound screening, we also seeded identical numbers of NHLFs on 2D tissue culture plastic in parallel. Cultures were stimulated with TGF-1 on day 1, and pharmacologic treatments were added on day 3, following extensive fibroblast spreading, cell-cell junction formation, and proliferation (Fig. 3A).

As in our earlier studies, TGF-1 supplementation promoted proliferation and -SMA expression within 3D constructs as well as on rigid tissue culture plastic (Fig. 5A). Nintedanib and pirfenidone had differential effects on NHLFs depending on culture format; NHLFs on 2D tissue culture plastic were resistant to pirfenidone/nintedanib treatment with no difference in proliferation or -SMA expression relative to vehicle controls, whereas modest but significant decreases in -SMA expression (pirfenidone and nintedanib) and proliferation (nintedanib) were detected in 3D (Fig. 5, A to E). Combined treatment with pirfenidone and nintedanib provided an antifibrotic effect only in fibrotic matrices, supporting ongoing clinical studies exploring their use as a combinatorial therapy (ClinicalTrials.gov identifier NCT03939520). Dimethyl fumarate abrogated cell proliferation and -SMA expression across all conditions, suggesting that inhibition of downstream mechanosensing inhibits MF differentiation in both 2D and 3D contexts in support of the general requirement for mechanosensing during MF differentiation independent of culture substrate (11). Inhibition of YAP activity in vivo has been shown to mitigate fibrosis and may be an advantageous therapeutic target (22). Blockade of MMP activity via marimastat treatment proved ineffectual in reducing -SMA expression or proliferation on 2D tissue culture plastic, but surprisingly fully abrogated the proliferation and differentiation response in 3D fibrotic matrices (Fig. 5, A to E). Given the role of protease activity in tissue remodeling in vivo (30) and in cellular outgrowth within 3D hydrogels (17, 31), our data suggest that degradative matrix remodeling is a requirement for MF differentiation in 3D, but not in more simplified 2D settings. To summarize, multiple antifibrotic agents (pirfenidone, nintedanib, dimethyl fumarate, and marimastat) demonstrating efficacy in clinical literature elicited an antifibrotic effect in our engineered fibrotic pulmonary interstitial matrices, but not in the 2D tissue culture plastic contexts traditionally used for compound screening.

(A) Representative confocal images stained for -SMA (magenta), F-actin (cyan), and nuclei (yellow) of NHLFs after 9 days of culture on tissue culture plastic (TCP) (top row) or 3D fibrotic matrices (bottom row) with pharmacologic treatment indicated from days 3 to 9 (scale bar, 100 m). Imaged regions were selected to maximize the number of -SMA+ cells per field of view within each sample. (B) Quantification of -SMA and (C) total cell count within 2D NHLF cultures. (D) Quantification of -SMA and (E) total cell count within 3D fibrotic matrices (n = 4 samples per group, n = 10 fields of view per group, and n > 50 cells per field of view). All data presented are means SDs with superimposed data points; asterisk denotes significance with P < 0.05 determined by one-way ANOVA; NS denotes nonsignificant comparison.

As the protease inhibitor marimastat fully ablated TGF-1induced -SMA expression and proliferation in our 3D fibrotic matrices, we leveraged bioinformatics methodologies to investigate the role of matrix proteases in patients with IPF on a network (Reactome) and protein (STRING) basis. Differential expression analysis of microarray data within the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) (dataset #GSE47460) was used to generate an uncurated/unbiased dataset composed of the top 1000 differentially regulated genes in IPF, revealing MMP1 as the most up-regulated gene in patients with IPF, with other matrix proteases (MMP1, MMP3, MMP7, MMP9, MMP10, MMP11, and MMP12) and matrix remodeling proteins (COL1A2, LOX, ACAN, DCN, and HS6ST2) similarly up-regulated (Fig. 6B, table S1, and data file S1). To focus on genes associated with MF differentiation for subsequent analyses, we performed Gene Ontology (GO) term enrichment (via GEO2R) to compile a curated dataset containing 188 key genes associated with MF differentiation (data file S1) and used Reactome and STRING analyses to investigate network signaling within both the uncurated and curated datasets. Analyses revealed 103 (uncurated) and 89 (curated) enriched signaling pathways in IPF (data file S1). The top 3/5 (uncurated) and 5/5 (curated) significantly enriched pathways in IPF involved matrix degradation and remodeling (Fig. 6C). Subsequent STRING protein-protein interaction analysis of datasets revealed that top signaling nodes were MMPs (uncurated: MMP1 and MMP3; Fig. 6D), fibrous collagens (uncurated: COL1A2 and COL3A1), or cytokines (curated: IL6, VEGFA, IL1B, and IGF1; Fig. 6D) known to increase MMP expression in fibroblasts (3235). These results emphasize the interdependence between MMP activity and systems-level pathogenic signaling in IPF and, in combination with our 3D drug screening results, highlight fibroblast-specific protease activity as a potential therapeutic target. Furthermore, given that protease inhibition had no effect on MF differentiation in 2D culture, these data also support the growing sentiment that simplified 2D screening models may be masking the identification of potentially viable antifibrotics.

(A) Schematic representation of bioinformatics workflow: Whole-genome transcriptomes from 91 healthy and 140 patients with lung fibrosis were fetched from the NCBI GEO. Differential expression analysis was used to assemble an uncurated list of the top 1000 differentially expressed genes. GO enrichment of choice biological pathways was used to assemble a curated list of genes associated with MF differentiation. Datasets were fed through a previous knowledgebased analysis pipeline to identify enriched signaling pathways (Reactome) and key protein signaling nodes (STRING) within patients with IPF. (B) Heatmaps of the top 20 differentially expressed genes within specified GO categories, which were manually selected for curated analysis. CN values indicate a high degree of interaction between proteins selected for curated analysis. Colors are based on differential expression values that were not log-normalized. (C) Summary of the top 5 significantly enriched pathways in the curated and uncurated gene set. (D) Representative STRING diagram depicting protein interactions within the curated dataset, with summary of the top 5 signaling nodes in the uncurated and curated gene set. Blue nodes and edges represent interactions within the top 5 signaling nodes for the curated dataset.

Despite fibrosis widely contributing to mortality worldwide, inadequate understanding of fibrotic disease pathogenesis has limited the development of efficacious therapies (12). Preclinical studies in vivo, while indispensable, often fail to translate to clinical settings as evidenced by the failure of ~90% of drugs identified in animal studies (36). In addition, limitations in current technologies (e.g., the embryonic lethality of many genetic ECM knockouts and the limited resolution/imaging depth of intravital microscopy) have hindered the application of preclinical in vivo models for the study of cell-ECM interactions that underlie fibrogenesis (37). In contrast, existing in vitro models use patient-derived cells that are affordable, scalable, and amenable to microscopy, but often fail to recapitulate the complex 3D matrix structure of the interstitial tissue regions where fibrotic diseases such as IPF originate. We leveraged electrospinning and bio-orthogonal chemistries to engineer novel pulmonary interstitial matrices that are 3D and have fibrous architecture with biomimetic ligand presentation. In the presence of profibrotic soluble factors, these settings reproduce hallmarks of fibrosis at cellular and tissue levels (Figs. 2 to 4). Examining the influence of physical microenvironmental cues (cross-linking/stiffness and fiber density) on MF differentiation, we find that cross-linking/stiffness has opposing effects on MF differentiation in 2D versus 3D (Fig. 1) and that incorporation of a fibrous architecture in 3D is a prerequisite to MF differentiation (Fig. 4). Furthermore, supported by the importance of protease signaling in IPF (Fig. 6), we performed proof-of-concept pharmacologic screening within our 3D fibrotic matrices (Fig. 5) and highlighted enhanced biomimicry as compared to traditional 2D drug screening substrates where matrix remodeling appears to be dispensable for MF differentiation.

While tunable synthetic hydrogels have identified mechanosensing pathways critical to MF differentiation in 2D, these observations have yet to be translated to 3D fibrous settings relevant to the interstitial spaces where fibrosis originates. Given that late-stage IPF progresses in the absence of external tissue damage, current dogma implicates fibrotic matrix stiffness as the continual driver of MF differentiation in vivo (10, 11, 38). While we cannot disregard this hypothesis, our work elucidates a contrasting MMP-dependent mechanism at play in 3D, whereby a compliant, degradable, and fibrous matrix architecture supports MF differentiation, with matrix contraction and stiffening occurring downstream of -SMA expression, nearly a week later. Given numerous 2D studies indicating matrix stiffness as a driver of MF differentiation, the finding that a compliant matrix promotes MF differentiation may appear counterintuitive (10, 11). However, MF accumulation has been documented before tissue stiffening in human disease (3), and a recent phase 2 clinical trial (ClinicalTrials.gov Identifier: NCT01769196) targeting the LOX pathway (the family of enzymes responsible for matrix stiffening in vivo) failed to prevent disease progression in patients with IPF and was terminated due to lack of efficacy (39). Furthermore, compelling recent work by Fiore et al. (3) combined immunohistochemistry with high-resolution AFM to characterize human IPF tissue mechanics and found that regions of active fibrogenesis were highly fibrous but had a similar Youngs modulus as healthy tissue. In concert with our in vitro data, these findings suggest that MF differentiation is possible within soft provisional ECM in vivo and that the initiation of fibrogenesis may not be dependent on heightened tissue stiffness so long as matrix fibers and appropriate soluble cues (e.g., TGF-1) are present.

Consequently, understanding the source of profibrotic soluble cues in vivo is of critical importance when identifying therapeutic targets for IPF. Luminex screening of supernatant from 3D fibrotic matrices revealed sixfold increases in cytokine secretion during fibrogenesis, most of which were potent inflammatory factors [e.g., granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-6 (IL-6), IL-8, and vascular endothelial growth factor A (VEGF-A)] and chemoattractants (e.g., CCL2, CCL7, CCL11, and CXCL1) (Fig. 4E). Furthermore, IL-6 and VEGF-A were found to be major signaling nodes in patients with IPF (Fig. 6D). While not typically regarded as an immunomodulatory cell population, these findings suggest that MFs may maintain localized inflammation to support continual fibrogenesis. Mitogens such as IL-6 and IL-8 promote endothelial- and epithelial-to-mesenchymal transition, a process that gives rise to matrix-producing MF-like cells in IPF (40). CCR2 (CCL2 and CCL7) and CXCR1 (CXCL1 and IL-8) ligation facilitates macrophage chemotaxis, potentially leading to a sustained influx of TGF-1producing cells in IPF, and glycoproteins such as GM-CSF inhibit caspase activity in mononuclear cells, potentially preventing apoptotic events required for the resolution of wound repair and return to homeostasis (23, 41). In addition, secretion of nearly all cytokines was increased as a function of fiber density, highlighting a potential feed-forward loop distinct from canonical TGF-1 signaling. Further model development (e.g., coculture platforms) will be required to examine these hypotheses and the role of MF-derived cytokines in persistent inflammation and fibrosis.

In addition to documenting the role of fibrotic matrix architecture in 3D fibrogenesis, we demonstrate proof-of-concept pharmacologic screening within our synthetic pulmonary interstitial matrices and highlight their improved relevance to human disease. Previous work in vitro has documented profound reductions in MF differentiation after treatment with clinically approved antifibrotics (pirfenidone and nintedanib), whereas in the clinic, pirfenidone and nintedanib impede disease progression but are far from curative (4, 28, 42, 43). Pirfenidone or nintedanib had insignificant effects in 2D settings in our hands and only modest effects in 3D (Fig. 5). One reason for this discrepancy may be the use of supraphysiologic pirfenidone and nintedanib concentrations in previous in vitro studies, whereas we selected dosages based on plasma concentrations in patients with IPF (44). Differences in pharmacokinetics, nutrient/growth factor diffusion, and cell metabolism between 2D and 3D tissue constructs likely also play a role. Furthermore, as evidenced by the preventative effect of the protease inhibitor marimastat in 3D hydrogels but not 2D settings (Fig. 5), pharmacologics that influence matrix degradation and remodeling are likely to have a minimized effect in 2D settings due to the less dynamic nature of tissue culture plastic and flat hydrogels (45). Nintedanib and pirfenidone have been shown to influence protease activity and matrix remodeling in vivo (16), and may be mediating their effects within fibrotic matrices through modulation of ECM remodeling. Given the identification of numerous potential antifibrotic agents (microRNA, TGF-1 inhibitors, IL-4, IL-13 neutralizing antibodies, and integrin blockers) in preclinical models, application of the system described here could elucidate how choice pharmacologics affect MF differentiation and matrix remodeling processes that are difficult to recapitulate in 2D culture. Further development of our interstitial matrices as an arrayed platform, as has been elegantly implemented with collagen matrices (42), is a critical next step to moving this technology toward high-throughput screening applications.

It is important to note that this work has several potential limitations. Our material approach allows facile control of initial microenvironmental conditions (e.g., dimensionality, fiber density, ligand density, and elastic modulus), and of note, composites of RGD-bearing nondegradable fibers and degradable bulk hydrogel decouple degradation-induced changes in matrix mechanics and ligand availability. However, we have no experimental control over subsequent dynamic cell-driven remodeling events (e.g., MMP-mediated hydrogel softening, fibronectin and collagen deposition, and hydrogel contraction/stiffening from resident cells) that likely affect local matrix mechanics, cellular mechanosensing, and MF differentiation. Exciting recent technologies such as 3D traction force microscopy (TFM) and magnetic bead microrheology could enable future examination of how these dynamic changes in cell-scale mechanics potentiate MF differentiation in 3D. Along similar lines, although our study suggests a requirement for initial adhesion to the surrounding matrix, how the dynamics of ligand presentation due to matrix remodeling regulates mechanosensing was not explored here. We present this platform as a reductionist approach to modeling the activation of fibroblasts within the 3D fibrous interstitia associated with fibrosis, a pathology that develops over years in vivo and involves multiple cell types. Human pulmonary tissue and fibrotic foci, in particular, also have viscoelastic and nonlinear mechanical behaviors (3, 46) that were not explored in our AFM measurements of murine lung or hydrogel composites. Given the important role such mechanical features can play in ECM mechanosensing, incorporating new synthetic material strategies in combination with cell-scale mechanical measurements will be essential to modeling physiologic complexity. Given that the development of lung organoids is still in its infancy, decellularized precision-cut lung slices currently represent the best culture platform to capture the full complexity of the lung microenvironment (5).

In summary, we designed a tunable 3D and fibrous hydrogel model that recapitulates dynamic physical (e.g., stiffening and contraction) and biochemical (e.g., secretion of fibronectin, collagen, and cytokines) alterations to the microenvironment observed during the progression of IPF. Implementation of our model allowed us to establish a developing mechanism for MF differentiation in 3D compliant environments, whereby cell spreading upon matrix fibers drives YAP activity, cytokine release, and proteolysis-dependent MF differentiation. Furthermore, we leveraged bioinformatics techniques to explore protease signaling in clinical IPF and, in concert with our therapeutic screening data, establish a strong role for proteases during IPF pathogenesis and in 3D MF differentiation. Whether protease activity promoted MF differentiation directly through modulation of intracellular signaling or indirectly through affects on the local matrix environment has yet to be explored in these settings but will be the focus of future efforts. Consequently, these results highlight critical design parameters (3D degradability and matrix architecture) frequently overlooked in established synthetic models of MF differentiation. Future work incorporating macrophages, endothelial cells, and epithelial cells may expand current understanding of how developing MF populations influence otherwise homeostatic cells and how matrix remodeling influences paracrine signaling networks and corresponding drug response. Given the low translation rate of drugs identified in high-throughput screening assays, we show that the application and development of engineered biomimetics, in combination with preclinical models, can improve drug discovery and pathophysiological understanding.

All reagents were purchased from Sigma-Aldrich and used as received, unless otherwise stated.

Dextran vinyl sulfone. A previously described protocol for vinyl sulfonating polysaccharides was adapted for use with linear highmolecular weight (MW) dextran (MW 86,000 Da; MP Biomedicals, Santa Ana, CA) (20). Briefly, pure divinyl sulfone (12.5 ml; Thermo Fisher Scientific, Hampton, NH) was added to a sodium hydroxide solution (0.1 M, 250 ml) containing dextran (5 g). This reaction was carried out at 1500 rpm for 3.5 min, after which the reaction was terminated by adjusting the pH to 5.0 via the addition of hydrochloric acid. A lower functionalization of DexVS was used for hydrogels, where the volume of divinyl sulfone reagent was reduced to 3.875 ml. All reaction products were dialyzed for 5 days against Milli-Q ultrapure water, with two water exchanges daily, and then lyophilized for 3 days to obtain the pure product. Functionalization of DexVS was characterized by 1H nuclear magnetic resonance (NMR) spectroscopy in D2O and was calculated as the ratio of the proton integral [6.91 parts per million (ppm)] and the anomeric proton of the glucopyranosyl ring (5.166 and 4.923 ppm); here, vinyl sulfone/dextran repeat unit ratios of 0.376 and 0.156 were determined for electrospinning and hydrogel DexVS polymers, respectively.

DexVS was dissolved at 0.6 g ml1 in a 1:1 mixture of Milli-Q ultrapure water and dimethylformamide with 0.015% Irgacure 2959 photoinitiator. Methacrylated rhodamine (0.5 mM; Polysciences Inc., Warrington, PA) was incorporated into the electrospinning solution to fluorescently visualize fibers under 555 laser. This polymer solution was used for electrospinning within an environment-controlled glovebox held at 21C and 30% relative humidity. Electrospinning was performed at a flow rate of 0.3 ml hour1, gap distance of 5 cm, and voltage of 10.0 kV onto a grounded collecting surface attached to a linear actuator. Fiber layers were collected on glass slabs and primary cross-linked under ultraviolet light (100 mW cm2) and then secondary cross-linked (100 mW cm2) in an Irgacure 2959 solution (1 mg ml1). After polymerization, fiber segments were resuspended in a known volume of phosphate-buffered saline (PBS) (typically 3 ml). The total volume of fibers was then calculated via a conservation of volume equation: total resulting solution volume = volume of fibers + volume of PBS (3 ml). After calculating total fiber volume, solutions were re-centrifuged, supernatant was removed, and fiber pellets were resuspended to create a 10 volume % fiber solution, which were then aliquoted and stored at 4C. To support cell adhesion, 2.0 mM RGD was coupled to vinyl sulfone groups along the DexVS backbone via Michael-type addition chemistry for 30 min, followed by quenching of excess VS groups in a 300 mM cysteine solution for 30 min.

DexVS gels were formed via a thiol-ene click reaction at 3.3% (w/v) (pH 7.4, 37C, 45 min) with VPMS cross-linker (12.5, 20, and 27.5 mM) (GCRDVPMSMRGGDRCG, GenScript, George Town, KY) in the presence of varying amounts of arginylglycylaspartic acid (RGD, CGRGDS, 2.0 mM; GenScript, George Town, KY), HBP (GCGAFAKLAARLYRKA, 1.0 mM; GenScript, George Town, KY), and fiber segments (0.0 to 5.0%, v/v). For experiments comparing hydrogels of varying ligand type (1 mM HBP versus 2 mM RGD), cysteine was added to precursor solutions to maintain a final vinyl sulfone concentration of 60 mM. All hydrogel and peptide precursor solutions were made in PBS containing 50 mM Hepes. To create fibrous hydrogels, a defined stock solution (10% v/v) of suspended fibers in PBS/Hepes was mixed into hydrogel precursor solutions before gelation. By controlling the dilution of the fiber suspension, fiber density was readily tuned within the hydrogel at a constant hydrogel weight percentage. For gel contraction experiments, DexVS was polymerized within a 5-mm-diameter polydimethylsiloxane (PDMS) gasket to ensure consistent hydrogel area on day 0.

NHLFs (University of Michigan Central Biorepository), normal human dermal fibroblasts (Lonza, Basel, Switzerland), and normal human mammary fibroblasts (Sciencal, Carlsbad, CA) were cultured in Dulbeccos modified Eagles medium containing 1% penicillin/streptomycin, l-glutamine, and 10% fetal bovine serum (Atlanta Biologicals, Flowery Branch, GA). NHLFs derived from three separate donors were used for experiments. Cells were passaged upon achieving 90% confluency at a 1:4 ratio and used for studies until passage 7. For all hydrogel studies, cells were trypsinized, counted and either encapsulated into or seeded onto 25-l hydrogels at a density of 1,000,000 cells ml1 of hydrogel, and subsequently cultured at 37C and 5% CO2 in serum-containing medium. For studies comparing 3D hydrogels to tissue culture plastic, the number of cells seeded into 2D conditions was analogous to the total cell number within hydrogel matrices. Medium was refreshed the day after encapsulation and every 2 days after. In selected experiments, recombinant human TGF-1 (5 ng/ml; PeproTech, Rocky Hill, NJ) was supplemented into the medium at 5 ng ml1. For pharmacological studies, nintedanib (50 nM; Thermo Fisher Scientific, Hampton, NH), pirfenidone (100 M; Thermo Fisher Scientific, Hampton, NH), marimastat (1.0 M), and dimethyl fumarate (100 nM) were supplemented in serum-containing medium and refreshed every 2 days.

Cultures were fixed with 4% paraformaldehyde for 30 min at room temperature. To stain the actin cytoskeleton and nuclei, samples were permeabilized in PBS solution containing Triton X-100 (5%, v/v), sucrose (10%, w/v), and magnesium chloride (0.6%, w/v); blocked in 1% bovine serum albumin (BSA); and stained simultaneously with phalloidin and 4,6-diamidino-2-phenylindole (DAPI). For immunostaining, samples were permeabilized, blocked for 8 hours in 1% (w/v) BSA, and incubated with mouse monoclonal anti-YAP antibody (1:1000; Santa Cruz Biotechnology, SC-101199), mouse monoclonal anti-fibronectin antibody (FN, 1:2000; Sigma-Aldrich, #F6140), rabbit monoclonal anti-Ki67 (1:500; Sigma-Aldrich #PIMA514520), or mouse monoclonal anti-SMA (1:2000; Sigma-Aldrich, #A2547) followed by secondary antibody for 6 hours each at room temperature with 3 PBS washes in between. High-resolution images of YAP, FN, and actin morphology were acquired with a 40 objective. Unless otherwise specified, images are presented as maximum intensity projections of 100-m Z-stacks. Hydrogel samples were imaged on a Zeiss LSM 800 laser scanning confocal microscope. SHG imaging of lung tissue was conducted on a Leica SPX8 laser scanning confocal microscope with an excitation wavelength of 820 nm and a collection window of 400 to 440 nm. Single-cell morphometric analyses (cell spread area) were performed using custom Matlab scripts with sample sizes >50 cells per group, while YAP, -SMA, Ki67, and FN immunostains were quantified on an image basis with a total of 10 frames of view. MFs were denoted as nucleated, F-actin+, -SMA+ cells. For cell density (number of nuclei) calculations, DAPI-stained cell nuclei were thresholded and counted in six separate 600 m 600 m 200 m image volumes, allowing us to calculate a total number of cells per mm3 of gel. Fiber recruitment analysis was conducted via a custom Matlab script; briefly, cell outlines were created via actin masking and total fiber fluorescence was quantified under each actin mask on a per-cell basis. A similar analysis method using Matlab was used for cell-cell junction analysis as published previously, with higher area:perimeter ratios and clusters/cell indicative as more pronounced network formation (47).

For all experiments, additional hydrogel replicates were finely minced and degraded in dextranase solution (4 IU/ml; Sigma-Aldrich) for 20 min and homogenized in buffer RLT (Qiagen, Venlo, The Netherlands), and RNA was isolated according to the manufacturers protocols. Complementary DNA (cDNA) was generated from deoxyribonuclease (DNase)free RNA and amplified, and gene expression was normalized to the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Experiments were run with technical triplicates across three individual biological experiments. For a complete list of primers, see table S2.

To determine the elastic modulus of lung tissue and DexVS hydrogels, indentation tests were used with a Nanosurf FlexBio AFM (Nanosurf, Liestal, Switzerland). Samples were indented via a 5-m bead at a depth of 10 m and an indentation rate of 0.333 m/s. Resulting force-displacement curves were fit to a spherical Hertz model using AtomicJ. Poissons ratios of 0.5 and 0.4 were used for hydrogels and lung tissue, respectively.

All animal studies were approved by the Animal Care and Use Committee at the University of Michigan. Bleomycin (0.025 U; Sigma-Aldrich) was instilled intratracheally in C57BL6 mice (8 weeks of age; The Jackson Laboratory, Bar Harbor, ME, USA) on day 0. Briefly, mice were anesthetized with sodium pentobarbital, the trachea was exposed and entered with a 30-gauge needle under direct visualization, and a single 30-l injection containing 0.025 U of bleomycin (Sigma-Aldrich) diluted in normal saline was injected. Lungs were collected on day 14 for mechanical and histological analysis. For histology samples, lungs were perfused with saline and inflated with 4% paraformaldehyde, sectioned, and stained with picrosirius red. For mechanical characterization via AFM, lungs were perfused with saline, infused with OCT (optimal cutting temperature) compound (Thermo Fisher Scientific), and flash-frozen in a slurry of dry ice and ethanol. Sections were mechanically tested via AFM nanoindentation immediately upon thawing.

To characterize the inflammatory secretome associated with various DexVS-VPMS environments, medium was collected from NHLF cultures 3, 5, 7, and 9 days after encapsulation. A Luminex FlexMAP 3D (Luminex Corporation, Austin, TX) systems technology was used to measure 41 cytokines/chemokines (HCTYMAG-60 K-PX41, Milliplex, EMD Millipore Corporation) in the medium samples according to the manufacturers instructions. Total secretion was reported as the sum of all 41 analytes measured for each respective condition. Cell-secreted collagen was measured using the established colorimetric Sircol assay in hydrogels cultured with serum-free medium in the presence of ascorbic acid (25 g ml1).

The NCBI GEO database was consulted [dataset GSE47460 (GP14550)] to fetch gene expression data from 91 healthy patients and 140 patients with IPF; patients with chronic obstructive pulmonary disease and nonidiopathic fibrotic lung diseases were excluded from the analysis (48). GEO2R (www.ncbi.nlm.nih.gov/geo/geo2r/) software was used for GO term enrichment, with keywords ECM, MMP, integrin, cytoskeleton, cytokine, chemokine, and MAPK used as search terms for dataset curation (48). Noncurated datasets were composed of the top 1000 differentially expressed genes between healthy and interstitial lung disease (ILD) conditions. Differential expression was calculated on the basis of subtracting normalized expression values between diseased and healthy patients. All genes were normalized before analysis with GEO2R via a pairwise cyclic losses approach. For pathway and protein-protein enrichment analyses, a curated pathway database [Reactome (49)] and Search Tool for Retrieval of Interacting Genes/Proteins [STRING (50)] methodology were consulted, respectively. For STRING analyses, up-regulated genes within the druggable genome were focused upon. A tabulated list of top genes, pathways, and nodes can be seen in data file S1.

Statistical significance was determined by one-way analysis of variance (ANOVA) or Students t test where appropriate, with significance indicated by P < 0.05. All data are presented as means SD.

Acknowledgments: We thank E. S. White (University of Michigan) for providing patient-derived lung fibroblasts used in these studies. Funding: This work was supported, in part, by the NIH (HL124322, R35HL144481). D.L.M. and C.D.D. acknowledge financial support from the NSF Graduate Research Fellowship Program (DGE1256260). Author contributions: D.L.M. and B.M.B. conceived and supervised the project. D.L.M. designed and performed the experiments. K.M.D. and K.B.A. performed and aided in analysis of the Luminex experiments. M.R.S. and C.D.D. helped with data analysis. R.P. and M.S. aided in polymer syntheses and microfiber fabrication. I.M.L. provided equipment for and assisted in polymerase chain reaction experiments. C.A.W. and B.B.M. helped perform the animal experiments for the bleomycin-induced lung fibrosis model. All authors edited and approved the manuscript. 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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Microengineered 3D pulmonary interstitial mimetics highlight a critical role for matrix degradation in myofibroblast differentiation - Science...

There is no silver bullet at the moment, and there might never be, said World Health Organization Director-General Tedros Adhanom at a virtual press conference at the beginning of August. While it was this bleak sound bite that made the headlines, Tedros also had words of praise for the progress made towards identifying treatments that aid the recovery of COVID-19 patients with the most serious forms of the disease.Research towards treatments for COVID-19 has been developing at a phenomenal speed, even though it feels as though solutions cant come soon enough; the widespread transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has had significant health, economic and social impacts across the globe, and as of September 8th more than 27 million cases and 890,000 deaths have been recorded in 188 countries.

Research groups across the world have set about identifying drugs for the treatment of COVID-19, by screening both novel and existing drugs for their ability to alleviate symptoms and stem viral replication. Here, we provide an update on ongoing global efforts to develop and test drugs for the treatment of COVID-19 and explore the range of strategies being employed.

COVID-19 is a disease which can leave you with anything between a mild sniffle to an unpleasant combination of high fever, heavy fatigue, and lung inflammation and damage. The drivers of clinical symptoms can be roughly divided into two categories: the virus itself and the hyperinflammatory response to the virus that occurs in the most severely ill people. Consequently, efforts to identify appropriate treatments are often focused on one category, and sometimes, a particular patient group or stage of disease. Given the nature of COVID-19, it is highly likely that a combination of drugs (drug cocktail) will be needed to both neutralize the virus and suppress the symptoms of COVID-19. Antiviral treatments may target viral components directly, or other cellular processes involved in viral infection or replication. To date, interventional studies for COVID-19 have attempted to achieve a wide range of goals, including:

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Meet the scientists on the frontline with coronavirus. Video credit: Sanford Burnham Prebys Medical Discovery Institute

Of the ~12,000 compounds screened, 100 inhibited SARS-CoV-2 replication in mammalian cells, 21 of which did so in a dose-response fashion. Achieving a sufficiently high dose concentration to elicit antiviral effects in vivo was predicted to be practical and possible for 13 of these compounds based on EC50 values in various cell lines. The most potent of these were evaluated for antiviral activity in human induced pluripotent stems cell (iPSC)-derived pneumocyte-like cells (five candidates) and in an ex vivo lung culture system (one candidate). The latter candidate is called apilimod, a small molecule inhibitor of an enzyme (phosphoinositide 5-kinase or PIKfyve, an endosomal lipid kinase) important to the endocytic pathway in which SARS-CoV-2 travels along during its journey through the cell. Encouragingly, apilimod potently antagonized viral replication in these tissues, and the findings are in agreement with those of another research group. This month, Kang et al. published an article in PNAS, describing the potent inhibition of SARS-CoV-2 by apilimod, providing further evidence to suggest PIKfyve-inhibition as a potential strategy that could limit infection and disease pathogenesis. The authors also noted that apilimod has passed safety tests in previous clinical trials for nonviral indications.

Chanda highlights the incredible pace at which this work was produced. Typically, a project like this would take years, rather than months. He points out that by wanting to do something quickly, there were sacrifices (and not just weekends). For example, they ran with the assay and the cell lines that allowed them to produce results quickly. This is the reason we put the entire dataset out there not one/three/20 molecules, we put all 100 molecules out there. These are the ones we found because of our experimental system, but please keep testing the others because youll probably find other things that work, said Chanda.

To design multiple peptide sequences that can competitively bind to the SARS-CoV-2 receptor binding domain, the University of Michigan research group used a protein design system called EvoDesign.EvoDesign is the first de novo protein design protocol developed in our lab; it performs design simulation by combining the evolution-based information collected from protein databases and an accurate physics- and knowledge-based energy function, namely EvoEF2, for computing atomic interactions such as van der Waals forces, electrostatics, hydrogen bonding, and desolvation energies, said Huang.

Overall, these sophisticated computational tools represent a promising new avenue for the de novo development of drug discovery studies.

Michele Wilson is a freelance science writer for Choice Science Writing.

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COVID-19 Drug Discovery and Development Why Diverse Strategies Are Critical - Technology Networks

A piercing smile that beams out of his photos is not the only legacy left behind by much-loved Joe Brown.

At just 29, his life was cut short, but not before he made a decision that would save the lives of three other people.

Prior to his untimely death, the avid gamer signed up to donate all his organs to help those waiting for a life-saving transplant.

For his mother and siblings, nothing could prepare them for losing "kind and generous" Joe so suddenly, but they have found solace in knowing part of him lives on.

Big sister, Louise Edwards, told Black Country Live: "He had opted to donate all his organs. Initially, my mum struggled with this decision but its what he wanted.

"He saved two 29-year-old men who had been waiting nearly two years for a life-saving transplant and he also helped a lady in her 50s.

Joes liver and kidneys were donated shortly after his death at Walsall Manor Hospital on July 23 this year.

Louise said: "My brother was a caring person and, even on his deathbed, he wanted to help. He chose to do it so we had to support it. He agreed to have his stem cells taken to help children. Although he is no longer here, he is still helping people.

"Its comforting to know he saved the lives of three people and his legacy lives on."

According to figures released by the NHS blood and transplant service, there are currently around 6,000 people on the UK transplant waiting list.

Last year, more than 350 people died while waiting for a transplant. Just eight per cent of organs donated were from those of African, Caribbean or Asian heritage.

While his final gesture represented the gift of life, Joe was hiding a silent battle with mental health.

An inquest hearing held at Black Country Coroners Court decided he had tragically taken his own life.

Louise said: "He didnt talk about his struggles with mental health, he always said he didnt want to be a burden. He had stopped talking to us, we only got him back two weeks before his death."

During the inquest, it was revealed that, in the weeks before Joe's death, he had tried to contact the emergency mental health crisis team but was denied a face-to-face appointment because of the COVID-19 outbreak.

"He only used to confide in his friends on the Xbox, he talked about his past, his troubles in his relationship and previous suicide attempts, his sister continued.

She added: "He was a family person, he was a kind and generous person, thats the legacy that he left behind. More than 200 people came to pay their respects at his funeral, he didnt know how loved he was.

"To those struggling with mental health, speak out. It doesnt make you weak to speak out. If he had told us how he was feeling we could have helped to get him the help he needed.

Samaritans (116 123) samaritans.org operates a 24-hour service available every day of the year. If you prefer to write down how youre feeling, or if youre worried about being overheard on the phone, you can email Samaritans at jo@samaritans.org , write to Freepost RSRB-KKBY-CYJK, PO Box 9090, STIRLING, FK8 2SA and visit http://www.samaritans.org/branches to find your nearest branch.

CALM (0800 58 58 58) thecalmzone.net has a helpline is for men who are down or have hit a wall for any reason, who need to talk or find information and support. They're open 5pm to midnight, 365 days a year.

Childline (0800 1111 ) runs a helpline for children and young people in the UK. Calls are free and the number wont show up on your phone bill. PAPYRUS (0800 068 41 41) is a voluntary organisation supporting teenagers and young adults who are feeling suicidal.

Depression Alliance is a charity for people with depression. It doesnt have a helpline, but offers a wide range of useful resources and links to other relevant information depressionalliance.org Students Against Depression is a website for students who are depressed, have a low mood or are having suicidal thoughts. Bullying UK is a website for both children and adults affected by bullying studentsagainstdepression.org The Sanctuary (0300 003 7029 ) helps people who are struggling to cope - experiencing depression, anxiety, panic attacks or in crisis. You can call them between 8pm and 6am every night.There are other depression charities.

"The family are distraught, my kids and the younger siblings dont understand why Joe isnt here. Im the oldest and I never thought he would be gone before me."

At the hearing, coroner, Joanne Lees, told the court that Joe had been found unresponsive in his room by his ex-girlfriend on July 20, 2020.

Paramedics were able to resuscitate him and he was rushed to Walsall Manor Hospital but, due to a cardiac arrest, he suffered a brain injury which led to his death.

Fondly recalling her sons warm character, Vicky Spriggs told the court: "He was a happy go lucky person who didnt believe in mental health, he would always say, 'suck it up' or 'man up'.

"He was bubbly and outgoing. Joe was always smiling and joking around.

Ms Lees recorded a verdict of suicide and offered her condolences to the family.

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Much-loved son Joe Brown saved three lives after tragic death at just 29 - Birmingham Live

The success of approved stem cell therapies has caused a surge in interest of biopharma developers in this field; many innovator companies are currently progressing proprietary leads across different phases of clinical development, with cautious optimism

Roots Analysis has announced the addition of Global Stem Cells Market: Focus on Clinical Therapies, 20202030 (Based on Source (Allogeneic, Autologous); Origin (Adult, Embryonic); Type (Hematopoietic, Mesenchymal, Progenitor); Lineage (Amniotic Fluid, Adipose Tissue, Bone Marrow, Cardiosphere, Chondrocytes, Corneal Tissue, Cord Blood, Dental Pulp, Neural Tissue Placenta, Peripheral Blood, Stromal Cells); and Potency (Multipotent, Pluripotent)) report to its list of offerings.

There is a growing body of evidence supporting the vast applicability and superiority of treatment outcomes of stem cell therapies, compared to conventional treatment options. In fact, the unmet needs within this domain have spurred the establishment of many start-ups in recent years.

To order this 500+ page report, which features 185+ figures and 220+ tables, please visit this link

Over 280 stem cell therapies are under development, most of which are allogeneic productsMore than 50% of the pipeline candidates are in the mid to late phase trials (phase II and above), and allogenic therapies (majority of which are derived from the bone marrow) make up 65% of the pipeline.

70% of pipeline candidates are based on mesenchymal stem cellsIt is worth highlighting that the abovementioned therapies are designed to treat musculoskeletal (22%), neurological (21%) and cardiovascular (15%) disorders. On the other hand, hematopoietic stem cell-based products are mostly being evaluated for the treatment of oncological disorders, primarily hematological malignancies.

Close to 85% stem cell therapy developers are based in North America and Asia-Pacific regionsWithin these regions, the US, China, South Korea and Japan, have emerged as key R&D hubs for stem cell therapies. It is worth noting that majority of the initiatives in this domain are driven by small / mid-sized companies

Over 1,500 grants were awarded for stem cell research, since 2015More than 45% of the total amount was awarded under the R01 mechanism (which supports research projects). The NCI, NHLBI, NICHD, NIDDK, NIGMS and OD emerged as key organizations that have offered financial support for time periods exceeding 25 years as well.

Outsourcing has become indispensable to R&D and manufacturing activity in this domainPresently, more than 80 industry / non-industry players, based in different regions across the globe, claim to provide contract development and manufacturing services to cater to the unmet needs of therapy developers. Examples include (in alphabetical order) Bio Elpida, Cell and Gene Therapy Catapult, Cell Tech Pharmed, GenCure, KBI Biopharma, Lonza, MEDINET, Nikon CeLL innovation, Roslin Cell Therapies, WuXi Advanced Therapies and YposKesi.

North America and Asia-Pacific markets are anticipated to capture over 80% share by 2030The stem cell therapies market is anticipated to witness an annualized growth rate of over 30% during the next decade. Interestingly, the market in China / broader Asia-Pacific region is anticipated to grow at a relatively faster rate.

To request a sample copy / brochure of this report, please visit this link

The USD 8.5 billion (by 2030) financial opportunity within the stem cell therapies market has been analyzed across the following segments:

The report features inputs from eminent industry stakeholders, according to whom stem cell therapies are currently considered to be a promising alternatives for the treatment of a myriad of disease indications, with the potential to overcome challenges associated with conventional treatment options. The report includes detailed transcripts of discussions held with the following experts:

The research covers brief profiles of several companies (including those listed below); each profile features an overview of the company, financial information (if available), stem cell therapy portfolio and an informed future outlook.

For additional details, please visithttps://www.rootsanalysis.com/reports/view_document/stem-cells-market/296.html

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Market Growth of Global Stem Cells to Remain Sluggish through 2020 2030 - The News Brok

Global Coronavirus pandemic has impacted all industries across the globe, Progenitor Cell Product market being no exception. As Global economy heads towards major recession post 2009 crisis, Cognitive Market Research has published a recent study which meticulously studies impact of this crisis on Global Progenitor Cell Product market and suggests possible measures to curtail them. This press release is a snapshot of research study and further information can be gathered by accessing complete report. To Contact Research Advisor Mail us @ [emailprotected] or call us on +1-312-376-8303.

Report is a detailed study of the Progenitor Cell Product market, which covers all the essential information required by a new market entrant as well as the existing players to gain a deeper understanding of the market. Report has been segmented into Geographical Segmentation, Key players, Key Topics Industry Value and Demand Analysis Forecast to 2027 and provides comprehensive investigation.

Global Progenitor Cell Product Market: Product analysis: Pancreatic progenitor cells, Cardiac Progenitor Cells, Intermediate progenitor cells, Neural progenitor cells (NPCs), Endothelial progenitor cells (EPC), Others

Global Progenitor Cell Product Market: Application analysis: Medical care, Hospital, Laboratory

Major Market Players with an in-depth analysis: NeuroNova AB, StemCells, ReNeuron Limited, Asterias Biotherapeutics, Thermo Fisher Scientific, STEMCELL Technologies, Axol Bio, R&D Systems, Lonza, ATCC, Irvine Scientific, CDI

Any query? Enquire Here For Discount (COVID-19 Impact Analysis Updated Sample): Click Here>Download Sample Report of Progenitor Cell Product Market Report 2020 (Coronavirus Impact Analysis on Progenitor Cell Product Market)

The research comprises primary information about the products. Similarly, it includes supply-demand statistics, and segments that constrain the growth of an industry. It also includes raw materials used and manufacturing process of Progenitor Cell Product market. Additionally, report provides market drivers and challenges & opportunities for overall market in the particular provincial sections.

The report gives detailed account on each segment which helps to understand market more effectively. The company profiling of key players include: business overview, product description, research and development investment, key development, business strategy, and SWOT analysis. It also involves sales revenue of each division and geographical coverage for two consecutive years.

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The industry intelligence study of the Progenitor Cell Product market covers the estimation size of the market each in phrases of value (Mn/Bn USD) and volume (x units). Further, report consists of Porters Five Forces and BCG matrix as well as product life cycle to help you in taking wise decisions. Additionally, this report covers the inside and out factual examination and the market elements and requests which give an entire situation of the business.

Regional Analysis for Progenitor Cell Product Market:North America (United States, Canada)Europe (Germany, Spain, France, UK, Russia, and Italy)Asia-Pacific (China, Japan, India, Australia, and South Korea)Latin America (Brazil, Mexico, etc.)The Middle East and Africa (GCC and South Africa)

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Global and Asia Pacific Progenitor Cell Product Market to Witness Huge Growth by 2027 Major Manufacturers included in report NeuroNova AB, StemCells,...

Global Autologous Stem Cell and Non-Stem Cell Based Therapies Market research report estimates a considerable growth of market in percentage during the forecast period of 2020-2026. This report also explains market definitions, classifications, applications, and engagements in the Healthcare industry. In addition, the scope of this market report can be broadened from market scenarios to comparative pricing between major players, cost & profit of the specified market regions. Autologous Stem Cell and Non-Stem Cell Based Therapies Market report is very consistent as all the data and information regarding the Healthcare industry is derived via authentic sources such as websites, journals, annual reports of the companies, and magazines.

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TheGlobalAutologous Stem Cell and Non-Stem Cell Based Therapies Marketis expected to reach USD113.04 billion by 2025, from USD 87.59 billion in 2017 growing at a CAGR of 3.7% during the forecast period of 2018 to 2025. The upcoming market report contains data for historic years 2015 & 2016, the base year of calculation is 2017 and the forecast period is 2018 to 2025.

Some of the major players operating in the globalautologous stem cell and non-stem cell based therapies marketareAntria (Cro), Bioheart, Brainstorm Cell Therapeutics, Cytori, Dendreon Corporation, Fibrocell, Genesis Biopharma, Georgia Health Sciences University, Neostem, Opexa Therapeutics, Orgenesis, Regenexx, Regeneus, Tengion, Tigenix, Virxsys and many more.

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Market Definition:Global Autologous Stem Cell and Non-Stem Cell Based Therapies Market

In autologous stem-cell transplantation persons own undifferentiated cells or stem cells are collected and transplanted back to the person after intensive therapy. These therapies are performed by means of hematopoietic stem cells, in some of the cases cardiac cells are used to fix the damages caused due to heart attacks. The autologous stem cell and non-stem cell based therapies are used in the treatment of various diseases such as neurodegenerative diseases, cardiovascular diseases, cancer and autoimmune diseases, infectious disease.

According to World Health Organization (WHO), cardiovascular disease (CVD) causes more than half of all deaths across the European Region. The disease leads to death or frequently it is caused by AIDS, tuberculosis and malaria combined in Europe. With the prevalence of cancer and diabetes in all age groups globally the need of steam cell based therapies is increasing, according to article published by the US National Library of Medicine National Institutes of Health, it was reported that around 382 million people had diabetes in 2013 and the number is growing at alarming rate which has increased the need to improve treatment and therapies regarding the diseases.

Market Segmentation:Global Autologous Stem Cell and Non-Stem Cell Based Therapies Market

Major Autologous Stem Cell and Non-Stem Cell Based Therapies Market Drivers and Restraints:

Introduction of novel autologous stem cell based therapies in regenerative medicine

Reduction in transplant associated risks

Prevalence of cancer and diabetes in all age groups

High cost of autologous cellular therapies

Lack of skilled professionals

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Autologous Stem Cell and Non-Stem Cell Based Therapies Market 2020-2025 | Major Giants Fibrocell, Genesis Biopharma, Georgia Health Sciences...

A health ministry panel on Thursday approved a Keio University clinical research project to transplant heart muscle cells made from induced pluripotent stem (iPS) cells into heart disease patients.

The research will be carried out by a team led by Prof. Keiichi Fukuda for three people between 20 and 74 suffering from dilated cardiomyopathy, which lowers the hearts power to pump blood. The first transplant will be conducted by the end of this year at the earliest.

The team will use iPS cells made by Kyoto University from the blood of a person who has a special immunological type with less risk of rejection.

The team will transform the iPS cells into heart muscle cells and inject about 50 million of them into the heart using a special syringe. Immunosuppressive drugs will be used for about half a year, and the team will spend a year checking to see whether the treatment leads to the development of tumors and irregular heartbeat or whether it restores heart function.

In January, Osaka University conducted the worlds first transplant of heart muscle cells made from iPS cells. The heart muscle cells were made into sheets and pasted on the surface of the patients heart so that a substance they emit can help regenerate the heart muscles. The cells themselves, however, disappear quickly.

Meanwhile, Keio University has confirmed in an experiment on monkeys that cells colonize after a transplant and heart function improves.

The university expects that transplanted cells will colonize over a long period also in the upcoming clinical research project.

According to the team, there are about 25,000 dilated cardiomyopathy patients in Japan.

A startup led by Fukuda is planning a clinical trial aimed at commercializing the iPS-derived cells, hoping they will also be used for the treatment of other cardiac diseases.

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Keio University gets OK for iPS-based heart cell transplant plan - The Japan Times

Michigan State University researchers have created for the first time a miniature human heart model in the laboratory, complete with all primary heart cell types and a functioning structure of chambers and vascular tissue.

Aitor Aguirre, assistant professor of biomedical engineering at MSUs Institute for Quantitative Health Science and Engineering.

These minihearts constitute incredibly powerful models in which to study all kinds of cardiac disorders with a degree of precision unseen before, said Aitor Aguirre, the studys senior author and assistant professor of biomedical engineering at MSUs Institute for Quantitative Health Science and Engineering.

This study, Generation of Heart Organoids Modeling Early Human Cardiac Development Under Defined Conditions, appears on the bioRxiv preprint server and was funded by grants from the American Heart Association and the National Institutes of Health. In the United States, heart disease is the No. 1 cause of death.

The human heart organoids, or hHOs for short, were created by way of a novel stem cell framework that mimics the embryonic and fetal developmental environments.

Organoids meaning resembling an organ are self-assembling 3D cell constructs that recapitulate organ properties and structure to a significant extent, said Yonatan Israeli, a graduate student in the Aguirre Lab and first author of the study.

The innovation deploys a bioengineering process that uses induced pluripotent stem cells adult cells from a patient to trigger embryonic-like heart development in a dish generating a functional mini heart after a few weeks. The stem cells are obtained from consenting adults and therefore free of ethical concerns.

This process allows the stem cells to develop, basically as they would in an embryo, into the various cell types and structures present in the heart, Aguirre said. We give the cells the instructions and they know what they have to do when all the appropriate conditions are met.

Because the organoids followed the natural cardiac embryonic development process, the researchers studied, in real time, the natural growth of an actual fetal human heart.

This technology allows for the creation of numerous hHOs simultaneously with relative ease, contrasting with existing tissue engineering approaches that are expensive, labor intensive and not readily scalable.

One of the primary issues facing the study of fetal heart development and congenital heart defects is access to a developing heart. Researchers have been confined to the use of mammalian models, donated fetal remains and in vitro cell research to approximate function and development.

Now we can have the best of both worlds, a precise human model to study these diseases a tiny human heart without using fetal material or violating ethical principles. This constitutes a great step forward, Aguirre said.

Whats next? For Aguirre, the process is twofold. First, the heart organoid represents an unprecedented look into the nuts and bolts of how a fetal heart develops.

In the lab, we are currently using heart organoids to model congenital heart disease the most common birth defect in humans affecting nearly 1% of the newborn population, Aguirre said. With our heart organoids, we can study the origin of congenital heart disease and find ways to stop it.

And second, while the hHO is complex, it is far from perfect. For the team, improving the final organoid is another key avenue of future research. The organoids are small models of the fetal heart with representative functional and structural features, Israeli said. They are, however, not as perfect as a human heart yet. That is something we are working toward.

Aguirre and team are excited about the wide-ranging applicability of these miniature hearts. They enable an unprecedented ability to study many other cardiovascular-related diseases from chemotherapy-induced cardiotoxicity to the effect of diabetes, during pregnancy, on the developing fetal heart.

Other researchers involved in this study were Aaron Wasserman, Mitchell Gabalski and Kristen Ball at MSU; and Chao Zhou, Jinyon Zhou and Guangming Ni at Washington University in St. Louis.

(Note for media: Please include a link to the original paper in online coverage: https://www.biorxiv.org/content/10.1101/2020.06.25.171611v2)

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Scientists grow the first functioning mini human heart model - MSUToday

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Researchers have created, for the first time, a miniature human heart model in the laboratory.

The mini-hearts are complete with all primary heart cell types and a functioning structure of chambers and vascular tissue.

The organoids are small models of the fetal heart with representative functional and structural features. They are, however, not as perfect as a human heart yet. That is something we are working toward.

These mini-hearts constitute incredibly powerful models in which to study all kinds of cardiac disorders with a degree of precision unseen before, says Aitor Aguirre, assistant professor of biomedical engineering at Michigan State Universitys Institute for Quantitative Health Science and Engineering and senior author of the study on the work on the bioRxiv preprint server. In the United States, heart disease is the leading cause of death.

The researchers created the human heart organoids, or hHOs for short,by way of a novel stem cell framework that mimics the embryonic and fetal developmental environments.

Organoidsmeaning resembling an organare self-assembling 3D cell constructs that recapitulate organ properties and structure to a significant extent, says first author Yonatan Israeli, a graduate student in Aguirres lab.

The innovation deploys a bioengineering process that uses induced pluripotent stem cellsadult cells from a patient to trigger embryonic-like heart development in a dishgenerating a functional mini-heart after a few weeks. The stem cells are obtained from consenting adults and therefore free of ethical concerns.

This process allows the stem cells to develop, basically as they would in an embryo, into the various cell types and structures present in the heart, Aguirre says. We give the cells the instructions and they know what they have to do when all the appropriate conditions are met.

Because the organoids followed the natural cardiac embryonic development process, the researchers studied, in real time, the natural growth of an actual fetal human heart.

This technology allows for the creation of numerous hHOs simultaneously with relative ease, contrasting with existing tissue engineering approaches that are expensive, labor intensive and not readily scalable.

One of the primary issues facing the study of fetal heart development and congenital heart defects is access to a developing heart. Researchers have been confined to the use of mammalian models, donated fetal remains, and in vitro cell research to approximate function and development.

Now we can have the best of both worlds, a precise human model to study these diseasesa tiny human heartwithout using fetal material or violating ethical principles. This constitutes a great step forward, Aguirre says.

Whats next? For Aguirre, the process is twofold. First, the heart organoid represents an unprecedented look into the nuts and bolts of how a fetal heart develops.

In the lab, we are currently using heart organoids to model congenital heart diseasethe most common birth defect in humans affecting nearly 1% of the newborn population, Aguirre says. With our heart organoids, we can study the origin of congenital heart disease and find ways to stop it.

And second, while the hHO is complex, it is far from perfect. For the team, improving the final organoid is another key avenue of future research.

The organoids are small models of the fetal heart with representative functional and structural features, Israeli says. They are, however, not as perfect as a human heart yet. That is something we are working toward.

The researchers are excited about the wide-ranging applicability of these miniature hearts. They enable an unprecedented ability to study many other cardiovascular-related diseasesincluding chemotherapy-induced cardiotoxicity and the effect of diabetes, during pregnancy, on the developing fetal heart.

Additional researchers from Michigan State and Washington University in St. Louis contributed to the work.

The American Heart Association and the National Institutes of Health funded the study.

Source: Michigan State University

Original Study DOI: 10.1101/2020.06.25.171611

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First lab-made 'mini-hearts' mimic the real thing - Futurity: Research News

KENILWORTH, N.J.--(BUSINESS WIRE)--Aug 19, 2020--

Merck (NYSE: MRK), known as MSD outside the United States and Canada, today announced that the pivotal Phase 3 KEYNOTE-590 trial evaluating KEYTRUDA, Mercks anti-PD-1 therapy, in combination with chemotherapy (cisplatin plus 5-fluorouracil [5-FU]), met its primary endpoints of overall survival (OS) and progression-free survival (PFS) for the first-line treatment of patients with locally advanced or metastatic esophageal cancer. Based on an interim analysis conducted by an independent Data Monitoring Committee, KEYTRUDA in combination with chemotherapy demonstrated a statistically significant and clinically meaningful improvement in OS and PFS compared with chemotherapy (cisplatin plus 5-FU), the current standard of care, in the intention-to-treat (ITT) population. The study also met the key secondary endpoint of objective response rate (ORR), with significant improvements for KEYTRUDA in combination with chemotherapy compared with chemotherapy alone. The safety profile of KEYTRUDA in this trial was consistent with that observed in previously reported studies. Results will be shared with global regulatory authorities and have been submitted for presentation at the European Society for Medical Oncology (ESMO) Virtual Congress 2020.

Esophageal cancer is a devastating malignancy with a high mortality rate and few treatment options in the first-line setting beyond chemotherapy, said Dr. Roy Baynes, senior vice president and head of global clinical development, chief medical officer, Merck Research Laboratories. In this pivotal study, KEYTRUDA plus chemotherapy resulted in superior overall survival compared with the current standard of care in the full study population and across all patient groups evaluated. These results build upon our research reinforcing the survival benefits of KEYTRUDA, and we look forward to engaging regulatory authorities as quickly as possible.

KEYTRUDA is currently approved in the U.S. and China as monotherapy for the second-line treatment of patients with recurrent locally advanced or metastatic squamous cell carcinoma of the esophagus whose tumors express PD-L1 (Combined Positive Score [CPS] 10). Merck is continuing to study KEYTRUDA across multiple settings and stages of gastrointestinal cancer including gastric, hepatobiliary, esophageal, pancreatic, colorectal and anal cancers through its broad clinical program.

About KEYNOTE-590

KEYNOTE-590 is a randomized, double-blind, Phase 3 trial (ClinicalTrials.gov, NCT03189719 ) evaluating KEYTRUDA in combination with chemotherapy compared with placebo plus chemotherapy for the first-line treatment of patients with locally advanced or metastatic esophageal carcinoma (adenocarcinoma or squamous cell carcinoma of the esophagus or Siewert type 1 adenocarcinoma of the esophagogastric junction). The primary endpoints are OS and PFS. The secondary endpoints include ORR, duration of response and safety. The study enrolled 749 patients who were randomized to receive:

About Esophageal Cancer

Esophageal cancer, a type of cancer that is particularly difficult to treat, begins in the inner layer (mucosa) of the esophagus and grows outward. The two main types of esophageal cancer are squamous cell carcinoma and adenocarcinoma. Esophageal cancer is the seventh most commonly diagnosed cancer and the sixth leading cause of death from cancer worldwide. Globally, it is estimated there were more than 572,000 new cases of esophageal cancer diagnosed and nearly 509,000 deaths resulting from the disease in 2018. In the U.S. alone, it is estimated there will be nearly 18,500 new cases of esophageal cancer diagnosed and more than 16,000 deaths resulting from the disease in 2020.

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 1,200 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

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 head and neck squamous cell carcinoma (HNSCC) with disease progression on or after platinum-containing chemotherapy.

Classical Hodgkin Lymphoma

KEYTRUDA is indicated for the treatment of adult and pediatric patients with refractory classical Hodgkin lymphoma (cHL), or who have relapsed after 3 or more prior lines 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 the confirmatory trials.

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. 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. 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 [combined positive score (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 (mut/Mb)] 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.

Selected Important Safety Information for KEYTRUDA

Immune-Mediated Pneumonitis

KEYTRUDA can cause immune-mediated pneumonitis, including fatal cases. Pneumonitis occurred in 3.4% (94/2799) of patients with various cancers receiving KEYTRUDA, including Grade 1 (0.8%), 2 (1.3%), 3 (0.9%), 4 (0.3%), and 5 (0.1%). Pneumonitis occurred in 8.2% (65/790) of NSCLC patients receiving KEYTRUDA as a single agent, including Grades 3-4 in 3.2% of patients, and occurred more frequently in patients with a history of prior thoracic radiation (17%) compared to those without (7.7%). Pneumonitis occurred in 6% (18/300) of HNSCC patients receiving KEYTRUDA as a single agent, including Grades 3-5 in 1.6% of patients, and occurred in 5.4% (15/276) of patients receiving KEYTRUDA in combination with platinum and FU as first-line therapy for advanced disease, including Grades 3-5 in 1.5% of patients.

Monitor patients for signs and symptoms of pneumonitis. Evaluate suspected pneumonitis with radiographic imaging. Administer corticosteroids for Grade 2 or greater pneumonitis. Withhold KEYTRUDA for Grade 2; permanently discontinue KEYTRUDA for Grade 3 or 4 or recurrent Grade 2 pneumonitis.

Immune-Mediated Colitis

KEYTRUDA can cause immune-mediated colitis. Colitis occurred in 1.7% (48/2799) of patients receiving KEYTRUDA, including Grade 2 (0.4%), 3 (1.1%), and 4 (<0.1%). Monitor patients for signs and symptoms of colitis. Administer corticosteroids for Grade 2 or greater colitis. Withhold KEYTRUDA for Grade 2 or 3; permanently discontinue KEYTRUDA for Grade 4 colitis.

Immune-Mediated Hepatitis (KEYTRUDA) and Hepatotoxicity (KEYTRUDA in Combination With Axitinib)

Immune-Mediated Hepatitis

KEYTRUDA can cause immune-mediated hepatitis. Hepatitis occurred in 0.7% (19/2799) of patients receiving KEYTRUDA, including Grade 2 (0.1%), 3 (0.4%), and 4 (<0.1%). Monitor patients for changes in liver function. Administer corticosteroids for Grade 2 or greater hepatitis and, based on severity of liver enzyme elevations, withhold or discontinue KEYTRUDA.

Hepatotoxicity in Combination With Axitinib

KEYTRUDA in combination with axitinib can cause hepatic toxicity with higher than expected frequencies of Grades 3 and 4 ALT and AST elevations compared to KEYTRUDA alone. With the combination of KEYTRUDA and axitinib, Grades 3 and 4 increased ALT (20%) and increased AST (13%) were seen. Monitor liver enzymes before initiation of and periodically throughout treatment. Consider more frequent monitoring of liver enzymes 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.

Immune-Mediated Endocrinopathies

KEYTRUDA can cause adrenal insufficiency (primary and secondary), hypophysitis, thyroid disorders, and type 1 diabetes mellitus. Adrenal insufficiency occurred in 0.8% (22/2799) of patients, including Grade 2 (0.3%), 3 (0.3%), and 4 (<0.1%). Hypophysitis occurred in 0.6% (17/2799) of patients, including Grade 2 (0.2%), 3 (0.3%), and 4 (<0.1%). Hypothyroidism occurred in 8.5% (237/2799) of patients, including Grade 2 (6.2%) and 3 (0.1%). The incidence of new or worsening hypothyroidism was higher in 1185 patients with HNSCC (16%) receiving KEYTRUDA, as a single agent or in combination with platinum and FU, including Grade 3 (0.3%) hypothyroidism. Hyperthyroidism occurred in 3.4% (96/2799) of patients, including Grade 2 (0.8%) and 3 (0.1%), and thyroiditis occurred in 0.6% (16/2799) of patients, including Grade 2 (0.3%). Type 1 diabetes mellitus, including diabetic ketoacidosis, occurred in 0.2% (6/2799) of patients.

Monitor patients for signs and symptoms of adrenal insufficiency, hypophysitis (including hypopituitarism), thyroid function (prior to and periodically during treatment), and hyperglycemia. For adrenal insufficiency or hypophysitis, administer corticosteroids and hormone replacement as clinically indicated. Withhold KEYTRUDA for Grade 2 adrenal insufficiency or hypophysitis and withhold or discontinue KEYTRUDA for Grade 3 or Grade 4 adrenal insufficiency or hypophysitis. Administer hormone replacement for hypothyroidism and manage hyperthyroidism with thionamides and beta-blockers as appropriate. Withhold or discontinue KEYTRUDA for Grade 3 or 4 hyperthyroidism. Administer insulin for type 1 diabetes, and withhold KEYTRUDA and administer antihyperglycemics in patients with severe hyperglycemia.

Immune-Mediated Nephritis and Renal Dysfunction

KEYTRUDA can cause immune-mediated nephritis. Nephritis occurred in 0.3% (9/2799) of patients receiving KEYTRUDA, including Grade 2 (0.1%), 3 (0.1%), and 4 (<0.1%) nephritis. Nephritis occurred in 1.7% (7/405) of patients receiving KEYTRUDA in combination with pemetrexed and platinum chemotherapy. Monitor patients for changes in renal function. Administer corticosteroids for Grade 2 or greater nephritis. Withhold KEYTRUDA for Grade 2; permanently discontinue for Grade 3 or 4 nephritis.

Immune-Mediated Skin Reactions

Immune-mediated rashes, including Stevens-Johnson syndrome (SJS), toxic epidermal necrolysis (TEN) (some cases with fatal outcome), exfoliative dermatitis, and bullous pemphigoid, can occur. Monitor patients for suspected severe skin reactions and based on the severity of the adverse reaction, withhold or permanently discontinue KEYTRUDA and administer corticosteroids. For signs or symptoms of SJS or TEN, withhold KEYTRUDA and refer the patient for specialized care for assessment and treatment. If SJS or TEN is confirmed, permanently discontinue KEYTRUDA.

Other Immune-Mediated Adverse Reactions

Immune-mediated adverse reactions, which may be severe or fatal, can occur in any organ system or tissue in patients receiving KEYTRUDA and may also occur after discontinuation of treatment. For suspected immune-mediated adverse reactions, ensure adequate evaluation to confirm etiology or exclude other causes. Based on the severity of the adverse reaction, withhold KEYTRUDA and administer corticosteroids. Upon improvement to Grade 1 or less, initiate corticosteroid taper and continue to taper over at least 1 month. Based on limited data from clinical studies in patients whose immune-related adverse reactions could not be controlled with corticosteroid use, administration of other systemic immunosuppressants can be considered. Resume KEYTRUDA when the adverse reaction remains at Grade 1 or less following corticosteroid taper. Permanently discontinue KEYTRUDA for any Grade 3 immune-mediated adverse reaction that recurs and for any life-threatening immune-mediated adverse reaction.

The following clinically significant immune-mediated adverse reactions occurred in less than 1% (unless otherwise indicated) of 2799 patients: arthritis (1.5%), uveitis, myositis, Guillain-Barr syndrome, myasthenia gravis, vasculitis, pancreatitis, hemolytic anemia, sarcoidosis, and encephalitis. In addition, myelitis and myocarditis were reported in other clinical trials, including classical Hodgkin lymphoma, and postmarketing use.

Treatment with KEYTRUDA may increase the risk of rejection in solid organ transplant recipients. Consider the benefit of treatment vs the risk of possible organ rejection in these patients.

Infusion-Related Reactions

KEYTRUDA can cause severe or life-threatening infusion-related reactions, including hypersensitivity and anaphylaxis, which have been reported in 0.2% (6/2799) of patients. Monitor patients for signs and symptoms of infusion-related reactions. For Grade 3 or 4 reactions, stop infusion and permanently discontinue KEYTRUDA.

Complications of Allogeneic Hematopoietic Stem Cell Transplantation (HSCT)

Immune-mediated complications, including fatal events, occurred in patients who underwent allogeneic HSCT after treatment with KEYTRUDA. Of 23 patients with cHL who proceeded to allogeneic HSCT after KEYTRUDA, 6 (26%) developed graft-versus-host disease (GVHD) (1 fatal case) and 2 (9%) developed severe hepatic veno-occlusive disease (VOD) after reduced-intensity conditioning (1 fatal case). Cases of fatal hyperacute GVHD after allogeneic HSCT have also been reported in patients with lymphoma who received a PD-1 receptorblocking antibody before transplantation. Follow patients closely for early evidence of transplant-related complications such as hyperacute graft-versus-host disease (GVHD), Grade 3 to 4 acute GVHD, steroid-requiring febrile syndrome, hepatic veno-occlusive disease (VOD), and other immune-mediated adverse reactions.

In patients with a history of allogeneic HSCT, acute GVHD (including fatal GVHD) has been reported after treatment with KEYTRUDA. Patients who experienced GVHD after their transplant procedure may be at increased risk for GVHD after KEYTRUDA. Consider the benefit of KEYTRUDA vs the risk of GVHD in these patients.

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 a PD-1 or PD-L1 blocking antibody 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-002, KEYTRUDA was permanently discontinued due to adverse reactions in 12% of 357 patients with advanced melanoma; the most common (1%) were general physical health deterioration (1%), asthenia (1%), dyspnea (1%), pneumonitis (1%), and generalized edema (1%). The most common adverse reactions were fatigue (43%), pruritus (28%), rash (24%), constipation (22%), nausea (22%), diarrhea (20%), and decreased appetite (20%).

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%).

In KEYNOTE-189, when KEYTRUDA was administered with pemetrexed and platinum chemotherapy in metastatic nonsquamous NSCLC, KEYTRUDA was discontinued due to adverse reactions in 20% of 405 patients. The most common adverse reactions resulting in permanent discontinuation of KEYTRUDA were pneumonitis (3%) and acute kidney injury (2%). The most common adverse reactions (20%) with KEYTRUDA were nausea (56%), fatigue (56%), constipation (35%), diarrhea (31%), decreased appetite (28%), rash (25%), vomiting (24%), cough (21%), dyspnea (21%), and pyrexia (20%).

In KEYNOTE-407, when KEYTRUDA was administered with carboplatin and either paclitaxel or paclitaxel protein-bound in metastatic squamous NSCLC, KEYTRUDA was discontinued due to adverse reactions in 15% of 101 patients. The most frequent serious adverse reactions reported in at least 2% of patients were febrile neutropenia, pneumonia, and urinary tract infection. Adverse reactions observed in KEYNOTE-407 were similar to those observed in KEYNOTE-189 with the exception that increased incidences of alopecia (47% vs 36%) and peripheral neuropathy (31% vs 25%) were observed in the KEYTRUDA and chemotherapy arm compared to the placebo and chemotherapy arm in KEYNOTE-407.

In KEYNOTE-042, KEYTRUDA was discontinued due to adverse reactions in 19% of 636 patients with advanced NSCLC; the most common were pneumonitis (3%), death due to unknown cause (1.6%), and pneumonia (1.4%). The most frequent serious adverse reactions reported in at least 2% of patients were pneumonia (7%), pneumonitis (3.9%), pulmonary embolism (2.4%), and pleural effusion (2.2%). The most common adverse reaction (20%) was fatigue (25%).

In KEYNOTE-010, KEYTRUDA monotherapy was discontinued due to adverse reactions in 8% of 682 patients with metastatic NSCLC; the most common was pneumonitis (1.8%). The most common adverse reactions (20%) were decreased appetite (25%), fatigue (25%), dyspnea (23%), and nausea (20%).

Adverse reactions occurring in patients with SCLC were similar to those occurring in patients with other solid tumors who received KEYTRUDA as a single agent.

In KEYNOTE-048, KEYTRUDA monotherapy was discontinued due to adverse events in 12% of 300 patients with HNSCC; the most common adverse reactions leading to permanent discontinuation were sepsis (1.7%) and pneumonia (1.3%). The most common adverse reactions (20%) were fatigue (33%), constipation (20%), and rash (20%).

In KEYNOTE-048, when KEYTRUDA was administered in combination with platinum (cisplatin or carboplatin) and FU chemotherapy, KEYTRUDA was discontinued due to adverse reactions in 16% of 276 patients with HNSCC. The most common adverse reactions resulting in permanent discontinuation of KEYTRUDA were pneumonia (2.5%), pneumonitis (1.8%), and septic shock (1.4%). The most common adverse reactions (20%) were nausea (51%), fatigue (49%), constipation (37%), vomiting (32%), mucosal inflammation (31%), diarrhea (29%), decreased appetite (29%), stomatitis (26%), and cough (22%).

In KEYNOTE-012, KEYTRUDA was discontinued due to adverse reactions in 17% of 192 patients with HNSCC. Serious adverse reactions occurred in 45% of patients. The most frequent serious adverse reactions reported in at least 2% of patients were pneumonia, dyspnea, confusional state, vomiting, pleural effusion, and respiratory failure. The most common adverse reactions (20%) were fatigue, decreased appetite, and dyspnea. Adverse reactions occurring in patients with HNSCC were generally similar to those occurring in patients with melanoma or NSCLC who received KEYTRUDA as a monotherapy, with the exception of increased incidences of facial edema and new or worsening hypothyroidism.

In KEYNOTE-087, KEYTRUDA was discontinued due to adverse reactions in 5% of 210 patients with cHL. Serious adverse reactions occurred in 16% of patients; those 1% included pneumonia, pneumonitis, pyrexia, dyspnea, GVHD, and herpes zoster. Two patients died from causes other than disease progression; 1 from GVHD after subsequent allogeneic HSCT and 1 from septic shock. The most common adverse reactions (20%) were fatigue (26%), pyrexia (24%), cough (24%), musculoskeletal pain (21%), diarrhea (20%), and rash (20%).

In KEYNOTE-170, KEYTRUDA was discontinued due to adverse reactions in 8% of 53 patients with PMBCL. Serious adverse reactions occurred in 26% of patients and included arrhythmia (4%), cardiac tamponade (2%), myocardial infarction (2%), pericardial effusion (2%), and pericarditis (2%). Six (11%) patients died within 30 days of start of treatment. The most common adverse reactions (20%) were musculoskeletal pain (30%), upper respiratory tract infection and pyrexia (28% each), cough (26%), fatigue (23%), and dyspnea (21%).

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Merck's KEYTRUDA (pembrolizumab) in Combination With Chemotherapy Significantly Improved Overall Survival and Progression-Free Survival Compared With...

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)

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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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.

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 an Analyst Brief, our team will help you create a revenue impact solution to achieve your desired goal.

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.

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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.

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.

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.

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Exosome Therapeutic Market (Covid 19 Impact Analysis) Data Highlighting Major Vendors, Promising Regions, Anticipated Growth Forecast To 2027 -...

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.

Know the Growth Opportunities in Emerging Markets

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.

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.

The regional analysis covers:

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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 Landscape Assessment By Type and Analysis Current Trends by Forecast To 2025 - The Daily Chronicle

August 17, 2020

Denver is known for its relatively mild climate and its four distinct seasons. Its also known for its temperature fluctuations over the course of a day or even hours. But what does that mean for the citys residents and for that matter, the rest of the inhabitants of the continental United States when it comes to temperature extremes?

Thats what Ashley Broadbentwanted to know. Specifically, he wanted to know how populations throughout the United States will experience heat and cold during the 21st century.

So, Broadbent, an assistant research professor in Arizona State Universitys School of Geographical Sciences and Urban Planning, used state-of-the-art modeling tools to analyze how three key variables would affect human exposure to extreme temperatures from the beginning of this century to its end.

He and his collaborator Matei Georgescu, an associate professor in the School of Geographical Sciences and Urban Planning, concentrated on the following three key factors: climate change brought about by greenhouse gas emissions, urban development-induced impacts arising from the growth of cities, and population change in individual cities.

The paper, "The motley drivers of heat and cold exposure in 21st century U.S. cities," was published onlineAug. 17 in the Proceedings of the National Academy of Sciences. It is the first study of its kind to consider population-weighted heat and cold exposure that directly and simultaneously account for greenhouse gas and urban development-induced warming.

Graphic by Alex Davis/ASU Media Relations and Strategic Communications

To describe how these three variables would affect temperatures, and in turn populations, Broadbent, Georgescu and co-author Eric Scott Krayenhoff, assistant professor at the University of Guelph, Ontario, in Canada, used a metric they dubbed person-hours, to describe humans exposure to extreme heat and cold.

Its an intuitive metric, Broadbent said. For example, when one person is exposed to one hour of an extreme temperature, that exposure equals one person-hour of exposure. Likewise, if 10 people are exposed to 10 hours of an extreme temperature, that exposure equals 100 person-hours.

I think this definition is more representative of what people experience, which is what this study is about versus a study that simply communicates temperature changes without any human element attached to it, Broadbent said.

Overall, the researchers found that the average annual heat exposure at the start of this century in the United States was about 5.2 billion person-hours. Assuming a worst-case scenario of peak global warming, population growth and urban development, the annual heat exposure would rise to 150 billion person-hours by the end of the century, a nearly 30-fold increase.

The combined effect of these three drivers will substantially increase the average heat exposure across the United States, but heat exposure is not projected to increase uniformly in all cities across the U.S., Broadbent said. There will be hot spots where heat exposure grows sharply.

To that end, the researchers defined heat thresholds based on local city definitions, something previous studies have not done. Instead, prior studies have used fixed-temperature thresholds that may be inappropriate for some cities. Afterall, a 90-degree day in Phoenix feels much different than a 90-degree day in New York City, given relative humidity differences.

Its well-known that cities have locally defined thresholds where heat and cold cause mortality and morbidity, Broadbent explained. In other words, people die at different temperatures in different cities because what is extreme in one city may be normal in another.

Importantly, areas of the United States where human exposure would increase the most is where climate change and population increase in tandem. Meanwhile, urban development has a smaller, yet not negligible effect.

According to the results of the study, the largest absolute changes in population heat exposure are projected to occur in major U.S. metropolitan regions, such as New York, Los Angeles and Atlanta.

The study also finds the largest relativechanges in person-hours related to heat exposure are projected to occur in rapidly growing cities located in the Sun Belt, including Austin, Texas; Orlando, Florida; and Atlanta.

The increase in exposure is quite large if you look at it relative to the start of the century, Broadbent said. Some cities across the Sun Belt, according to our projections, will have 90 times the number of person-hours of heat exposure. For example, cities in Texas that see substantial population growth and strong greenhouse gas-induced climate warming could be markedly affected.

One way to prepare for increased heat exposure is to reduce greenhouse gas emissions on a global scale, which would reduce the number of hours people are exposed to extreme temperatures. Other options include localized infrastructure adaptation that provides buffering effects against rising temperatures such as planting trees, providing shade and cooling areas and constructing buildings using materials that absorb less heat.

Although the average temperature in the United States will be warmer in the future, the study finds that cold exposure will increase slightly compared with the start of the century, primarily because of population growth. While there is a generaldecreasein the number of projected extreme cold events by the end of this century, the number of individuals exposed to extreme cold is projected toincrease,as population growth means that the total number of person-hours of cold exposure will go up, Broadbent said.

Cold is currently more of a national health problem than heat, but our results suggest that by the end of the century heat exposure may become a larger health problem than cold exposure, Broadbent said. However, cold exposure will not disappear completely as the climate warms. In fact, according to one of the teams simulations, Denver is projected to have more extreme cold at the end of the century compared with the beginning, according to the study.

Thats the interesting thing about climate change. We know the average temperature is going to increase, said Broadbent. But we know less about how the extremes are going to change, and often the extremes are the most important part of our daily lives.

There are several takeaway messages from this work, but one of the central ones concerns the future resiliency of our cities, Georgescu said.

The successful steps taken will require holistic thinking that embraces contributions from urban planners, engineers, social scientists and climate scientists with a long-range vision of how we want our cities to be.

"We therefore call on cities to start asking some very foundational questions regarding the projected exposure of their constituents to future environmental change," Georgescu said. "Is the work of the urban climate modeling community being integrated into their environmental adaptation plans? If so, how, and if not, why not?

This work was funded by the National Science Foundation.

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ASU engineers get to the heart of organs-on-a-chip - ASU Now

CAMBRIDGE, Mass.--(BUSINESS WIRE)--bluebird bio, Inc. (Nasdaq: BLUE) today announced that new data from the clinical development program for its investigational elivaldogene autotemcel (eli-cel, Lenti-D) gene therapy in patients with cerebral adrenoleukodystrophy (CALD), including data from the Phase 2/3 Starbeam study (ALD-102) and available data from the Phase 3 ALD-104 study, will be presented at the 46th Annual Meeting of the European Society for Blood and Marrow Transplantation (EBMT 2020), taking place virtually from August 29 - September 1, 2020.

New Cerebral Adrenoleukodystrophy (CALD) Data at EBMT 2020

Lenti-D hematopoietic stem cell gene therapy stabilizes neurologic function in boys with cerebral adrenoleukodystrophy (ALD-102 and ALD-104)Presenting Author: Dr. Jrn-Sven Khl, Department of Pediatric Oncology, Hematology and Hemostaseology, Center for Womens and Childrens Medicine, University Hospital LeipzigPoster Session & Number: Gene Therapy; ePoster O077

Additional bluebird bio data at EBMT 2020 includes encore presentations from the companys CALD, sickle cell disease (SCD), transfusion-dependent -thalassemia (TDT) and multiple myeloma programs.

Cerebral Adrenoleukodystrophy (CALD) Encore Data at EBMT 2020

Outcomes of allogeneic hematopoietic stem cell transplant in patients with cerebral adrenoleukodystrophy vary by donor cell source, conditioning regimen, and stage of cerebral disease status (ALD-103)Presenting Author: Dr. Jaap Jan Boelens, Chief, Pediatric Stem Cell Transplantation and Cellular Therapies Service, Memorial Sloan Kettering Cancer CenterPoster Session & Number: Haemoglobinopathy and inborn errors; ePoster O106

Multiple Myeloma Correlative Encore Data at EBMT 2020

Markers of initial and long-term responses to idecabtagene vicleucel (ide-cel; bb2121) in the CRB-401 study in relapsed/refractory multiple myelomaPresenting Author: Dr. Ethan G. Thompson, Bristol Myers SquibbPoster Session & Number: CAR-based Cellular Therapy clinical; ePoster A089

Sickle Cell Disease (SCD) Encore Data at EBMT 2020

LentiGlobin for sickle cell disease (SCD) gene therapy (GT): updated results in Group C patients from the Phase 1/2 HGB-206 studyPresenting Author: Dr. Markus Y. Mapara, Director, Adult Blood and Marrow Transplantation Program, Columbia University Medical CenterOral Session & Number: Inborn Errors; O080Date & Time: September 1, 2020; 4:35 4:42 PM CET/10:35 10:42 AM ET

Transfusion-Dependent -Thalassemia (TDT) Encore Data at EBMT 2020

Clinical outcomes following autologous hematopoietic stem cell transplantation with LentiGlobin gene therapy in the Phase 3 Northstar-2 and Northstar-3 studies for transfusion-dependent -thalassemiaPresenting Author: Professor Franco Locatelli, Director, Department of Pediatric Hematology and Oncology, Ospedale Pediatrico Bambino GesPoster Session & Number: Gene Therapy; ePoster O074

LentiGlobin gene therapy treatment of two patients with transfusion-dependent -thalassemia (case report)Presenting Author: Dr. Mattia Algeri, Department of Pediatric Oncohematology - Transplantation Unit and Cell Therapies, Ospedale Pediatrico Bambino GesPoster Session & Number: Haemoglobinopathy and inborn errors; ePoster A328

Cross Indication Encore Data at EBMT 2020

Safety of autologous hematopoietic stem cell transplantation with gene addition therapy for transfusion-dependent -thalassemia, sickle cell disease, and cerebral adrenoleukodystrophyPresenting Author: Dr. Evangelia Yannaki, Director, Gene and Cell Therapy Center, Hematology Department, George Papanicolaou HospitalPoster Session & Number: Gene Therapy; ePoster O078

Abstracts outlining bluebird bios accepted data at EBMT 2020 are available on the Annual Meeting website. On August 29, 2020, at 12:30 PM CET/6:30 AM ET, the embargo will lift for ePosters and oral presentations accepted for EBMT 2020. Presentations will be available for virtual viewing throughout the duration of the live meeting and content will be accessible online following the close of the meeting until November 1, 2020.

About elivaldogene autotemcel (eli-cel, Lenti-D gene therapy)In July 2020, the Committee for Medicinal Products for Human Use (CHMP) of the European Medicines Agency (EMA) granted an accelerated assessment to eli-cel gene therapy for cerebral adrenoleukodystrophy (CALD). bluebird bio is currently on track to submit the Marketing Authorization Application (MAA) in the EU for eli-cel for CALD by year-end 2020, and the Biologics License Application (BLA) in the U.S. in mid-2021.

bluebird bio is currently enrolling patients for a Phase 3 study (ALD-104) designed to assess the efficacy and safety of eli-cel after myeloablative conditioning using busulfan and fludarabine in patients with CALD. Contact clinicaltrials@bluebirdbio.com for more information and a list of study sites.

Additionally, bluebird bio is conducting a long-term safety and efficacy follow-up study (LTF-304) for patients who have been treated with eli-cel for CALD and completed two years of follow-up in bluebird bio-sponsored studies.

The Phase 2/3 Starbeam study (ALD-102) has completed enrollment. For more information about the ALD-102 study visit: http://www.bluebirdbio.com/our-science/clinical-trials or clinicaltrials.gov and use identifier NCT01896102.

Adrenoleukodystrophy (ALD) is a rare, X-linked metabolic disorder that is estimated to affect one in 21,000 male newborns worldwide. Approximately 40 percent of boys with ALD will develop CALD, the most severe form of ALD. CALD is a progressive neurogenerative disease that involves breakdown of myelin, the protective sheath of the nerve cells in the brain that are responsible for thinking and muscle control. Symptoms of CALD usually occur in early childhood and progress rapidly, if untreated, leading to severe loss of neurologic function, and eventual death, in most patients.

The European Medicines Agency (EMA) accepted eli-cel gene therapy for the treatment of CALD into its Priorities Medicines scheme (PRIME) in July 2018, and previously granted Orphan Medicinal Product designation to eli-cel.

The U.S. Food and Drug Administration (FDA) granted eli-cel Orphan Drug status, Rare Pediatric Disease designation, and Breakthrough Therapy designation for the treatment of CALD.

Eli-cel is not approved for any indication in any geography.

About idecabtagene vicleucel (ide-cel; bb2121)Ide-cel is a B-cell maturation antigen (BCMA)-directed genetically modified autologous chimeric antigen receptor (CAR) T cell immunotherapy. The ide-cel CAR is comprised of a murine extracellular single-chain variable fragment (scFv) specific for recognizing BCMA, attached to a human CD8 hinge and transmembrane domain fused to the T cell cytoplasmic signaling domains of CD137 4-1BB and CD3- chain, in tandem. Ide-cel recognizes and binds to BCMA on the surface of multiple myeloma cells leading to CAR T cell proliferation, cytokine secretion, and subsequent cytolytic killing of BCMA-expressing cells.

In addition to the pivotal KarMMa trial evaluating ide-cel in patients with relapsed and refractory multiple myeloma, bluebird bio and Bristol Myers Squibbs broad clinical development program for ide-cel includes clinical studies (KarMMa-2, KarMMa-3, KarMMa-4) in earlier lines of treatment for patients with multiple myeloma, including newly diagnosed multiple myeloma. For more information visit clinicaltrials.gov.

In July 2020, Bristol Myers Squibb (BMS) and bluebird bio submitted the Biologics License Application for ide-cel to the U.S. Food and Drug Administration for the treatment of adult patients with multiple myeloma who have received at least three prior therapies, including an immunomodulatory agent, a proteasome inhibitor and an anti-CD38 antibody. Ide-cel is the first CAR T cell therapy submitted for regulatory review to target BCMA and for multiple myeloma.

Ide-cel was granted Breakthrough Therapy Designation (BTD) by the U.S. Food and Drug Administration (FDA) and PRIority Medicines (PRIME) designation, as well as Accelerated Assessment status, by the European Medicines Agency for relapsed and refractory multiple myeloma.

Ide-cel is being developed as part of a Co-Development, Co-Promotion and Profit Share Agreement between BMS and bluebird bio.

Ide-cel is not approved for any indication in any geography.

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

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 betibeglogene autotemcel and LentiGlobin for SCD. For more information visit: https://www.bluebirdbio.com/our-science/clinical-trials or clinicaltrials.gov and use identifier NCT02633943 for LTF-303.

SCD is a serious, progressive and debilitating genetic disease caused by a mutation in the -globin gene that leads to the production of abnormal sickle hemoglobin (HbS). HbS causes red blood cells (RBCs) to become sickled and fragile, resulting in chronic hemolytic anemia, vasculopathy and painful vaso-occlusive crises (VOCs). For adults and children living with SCD, this means painful crises and other life-altering or life-threatening acute complicationssuch as acute chest syndrome (ACS), stroke and infections. If patients survive the acute complications, vasculopathy and end-organ damage, resulting complications can lead to pulmonary hypertension, renal failure and early death; in the U.S. the median age of death for someone with sickle cell disease is 43 - 46 years.

LentiGlobin for SCD received Orphan Medicinal Product designation from the European Commission for the treatment of SCD.

The U.S. Food and Drug Administration (FDA) granted Orphan Drug status and Regenerative Medicine Advanced Therapy (RMAT) designation and rare pediatric disease designation for LentiGlobin for the treatment of SCD.

bluebird bio reached general agreement with the U.S. Food and Drug Administration (FDA) that the clinical data package required to support a Biologics Licensing Application (BLA) submission for LentiGlobin for SCD will be based on data from a portion of patients in the HGB-206 study Group C that have already been treated. The planned submission will be based on an analysis using complete resolution of severe vaso-occlusive events (VOEs) as the primary endpoint with at least 18 months of follow-up post-treatment with LentiGlobin for SCD. Globin response will be used as a key secondary endpoint.

bluebird bio anticipates additional guidance from the FDA regarding the commercial manufacturing process, including suspension lentiviral vector. bluebird bio announced in a May 11, 2020 press release it plans to seek an accelerated approval and expects to submit the U.S. BLA for SCD in the second half of 2021.

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

About betibeglogene autotemcel (beti-cel; formerly LentiGlobin gene therapy for -thalassemia)The European Commission granted conditional marketing authorization (CMA) for betibeglogene autotemcel, 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. On April 28, 2020, the European Medicines Agency (EMA) renewed the CMA for ZYNTEGLO, supported by data from 32 patients treated with ZYNTEGLO, including three patients with up to five years of follow-up.

In the HGB-207 clinical study supporting the conditional marketing approval of ZYNTEGLO, the primary endpoint was transfusion independence (TI) by Month 24, defined as a weighted average Hb 9 g/Dl without any RBC transfusions for a continuous period of 12 months at any time during the study after infusion of ZYNTEGLO. Ten patients were evaluable for assessment of TI. Of these, 9/10 (90.0%, 95% CI 55.5-99.7%) achieved TI at last follow-up. Among these nine patients, the median (min, max) weighted average Hb during TI was 12.22 (11.4, 12.8) g/dLl.

TDT is a severe genetic disease caused by mutations in the -globin gene that result in reduced or significantly reduced hemoglobin (Hb). In order to survive, people with TDT maintain Hb levels through lifelong chronic blood transfusions. These transfusions carry the risk of progressive multi-organ damage due to unavoidable iron overload.

Beti-cel 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 hemoglobin, at levels that may eliminate or significantly reduce the need for transfusions.

Non-serious adverse events (AEs) observed during the clinical studies that were attributed to betibeglogene autotemcel included abdominal pain, thrombocytopenia, leukopenia, neutropenia, hot flush, dyspnoea, pain in extremity, and non-cardiac chest pain. Two serious adverse events (SAE) of thrombocytopenia were 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.

The CMA for beti-cel is valid in the 27 member states of the EU as well as UK, Iceland, Liechtenstein and Norway. For details, please see the Summary of Product Characteristics (SmPC).

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 United States.

Beti-cel continues to be evaluated in the ongoing Phase 3 Northstar-2 and Northstar-3 studies. For more information about the ongoing clinical studies, visit http://www.northstarclinicalstudies.com or clinicaltrials.gov and use identifier NCT02906202 for Northstar-2 (HGB-207), NCT03207009 for Northstar-3 (HGB-212).

About bluebird bio, Inc.bluebird bio is pioneering gene therapy with purpose. From our Cambridge, Mass., headquarters, were developing gene 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 including cerebral adrenoleukodystrophy, sickle cell disease, -thalassemia and multiple myeloma, using three gene therapy technologies: gene addition, cell therapy 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.

Lenti-D and bluebird bio are trademarks of bluebird bio, Inc.

Forward-Looking StatementsThis release contains forward-looking statements within the meaning of the Private Securities Litigation Reform Act of 1995, including statements regarding the companys financial condition, results of operations, as well as statements regarding the plans for regulatory submissions for beti-cel (marketed as ZYTENGLO in the European Union), eli-cel, ide-cel, and LentiGlobin for SCD, including anticipated endpoints to support regulatory submissions and timing expectations; the companys expectations regarding the potential for the suspension manufacturing process for lentiviral vector; its expectations for commercialization efforts for ZYNTEGLO in Europe; as well as the companys intentions regarding the timing for providing further updates on the development and commercialization of ZYNTEGLO and the companys product candidates. 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. These risks and uncertainties include, but are not limited to, the risks that the COVID-19 pandemic and resulting economic conditions will have a greater impact on the companys operations and plans than anticipated; that our amended collaboration with BMS will not continue or be successful; that preliminary positive efficacy and safety results from our prior and ongoing clinical trials will not continue or be repeated in our ongoing or future clinical trials; the risk that our plans for submitting a BLA for LentiGlobin for SCD may be delayed if the FDA does not accept our comparability plans for the use of the suspension manufacturing process for lentiviral vector; the risk that the submission of BLA for ide-cel is not accepted for filing by the FDA or approved in the timeline we expect, or at all; the risk of cessation or delay of any of the ongoing or planned clinical studies and/or our development of our product candidates, including due to delays from the COVID-19 pandemics impact on healthcare systems; the risk that the current or planned clinical trials of our product candidates will be insufficient to support regulatory submissions or marketing approval in the United States and European Union; the risk that regulatory authorities will require additional information regarding our product candidates, resulting in delay to our anticipated timelines for regulatory submissions, including our applications for marketing approval; the risk that we will encounter challenges in the commercial launch of ZYNTEGLO in the European Union, including in managing our complex supply chain for the delivery of drug product, in the adoption of value-based payment models, or in obtaining sufficient coverage or reimbursement for our products; and the risk that any one or more of our product candidates, will not be successfully developed, approved or commercialized. 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-K, 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.

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bluebird bio to Present New Data from Clinical Studies of elivaldogene autotemcel (eli-cel, Lenti-D) Gene Therapy for Cerebral Adrenoleukodystrophy...

Dear EditorExcellent review and so needed and well-timedThe only issue that did not get the attention it needs are the neuropsychiatric symptoms of mild COVID-19. This is important for medical professionals to know, to avoid labeling the patients' problems as psychiatric and even hysterical as some recently did in a major newspaper here in Belgium.There are two sides to the mental sequelae of mild COVID.a) the consequences of the impact of going through a global pandemic, of lockdown, of COVID patients in their immediate environment, of the fear of infection or infecting others, of losing their job, and finally of their own infection.b) the mental symptoms of an organic disorder.In the subject literature about COIVD-19 (and MERS, SARS and other infections) several mechanisms are mentioned.-A direct neurotropic impact of the virus, especially, but not only via ACE2, both in neurons and glial cells, especially targeting the brain stem which plays a role in emotions. and brought there, among other things, via the direct connection of the olfactory bulb.-Inflammatory and immune reactions that result in cognitive and psychiatric symptoms:(the "misty brain" cited by many patients)-Reactions of the autonomic nervous system, eg cardiac arrhythmias can also be very scary.-Alteration of the gas exchange -oxygen nd carbon dioxide- due to damage to the alveoli resulting in a suboptimal pH.These results in mental symptoms of an organic disorder: memory problems, word finding disorders, confusion, major sleeping problems, insecure motor skills, anorexia, etc. and of course very often chronic fatigue, muscle weakness and anxiety.Of course, fear or anger of the patient are amplified when the doctor labels this as purely psychological, while the patient who has never been ill before, clearly experiences its not.

Because we have only known the disease for six months and we still know so little about it, it is therefore better to take the experiences of the patients seriously, instead of brushing them off as purely psychological or psychiatric.

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Re: Management of post-acute covid-19 in primary care - The BMJ

Shahid Akhter, editor, ETHealthworld, spoke to. Dr A. Velumani, Promoter, Chairman, Managing Director and Chief Executive Officer, Thyrocare Technologies, to know more about the challenges and opportunities associated with Covid diagnostics business.

Covid-19 : ChallengesBecause of the lockdown, the existing non-covid healthcare and diagnostics business collapsed. It collapsed to 2% suddenly within a week and it didn't allow it to resume for three months.Multiple advisory's multiple tests, whom to test and whom not to, along with plenty of show cause notice because a lot of administrators wanted to under-report positivity and some probably wanted the professional gains, so a lot of time they took in the review. These were the challenges but there were a lot of opportunities as well.

Covid-19: LearningsIn my opinion, Lockdown is not the solution, repeating lockdowns, having every different strict guideline for every different state is not the solution. It won't help to reduce. Secondly, Rapid antigen kits are useless, it doesn't solve anything so we've learned the RT-PCR is the most reliable one in the covid diagnosis.Covid-19: Government's InitiativeAlso, the government labs have contributed significantly which wasn't expected, we were all thinking it is just the private labs who are truly scaling up but government labs too scaled up and contributed more in more than 50% of the testing.

Covid-19: Immunity and antibodyImmunity matters, I don't think lockdown can stop Covid. It is the antibodies that can stop the Covid and India is blessed as only 30% tests are there per million whereas in the US there are 600 tests per million. The antibody power is important to be seen as well if not then there is a problem.

Covid-19: Towards a new normalWork from home will continue, even in healthcare, it is just 17% which is working from home. Also, there will be two different religions in healthcare that will be Covid and Non-Covid so that infection will not pass on to one covid patient to another and non-covid will not move to covid hospitals. The spending on hygiene needs to be high because the general population is scared, medical doctors are scared and the patients are scared.

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RT-PCR is the most reliable test in the covid diagnosis: Dr A. Velumani, CEO, Thyrocare Technologies Ltd. - ETHealthworld.com

London: In a major study, researchers have found that Covid-19 patients with cardiovascular comorbidities or risk factors are more likely to develop heart complications while hospitalised, and more likely to die from the virus.

According to the study, published in the journal PLOS ONE, it is crucial for clinicians working with cardiovascular patients to understand the clinical presentation and risk factors for Covid-19 infection in this group.

"For most people, the Novel Coronavirus Disease 2019 (Covid-19) causes mild illness, however, it can generate severe pneumonia and lead to death in others," said study authors from the Magna Graecia University in Italy.

At the time they were admitted to the hospital, 12.89 per cent of the patients had cardiovascular comorbidities, 36.08 per cent had hypertension and 19.45 per cent had diabetes.

The findings showed that cardiovascular complications were documented during the hospital stay of 14.09 per cent of Covid-19 patients.

According to the researchers, the most common of these complications were arrhythmias or palpitations; significant numbers of patients also had myocardial injury.

Myocardial injury is considered acute if there is a rise and fall of cardiac troponin concentrations exceeding biological and analytical variation.

When the researchers analysed the data, they found that pre-existing cardiovascular comorbidities or risk factors were significant predictors of cardiovascular complications, but age and gender were not.

The study showed that both age and pre-existing cardiovascular comorbidities or risk factors were significant predictors of death.

"Cardiovascular complications are frequent among Covid-19 patients and might contribute to adverse clinical events and mortality," the study author concluded.

Link:
Covid-19 patients with heart problems more likely to die: Study - ETHealthworld.com

Global Stem Cell Therapy Market is expected to reach an approximate CAGR of 10.3% during the forecast period. The use of stem cell for treating medical conditions is referred to as stem cell therapy. Stem cells are undifferentiated cells and differentiate into specialized cell types.

This ability of stem cells to differentiate into cells of interest is used to treat diseases like diabetes, heart disease, hematopoietic disorders (for example leukemia, thalassemia, and others), degenerative disorders (osteoarthritis, Alzheimers disease, Parkinsons disease, chronic renal failure, congestive cardiac failure,) and others.

Some of the key players are Osiris Therapeutics, Inc. (US), MEDIPOST Co., Ltd. (South Korea), Anterogen Co., Ltd. (South Korea), Pharmicell Co., Ltd. (South Korea), Holostem Terapie Avanzate S.r.l. (Italy), JCR Pharmaceuticals Co., Ltd. (Japan), NuVasive, Inc. (US), RTI Surgical, Inc. (US), and AlloSource (US), Thermo Fisher Scientific are some of the key players operating in the global stem cell therapy market.

Avail Sample copy For this Report at: https://www.marketresearchfuture.com/sample_request/6422

Global Stem Cell Therapy Market, by Technique,

Global Stem Cell Therapy Market, by Product Type

Global Stem Cell Therapy Market, by Application

Global Stem Cell Therapy Market, by End-User

Geographically, Americas is the largest in the market owing to the increasing prevalence of heart diseases and growing healthcare expenditure. According to the Centers for Disease Control and Prevention in November 2017, report every year 735,000 Americans have a heart problem. Such a high number of heart patients in the Americas drives the market growth in this region.

Brows Full Report at: https://www.marketresearchfuture.com/reports/stem-cell-therapy-market-6422

Europe (UK, Belgium, France, and Netherlands) is the second largest global stem cell therapy market during the forecast period. The increasing occurrence of stroke, cancer, and osteoarthritis drives the market in this region. According to Anthony Nolan organization 2017, annual review 1.4million people register for donating stem cell in 2017. Also, more than 2,200 searches for a lifesaving stem cell transplant were made in 2017 by UK people. Such a high demand for Stem cell transplantation in this region promotes the market.

Asia-Pacific was projected to be the fastest growing region for the global stem cell therapy market in 2017. The market is expected to witness growth owing to the rising prevalence of smoking in this region.

According to the American Cancer Society, Inc 2018, report China 48.9%, India 16.2%, Japan 11.2% accounts of cancer cases in this region. Such a high cancer rate in this region favors the stem cell therapy market in this region.

The Middle East and Africa accounts for the least share due to low per capita income and lack of availability of well-trained healthcare professionals. However, the rising oncology and technology both at the hospital level and in the community are expected to influence the market in a positive way.

Enquire Discount for this Report at: https://www.marketresearchfuture.com/check-discount/6422

Key factors responsible for the market growth are the rising awareness for therapeutic application of stem cells in disease management, rising research for stem cell therapy applications, development of advanced genetic analysis techniques, increasing public-private investments for stem cell research, growing research in identification of new stem cell lines, and new developments in stem cell banking infrastructure are driving the growth of the global stem cell therapy market. Stem cells are used in the treatment of Alzheimers by replacing the diseased cells with stem cells

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Stem Cell Therapy Market Application Growth, Technology, Trends and Key Players Developments on Regional Industry Size Till 2023 - eRealty Express

This briefing has ended. Read live coronavirus updates here.

At least 97,000 children in the United States tested positive for the coronavirus the last two weeks of July alone, according to a new report from the American Academy of Pediatrics and the Childrens Hospital Association. The report says that at least 338,000 children have tested positive since the pandemic began, meaning more than a quarter have tested positive in just those two weeks.

The report comes as parents and education leaders grapple with the challenges of resuming schooling as the virus continues to surge in parts of the country.

More than seven out of 10 infections were from states in the South and West, according to the report, which relied on data from 49 states along with Washington, D.C., Puerto Rico and Guam. The count could be higher because the report did not include complete data from Texas and information from parts of New York State outside of New York City.

Missouri, Oklahoma, Alaska, Nevada, Idaho and Montana were among the states with the highest percent increase of child infections during that period, according to the report.

New York City, New Jersey and other states in the Northeast, where the virus peaked in March and April, had the lowest percent increase of child infections, according to the report.

In total, 338,982 children have been infected, according to the report.

Not every locality where data was collected categorized children in the same age range. Most places cited in the report considered children to be people no older than 17 or 19. In Alabama, though, the age limit was 24; in Florida and Utah the age limit was 14.

The report noted that children rarely get severely sick from Covid-19, but another report, from the Centers for Disease Control and Prevention, highlighted how the threat from a new Covid-19-related condition, called Multisystem Inflammatory Syndrome in Children or MIS-C, has disproportionately affected people of color.

The C.D.C. said that from early March through late July, it received reports of 570 young people ranging from infants to age 20 who met the definition of MIS-C. Most of those patients were previously healthy, the report said.

About 40 percent were Hispanic or Latino; 33 percent were Black and 13 percent were white, the report said. Ten died and nearly two-thirds were admitted to intensive care units, it said. Symptoms include a fever, rash, pinkeye, stomach distress, confusion, bluish lips, muscle weakness, racing heart rate and cardiac shock.

A reopened high school in Georgia that drew national attention over images of its crowded hallways has had at least nine coronavirus cases reported in the last week, and is switching to online-only instruction for at least the next two days while the school is disinfected and officials assess the situation.

At this time, we know there were six students and three staff members who were in school for at least some time last week who have since reported to us that they have tested positive, Gabe Carmona, the principal of North Paulding High School in Dallas, Ga., said in a letter to parents and guardians of the schools students on Saturday.

The superintendent of the Paulding County School District, Brian Otott, sent them another letter Sunday advising them about the switch to online instruction for Monday and Tuesday, at the least.

Both letters encouraged parents to check their childrens temperature twice daily and to monitor them for symptoms. Neither letter made any mention of social distancing or wearing masks, which the school has said are encouraged but not mandatory.

Photos circulated widely online last week showed North Paulding students crowded in a school hallway with few in masks. Hannah Watters, a 15-year-old student who posted one of the images, was initially suspended for doing so but the suspension was rescinded.

After the photo spread over social media, Mr. Otott said masks were not required at the school, which has about 2,000 students, because there is no practical way to enforce a mandate to wear them.

But Dr. Gary Voccio, the health director for Paulding County and nine other counties in Northwest Georgia, said that masks were crucial to containing the spread of the virus.

Of course you have to change classrooms, and its going to be very difficult to physically distance when that occurs, Dr. Voccio said in a video posted on Facebook. But the masks, again, are the important part of this problem. And we will have significant cases within the schools, Im sure.

At least 66 new coronavirus deaths and 4,032 new cases were reported in Georgia on Saturday, according to a New York Times database.

Governor DeWine got the positive result when he was screened before President Trump arrived in Ohio for campaign appearances.

That test was an antigen test manufactured by the diagnostic health care company Quidel, one of two such tests given emergency use authorization by the Food and Drug Administration. These tests, while fast and convenient, are known to be less accurate than PCR tests, which were used to retest Governor DeWine twice on Thursday and once more on Saturday. All three PCR tests came back negative, confirming that the governor is not infected.

His experience could raise concerns about how much states will rely on antigen tests to augment other forms of testing that are in short supply. Ohio is one of seven states that said this week that they were banding together to purchase a total of 3.5 million rapid coronavirus tests, including antigen tests, along with other vital supplies. Governor DeWine said on CNN Sunday that he had already been in touch with Gov. Larry Hogan of Maryland to talk about the tests and the seven-state agreement.

If anyone needed a wake-up call with antigens, how careful you have to be, we certainly saw that with my test, Governor DeWine said. And were going to be very careful in how we use it.

He added that he would direct any funding from a new federal relief package to expanded testing and helping schools adapt.

We have doubled our testing in the last four weeks, he said. We need to double it again and then double it again. And so that is not going to be cheap to do.

PCR tests are in short supply nationwide, and turnaround times for results have stretched past two weeks in some parts of the country, rendering the information useless.

Compared with PCR tests, Quidels antigen test is more likely to return a false negative result, missing up to 20 percent of cases that PCR detects, though the figure may drop below 5 percent for patients with high virus levels. But Governor DeWines antigen test produced the opposite error: a false positive.

He noted on Sunday that antigen tests function especially well as screening tests, delivering a quick preliminary indication that can be confirmed by the more accurate but slower PCR tests.

Virus cases have surged in the United States in recent weeks, particularly in the Sun Belt states and in communities where officials moved quickly to reopen. According to a New York Times database, the United States leads the world in confirmed cases with more than five million a milestone reached on Saturday followed by Brazil and India. Experts have warned that the actual number of people infected is far greater than the confirmed case count. Brazil also reached a milestone of 100,000 deaths on Saturday.

Trumps moves on economic aid draw fire on the Sunday news shows.

Administration officials struggled in television appearances on Sunday to explain President Trumps attempts to circumvent Congress in the absence of an agreement on a coronavirus aid package, sowing further confusion over whether tens of millions of Americans will receive the promised relief.

The president announced executive steps on Saturday that he said were intended to address lapsed unemployment benefits, reinstate an eviction ban, provide relief for student borrowers and suspend collection of payroll taxes. They came after crucial benefits provided under earlier aid bills had lapsed, and after two weeks of talks between congressional Democrats and administration officials failed to yield an agreement on a broader relief package.

But Mr. Trumps steps appeared unlikely to have a meaningful impact on the sputtering economy, raising questions about whether Mr. Trump had taken them mainly to gain more leverage in his face-off with Congress.

Democrats criticized the actions on Sunday as executive overreach, and warned that the nations social safety net could be jeopardized.

The presidents meager, weak and unconstitutional actions further demand that we have an agreement, Speaker Nancy Pelosi of California said on Fox News Sunday.

She, along with Senator Chuck Schumer of New York, the minority leader, urged administration officials to resume talks and seek a compromise on a broad relief package.

The presidents executive orders, described in one word, could be paltry; in three words, unworkable, weak and far too narrow, Mr. Schumer said on the ABC program This Week.

Mr. Trumps top economic advisers were on the defensive Sunday about whether the president had the authority to bypass Congress, which retains the constitutional power of the purse, and redirect billions of dollars in spending. But there was some acknowledgment that the measures were not as potent as congressional action would be.

The downside of executive orders is, you cant address some of the small business incidents that are there, said Mark Meadows, the White House chief of staff, in a prerecorded interview that was broadcast Sunday on Gray Television. You cant necessarily get direct payments, because it has to do with appropriations. Thats something that the president doesnt have the ability to do. So, you miss on those two key areas. You miss on money for schools. You miss on any funding for state and local revenue needs that may be out there.

Kansas City, a potential hot spot, needs federal aid to cope, the mayor says.

Like many communities, Kansas City, Mo., has been having a tough time lately, and it will get tougher if Congress and the White House cant reach a deal on more aid, the citys mayor, Quinton Lucas, said on Sunday.

Dr. Deborah Birx, the White House coronavirus coordinator, recently named Kansas City as one of 10 potential coronavirus hot spots around the country because of troubling signs in its testing data. Daily case counts have been declining, but the city is experiencing huge backlogs in testing that are delaying results by as much as two weeks, making them nearly useless in heading off the spread of the disease.

What will it take to address the problem? Money, Mr. Lucas said Sunday on the CBS program Face the Nation. We need more resources to get more testing, to get faster testing through.

Kansas Citys Covid-19 response has already cost the city millions, and without federal aid, a growing budget deficit may soon force officials to start furloughing workers and eliminating jobs.

This isnt just theoretical for us, he said. These are issues that are significant and in the now, and so we are looking for a deal.

Kansas City has delayed the start of its school year until after Labor Day to buy more time to make its schools safe to reopen. But the schools need more money to buy protective equipment and implement social distancing measures, Mr. Lucas said.

He said it was difficult to make the right decisions locally without clear guidance from the federal government: Im a lawyer by training. I talk to doctors and health care professionals here, but these are calls necessarily that sometimes mayors may not be equipped to make, or some governors.

With the number of severely ill patients rising and with remdesivir, the only drug shown to speed recovery, in short supply, an urgent need to quickly increase remdesivir production has arisen. Some U.S. hospitals have been forced to ration the drug, using various systems to decide who should get it.

Now, in a rare agreement between drug companies, Pfizer has entered into an agreement with Gilead Sciences, the maker of remdesivir, to manufacture the drug at a facility in Kansas. It is meant to be part of an effort to quickly increase the drugs supply.

Pfizer will be one of 40 companies in North America, Europe and Asia that will be making the drug. Gilead says it plans to produce more than two million courses of treatment by the end of 2020. It says it also will produce another several million doses of remdesivir in 2021 if they are needed.

Remdesivir is an anti-viral drug that failed as a treatment for hepatitis C but was tested in Covid-19 patients because it seemed effective against the virus in laboratory studies and because its safety had already been determined. It is supplied intravenously.

The evidence of its effectiveness against the new coronavirus comes from a federal study of 1,000 hospitalized patients who received remdesivir or a placebo. Preliminary results were announced on April 27, and on May 1, the Food and Drug Administration gave the drug emergency use authorization, allowing Gilead to sell remdesivir even though it has not yet been approved. The price for a five-day course is $3,120.

Gilead explains the supply problems by saying it is difficult and time consuming to make remdesivir. The company says manufacturing is a long, linear chemical synthesis process that must be completed sequentially and includes several specialized chemistry steps and novel substances with limited global availability.

Your immune system may already recognize the coronavirus.

Eight months ago, the new coronavirus was unknown. But to some human immune cells, it was already something of a familiar foe.

A flurry of recent studies has revealed that a large proportion of the population in some places, 20 to 50 percent of people may harbor immunity assassins called T cells that recognize the new coronavirus despite having never encountered it before.

These T cells, which lurked in the bloodstreams of people long before the pandemic began, are most likely stragglers from past scuffles with other related coronaviruses, including four that frequently cause common colds. Its a case of family resemblance: In the eyes of the immune system, germs with common roots can look alike, such that when a cousin comes to call, the body may already have an inkling of its intentions.

The presence of these T cells has intrigued experts, who say it is too soon to tell whether the cells will play a helpful, harmful or entirely negligible role against the current coronavirus.

Updated August 12, 2020

But should these cross-reactive T cells exert even a modest influence on the bodys immune response, they might make the disease milder and perhaps partly explain why some people who catch the germ become very sick while others are dealt only a glancing blow.

This contact tracer is fighting two contagions: the virus and fear.

Radhika Kumar goes to work every morning hoping to save lives. As a contact tracer for Los Angeles County, her job, at least on paper, entails phoning people who have tested positive for the coronavirus, along with others they may have exposed, and providing them with guidance on how to isolate so as not to infect others.

If that sounds easy, it is not.

To persuade people to cooperate, she has to get them to trust her. She has to convince them that they might be infected, even if they have no symptoms. She has to let people curse at her and hang up, then she has to call them back the next day.

And if she wants them to heed her advice, she has to listen, really listen, to how scared they are that if they stay home from their jobs, they might not be able to feed their families.

Sometimes it can really get to you, she said. The other day I had one young lady, and she was screaming on the phone, You dont understand I have three kids. I have to go to work.

I kept calling back and calling back, Ms. Kumar said. Im very relentless like that. I thought about it all night what am I going to do? I called her again first thing in the morning, and I was so relieved when she picked up.

Even as officials at the Centers for Disease Control and Prevention continue to tout the effectiveness of contact tracing, and state and local health agencies across the United States deploy new armies of tracers, tracking down everyone with the coronavirus is proving to be a Sisyphean task.

France is imposing a new requirement that people wear face masks outdoors in crowded areas of Paris and other major cities beginning on Monday as the number of coronavirus infections rises at the fastest rate since a national quarantine ended in mid-May.

The country had gotten the number of infections under control, but the pandemic is creeping back, with 2,288 new Covid-19 cases reported on Friday the third consecutive day of sharp increase. In the Paris Ile-de-France region, the rate of infections reached 2.4 percent on Friday, compared with a 1.6 percent national average.

The rise of new clusters has led the government to warn of the possibility of a second wave of infections in the autumn. In an effort to stem the spread of the virus, masks will now be mandatory for people age 11 and above in high-traffic areas, from the tourist havens of Saint Tropez and Biarritz to the Seine river in Paris, Montmartre and other popular sites, as well as at outdoor food markets and in Pariss crowded suburbs.

The police will be enforcing the measures which will be in place for at least a month in Paris and are subject to review in other areas with a fine of 135 euros ($159).

The authorities are especially concerned about the popularity of free parties, in which hundreds of young people gather in the Parisien woods and other areas, often without wearing masks.

Wearing a mask in crowded enclosed spaces, including museums, shopping malls and on public transportation, has been compulsory in France since mid-July.

Here is what else is happening around the world:

At least nine people were killed after a fire broke out on Sunday at a hotel in southern India that was being used as a makeshift facility for Covid-19 patients, officials said. The police attributed the accident to a short circuit in an air-conditioner on the ground floor of the Swarna Palace.

New Zealand on Sunday marked 100 days without any new reported cases of local transmission of the coronavirus, a milestone as the pandemic continues to devastate countries across the world. New Zealand, a nation of five million people, reported in March that it had stamped out the virus after strict lockdown measures were implemented. Dr. Ashley Bloomfield, the countrys top health official, said it was a significant milestone but added we cant afford to be complacent.

BUSINESS ROUNDUP

In 2009, when H1N1, better known as swine flu, was stoking fears of a devastating pandemic, a small biotech company named Inovio Pharmaceuticals rushed to create a vaccine. After announcing promising early results, the companys stock soared more than 1,000 percent.

In the years since, Inovio has announced encouraging news about its work on vaccines for malaria, the Zika virus and even a cancer vaccine. The declarations have caused the companys stock price to leap, enriching investors and senior executives.

There is a catch, though: Inovio has never brought a vaccine to market.

Now, Inovio is working on a vaccine for the coronavirus, and a flurry of positive news releases about its funding and preliminary results have helped the company attract money from the U.S. government and investors.

But some scientists and financial analysts question the viability of Inovios technology. While there are some early signs of promise with its vaccine, Inovio has released only bare-bones data from the first phase of clinical trials. It is locked in a legal battle with a key manufacturing partner that claims Inovio stole its technology.

And while the company has said that it is part of Operation Warp Speed the flagship federal effort to quickly produce treatments and vaccines for the coronavirus Inovio is not on the list of companies selected to receive financial support to mass-produce vaccines.

The absence of that funding, coupled with their ongoing litigation, coupled with the need to scale a device, coupled with the absence of complete Phase 1 data, makes people skeptical, said Stephen Willey, an analyst at Stifel, an investment firm.

Inovio could provide an update on its progress with the vaccine when it releases its second-quarter financial results on Monday.

In other business news:

Saudi Aramco, the worlds largest oil company, said on Sunday that its quarterly earnings had plunged more than 73 percent compared with a year ago, as lockdowns imposed to curb the pandemic drastically cut the demand for oil. Despite the steep fall in earnings, to $6.6 billion from $24.7 billion, the company said it would continue paying a quarterly dividend of $18.75 billion nearly all of which will go to the Saudi government.

Meet the people at the big biker rally, undaunted by the virus.

Despite the coronavirus pandemic, tens of thousands of motorcycle enthusiasts converged over the weekend outside the small South Dakota community of Sturgis for the 80th annual Sturgis Motorcycle Rally. Officials said about 250,000 enthusiasts were expected this year about half the number who attended last year, but a figure that would still make the rally one of the countrys largest public gatherings since the first coronavirus cases emerged in the spring.

Many in attendance said they were not concerned about the virus as they walked around without masks.

A New Hampshire poet laureate brightens up her citys Covid-19 advisories.

On Sundays, thousands of residents of Portsmouth, N.H., find a poem nestled inside the citys Covid-19 newsletter.

The poems, written by Tammi J. Truax, the citys poet laureate, help offset the gloom of the pandemic while giving residents a chance to pause briefly and reflect on something other than the coronavirus.

Since the beginning of the pandemic, there have been more than 6,800 cases and at least 419 deaths in New Hampshire, according to a New York Times database, with a recent average of 28 cases per day.

The idea for featuring the poems came from Stephanie Seacord, the public information officer in Portsmouth, a city of about 21,000 residents about 60 miles north of Boston. Ms. Seacord was compiling information about the virus and health updates in a weekly city newsletter sent to about 5,000 email subscribers and circulated on social media.

When the pandemic hit, it became quickly clear that people needed information more than once a week, Ms. Seacord recalled in an interview last week, adding that things were changing almost on a daily basis.

In mid-March, the newsletter turned into a daily advisory of coronavirus cases and tips, like where to find personal protective equipment. Around that time, Ms. Seacord had the idea that including a poem in the Sunday newsletter would be a good calm moment in the middle of the intensity, she said.

If you are feeling ready to go somewhere slightly out of the way, you may be worried about the buses, trains or planes you need to get there. Heres some information to help ease your anxiety and remain safe on mass transit.

Original post:
At Least 97,000 Children in the U.S. Tested Positive in Last 2 Weeks of July - The New York Times

In the 1960s, Milford Graves became a groundbreaking drummer in avant-garde jazz, but intertwined with his career had been his constant study of musics impact on the human heart.

Now Mr. Graves, a 78-year-old who lives in Jamaica, Queens, has become his own subject: He has amyloid cardiomyopathy, sometimes called stiff heart syndrome.

Doctors have informed him that the condition, also called cardiac amyloidosis, has no cure. When he received the diagnosis in 2018, he was told he had six months to live.

Since then, Mr. Graves said, he has come close to death several times because of fluid filling his lungs. His legs too weakened to walk, he remains in a recliner in his living room with a tube feeding medicine to his heart and another draining fluid from his midsection.

But he has hardly surrendered to the illness. Although he is under the care of a cardiologist, he is also treating himself with the alternative techniques he has spent decades researching.

Since the 1970s, Mr. Graves has studied the heartbeat as a source of rhythm and has maintained that recording musicians most prevalent heart rhythms and pitches, and then incorporating those sounds into their playing, would help them produce more personal music.

He also believes that heart problems can be helped by recording a patients unhealthy heart and musically tweaking it into a healthier rhythm to use as biofeedback.

In recent months, Mr. Graves has been listening constantly to his own heart with a stethoscope and monitoring it with an ultrasound device he bought on eBay.

It turns out, I was studying the heart to prepare for treating myself, he said.

His diagnosis has only invigorated his research, musical explorations and creative output as a visual artist, said Mr. Graves, whose daily fight against the disease has become something of a performance art project.

He said he is rushing to further his research and organize it, so that it can be continued after his death by his students, who are fastidiously documenting and videotaping his daily activity, both for his archives and for an exhibition in September at the Institute of Contemporary Art in Philadelphia.

The shows curator, Mark Christman, visits Mr. Graves and gathers his latest work, from sculptures to customized drums to new videos of Mr. Graves playing.

Mr. Graves has no idea how long he will live It could be three days, it could be a month, or longer but he is adamant that he will be strong enough to play live for the show, perhaps streamed from his recliner.

Where some might see cruel irony in being afflicted by heart disease, which he has studied for 45 years, he sees a challenge.

Its like some higher power saying, OK, buddy, you wanted to study this, here you go, he said. Now the challenge is inside of me.

He wonders if he has somehow internalized the subject of his study.

I ask myself, Why did I get something that, in my research, Ive been trying to rectify? he said. Its a rare disease with very little research on it. The experts say theres nothing to be done, so I have to look inward for answers.

Mr. Graves has long said that a healthy heart beats with flexible, varying rhythms that respond to stimuli from the body. The rhythms, he said, bear similarities to some traditional Nigerian drumming styles, and he has fashioned some of his drumming approaches along these lines.

Because of the abnormal heartbeats caused by his disease, which stiffens the heart muscle and can lead to heart failure, what he hears now in his own heart is the sound of survival, he said.

It sounds less elastic and more plodding than before the diagnosis, he said, with a more metronomic regularity that he has called a rigid, unhealthy quality in a heartbeat.

He is practicing his biofeedback techniques by listening to his heart with a stethoscope and mimicking the rhythm and melody by singing and playing on a drum near his recliner. He also plays recordings of his own hearts sounds on the drumhead with the help of electronic transducers, effectively turning the drumhead into a speaker.

That has helped him come up with drumming techniques, including adjustments in drumhead tensions and new stick styles. Its still drum practice, but with higher stakes.

Mr. Graves has seen a resurgence in popularity in recent years, with exhibitions of his art and research, festival performances and an acclaimed full-length documentary, Milford Graves Full Mantis.

Instead of going into despair, his response was, Ive been asked to look deeper at this, said Jake Meginsky, the films co-director and a longtime assistant of Mr. Graves. Hes surviving this prognosis, and through his creative process hes offering us a record on what that survival is like.

Mr. Graves approach is no surprise to those familiar with his unconventional life path.

He grew up in the South Jamaica housing projects and in the 1960s played with such avant-garde musicians as Cecil Taylor and Albert Ayler, with whom he performed at John Coltranes funeral in 1967. He turned down offers from Miles Davis to join Daviss band.

In more recent years, he has also collaborated with the rocker Lou Reed, the pianist Jason Moran and the avant-garde saxophonist John Zorn.

Mr. Graves became a largely self-taught musician and scientific researcher, delving into herbal medicine, holistic healing, acupuncture, martial arts and other disciplines.

With only a high school diploma and minimal formal medical training, he taught music healing and drumming classes at Bennington College in Vermont for nearly 40 years before retiring in 2012.

He developed a martial-arts style modeled after the movements of the praying mantis and dance traditions from West African styles and the Lindy Hop.

He did pretty much everything on his own, and its very important that his work continue, so he wants to leave everything in the right places with the right people, his wife, Lois, said. He knows he has more work to do and hes going to get it done.

Since 1970, Mr. and Ms. Graves have lived in a home in Queens that he has decorated with a Gaudesque mosaic of stones and colored glass. The Graveses have turned the yard into a lush garden, dense with citrus trees, herbs and exotic plants. He converted a free-standing garage into an ornate temple that was often used as a dojo for martial arts.

But it is the basement where his heart research was mainly conducted. The space is packed with African idols, anatomical models, herbal extracts, African drums and a hodgepodge of heart-monitoring equipment displaying intricate electrocardiogram readouts.

Here, he said, he has treated students, neighbors and colleagues, and since 1990 has recorded perhaps 5,000 heartbeats. Mr. Graves created programs to analyze the hearts rhythms and pitches caused by muscle and valve movement. He found ways to amplify the more obscure patterns and complex melody lines in the vibration frequencies underneath the basic thump-THUMP heartbeat, and use them for both musical and medical analysis.

In 2000, he received a Guggenheim grant to purchase heart-monitoring equipment. And in 2017, he co-patented technology for using heart melodies to regenerate stem cells.

Dr. Baruch Krauss, who teaches pediatrics at Harvard Medical School and is an emergency physician at Boston Childrens Hospital, said Mr. Gravess work has a lot of potential and possibility if it were to be furthered in a clinical setting.

Theres a lot there to be studied and used as a basis for further research, said Dr. Krauss, who follows Mr. Gravess work.

Hes continuously inquisitive and creative and interested, he added, and this condition really hasnt slowed him down.

In his living room on a recent Sunday, one of Mr. Gravess students, Peyton Pleninger, 24, helped him set up a device to play heart sounds and assisted him with making an assemblage for the art show.

I dont want to leave the planet with things undone, Mr. Graves said.

Go here to read the rest:
A Jazz Drummers Fight to Keep His Own Heart Beating - The New York Times

Abstract:

Altered olfactory function is a common symptom of COVID-19, but its etiology is unknown. A key question is whether SARS-CoV-2 (CoV-2) the causal agent in COVID-19 affects olfaction directly, by infecting olfactory sensory neurons or their targets in the olfactory bulb, or indirectly, through perturbation of supporting cells. Here we identify cell types in the olfactory epithelium and olfactory bulb that express SARS-CoV-2 cell entry molecules. Bulk sequencing demonstrated that mouse, non-human primate and human olfactory mucosa expresses two key genes involved in CoV-2 entry, ACE2 and TMPRSS2. However, single cell sequencing revealed that ACE2 is expressed in support cells, stem cells, and perivascular cells, rather than in neurons. Immunostaining confirmed these results and revealed pervasive expression of ACE2 protein in dorsally-located olfactory epithelial sustentacular cells and olfactory bulb pericytes in the mouse. These findings suggest that CoV-2 infection of non-neuronal cell types leads to anosmia and related disturbances in odor perception in COVID-19 patients.

SARS-CoV-2 (CoV-2) is a pandemic coronavirus that causes the COVID-19 syndrome, which can include upper respiratory infection (URI) symptoms, severe respiratory distress, acute cardiac injury and death (1-4). CoV-2 is closely related to other coronaviruses, including the causal agents in pandemic SARS and MERS (SARS-CoV and MERS-CoV, respectively) and endemic viruses typically associated with mild URI syndromes (hCoV-OC43, hCoV-HKU1, hCoV-229E and hCoV-NL63) (5-7). Clinical reports suggest that infection with CoV-2 is associated with high rates of disturbances in smell and taste perception, including anosmia (8-13). While many viruses (including coronaviruses) induce transient changes in odor perception due to inflammatory responses, in at least some cases COVID-related anosmia has been reported to occur in the absence of significant nasal inflammation or coryzal symptoms (11, 14-16). Furthermore, recovery from COVID-related anosmia often occurs over weeks (11, 17, 18), while recovery from typical post-viral anosmia which is often caused by direct damage to olfactory sensory neurons (OSNs) frequently takes months (19-21). These observations suggest that CoV-2 might target odor processing through mechanisms distinct from those used by other viruses, although the specific means through which CoV-2 alters odor perception remains unknown.

CoV-2 like SARS-CoV infects cells through interactions between its spike (S) protein and the ACE2 protein on target cells. This interaction requires cleavage of the S protein, likely by the cell surface protease TMPRSS2, although other proteases (such as Cathepsin B and L, CTSB/CTSL) may also be involved (4-6, 22-25). Other coronaviruses use different cell surface receptors and proteases to facilitate cellular entry, including DPP4, FURIN and HSPA5 for MERS-CoV, ANPEP for HCoV-229E, TMPRSS11D for SARS-CoV (in addition to ACE2 and TMPRSS2), and ST6GAL1 and ST3GAL4 for HCoV-OC43 and HCoV-HKU1 (6, 26-28).

We hypothesized that identifying the specific cell types susceptible to direct CoV-2 infection (due to e.g., ACE2 and TMPRSS2 expression) would provide insight into possible mechanisms through which COVID-19 alters smell perception. The nasal epithelium is divided into a respiratory epithelium (RE) and olfactory epithelium (OE), whose functions and cell types differ. The nasal RE is continuous with the epithelium that lines much of the respiratory tract and is thought to humidify air as it enters the nose; main cell types include basal cells, ciliated cells, secretory cells (including goblet cells), and brush/microvillar cells (29, 30) (Fig. 1). The OE, in contrast, is responsible for odor detection, as it houses mature OSNs that interact with odors via receptors localized on their dendritic cilia. OSNs are supported by sustentacular cells, which act to structurally support sensory neurons, phagocytose and/or detoxify potentially damaging agents, and maintain local salt and water balance (31-33); microvillar cells and mucus-secreting Bowmans gland cells also play important roles in maintaining OE homeostasis and function (29, 34) (Fig. 1). In addition, the OE contains globose basal cells (GBCs), which are primarily responsible for regenerating OSNs during normal epithelial turnover, and horizontal basal cells (HBCs), which act as reserve stem cells activated upon tissue damage (35-37). Although studies defining the lineage relationships between GBCs, HBCs and their progeny have necessarily been performed in rodents, basal progenitor populations with similar transcriptional profiles are present in adult human olfactory epithelium, suggesting closely related homeostatic and injury-response mechanisms (37, 38). Odor information is conveyed from the OE to the brain by OSN axons, which puncture the cribriform plate at the base of the skull and terminate in the olfactory bulb (OB). Within the OB local circuits process olfactory information before sending it to higher brain centers (Fig. 1).

Sagittal view of the human nasal cavity, in which respiratory and olfactory epithelia are colored (left). For each type of epithelium, a schematic of the anatomy and known major cell types are shown (right). In the olfactory bulb in the brain (tan) the axons from olfactory sensory neurons coalesce into glomeruli, and mitral/tufted cells innervate these glomeruli and send olfactory projections to downstream olfactory areas. Glomeruli are also innervated by juxtaglomerular cells, a subset of which are dopaminergic.

It has recently been demonstrated through single cell RNA sequencing analysis (referred to herein as scSeq) that cells from the human upper airway including nasal RE goblet and ciliated cells express high levels of ACE2 and TMPRSS2, suggesting that these RE cell types may serve as a viral reservoir during CoV-2 infection (39, 40). However, analyzed samples in these datasets did not include any OSNs or sustentacular cells, indicating that tissue sampling in these experiments did not include the OE (41, 42). Here we query both new and previously published bulk RNA-Seq and scSeq datasets from the olfactory system for expression of ACE2, TMRPSS2 and other genes implicated in coronavirus entry. We find that non-neuronal cells in the OE and olfactory bulb, including support, stem and perivascular cells, express CoV-2 entry-associated transcripts and their associated proteins, suggesting that infection of these non-neuronal cell types contributes to anosmia in COVID-19 patients.

To determine whether genes relevant to CoV-2 entry are expressed in OSNs or other cell types in the human OE, we queried previously published bulk RNA-Seq data derived from the whole olfactory mucosa (WOM) of macaque, marmoset and human (43), and found expression of almost all CoV-entry-related genes in all WOM samples (Figure S1A). To identify the specific cell types in human OE that express ACE2, we quantified gene expression in scSeq derived from four human nasal biopsy samples recently reported by Durante et al. (38). Neither ACE2 nor TMPRSS2 were detected in mature OSNs, whereas these genes were detected in both sustentacular cells and HBCs (Fig. 2A-D and S1B-E). In contrast, genes relevant to cell entry of other CoVs were expressed in OSNs, as well as in other OE cell types. We confirmed the expression of ACE2 proteins via immunostaining of human olfactory epithelium biopsy tissue, which revealed expression in sustentacular and basal cells, and an absence of ACE2 protein in OSNs (Figs. 2E and S2). Together, these results demonstrate that sustentacular and olfactory stem cells, but not mature OSNs, are potentially direct targets of CoV-2 in the human OE.

Coronavirus cell entry-related genes are expressed in human respiratory and olfactory epithelium but are not detected in human OSNs. (A) UMAP representation of cell types in human nasal biopsy scSeq data from Durante et al. 2020 (38). Each dot represents an individual cell, colored by cell type (HBC = horizontal basal cell, OSN = olfactory sensory neuron, SUS = sustentacular cell, MV: microvillar cell, Resp.: respiratory, OEC = olfactory ensheathing cell, SMC=smooth muscle cell). (B) UMAP representations of 865 detected immature (GNG8) and mature (GNG13) OSNs. Neither ACE2 nor TMPRSS2 are detected in either population of OSNs. The color represents the normalized expression level for each gene (number of UMIs for a given gene divided by the total number of UMIs for each cell). (C) UMAP representations of all cells, depicting the normalized expression of CoV-2 related genes ACE2 and TMPRSS2, as well as several cell type markers. ACE2 and TMPRSS2 are expressed in respiratory and olfactory cell types, but not in OSNs. ACE2 and TMPRSS2 are detected in HBC (KRT5) and sustentacular (CYP2A13) cells, as well as other respiratory epithelial cell types, including respiratory ciliated (FOXJ1) cells. (D) Percent of cells expressing ACE2 and TMPRSS2. ACE2 was not detected in any OSNs, but was observed in sustentacular cells and HBCs, among other olfactory and respiratory epithelial cell types. Olfactory and respiratory cell types are shown separately. ACE2 and TMPRSS2 were also significantly co-expressed (Odds ratio 7.088, p-value 3.74E-57, Fishers exact test). (E) ACE2 immunostaining of human olfactory mucosal biopsy samples (taken from a 28-year old female). ACE2 protein (green) is detected in sustentacular cells and KRT5-positive HBCs (red; white arrowhead). Nuclei were stained with DAPI (blue). Bar = 25 m. The ACE2 and KRT5 channels from the box on the left are shown individually on the right.

Given that the nasopharynx is a major site of infection for CoV-2 (10), we compared the frequency of ACE2 and TMPRSS2 expression among the cell types in the human RE and OE (38). Sustentacular cells exhibited the highest frequency of ACE2 expression in the OE (2.9% of cells) although this frequency was slightly lower than that observed in respiratory ciliated and secretory cells (3.6% and 3.9%, respectively). While all HBC subtypes expressed ACE2, the frequency of expression of ACE2 was lower in olfactory HBCs (0.8% of cells) compared to respiratory HBCs (1.7% of cells) (Fig. 2D). In addition, all other RE cell subtypes showed higher frequencies of ACE2 and TMPRSS2 expression than was apparent in OE cells.

These results demonstrate the presence of key CoV-2 entry-related genes in specific cell types in the OE, but at lower levels of expression than in RE isolated from the human nasal mucosa. We wondered whether these lower levels of expression might nonetheless be sufficient for infection by CoV-2. It was recently reported that the nasal RE has higher expression of CoV-2 entry genes than the RE that lines the trachea or lungs (44), and we therefore asked where the OE fell within this previously established spectrum of expression. To address this question, we developed a two step alignment procedure in which we first sought to identify cell types that were common across the OE and RE, and then leveraged gene expression patterns in these common cell types to normalize gene expression levels across all cell types in the OE and RE (Figs. 3 and S3). This approach revealed a correspondences between submucosal gland goblet cells in the RE and Bowmans gland cells in the OE (96% mapping probability, see Methods), and between pulmonary ionocytes in the RE and a subset of microvillar cells in the OE (99% mapping probability, see Methods and Figure S3); after alignment, human OE sustentacular cells were found to express ACE2 and TMPRSS2 at levels similar to those observed in the remainder of the non-nasal respiratory tract (44) (Fig. 3C). As CoV-2 can infect cells in the lower respiratory tract (40, 45), these results are consistent with the possibility that specific cell types in the human olfactory epithelium express ACE2 at a level that is permissive for direct infection.

Coronavirus cell entry-related genes are expressed at comparable levels across respiratory and olfactory epithelial datasets. (A) Schematic of the mapping strategy used to identify similar cell types across datasets, applied to a toy example. Each cell type from Dataset 1 dataset is mapped to cell types from the Dataset 2. From left to right: Each Dataset 1 cell voted on its 5 most similar cells in Dataset 2; the total number of votes cast for each Dataset 2 cell type was quantified; and vote totals were Z-scored against 1000 shuffles where cell type labels were permutated. (B) Mapping was performed bi-directionally between the Deprez et al. (41) and Durante et al. (38) datasets, and the mapping Z-scores in each direction are compared. The set of cell type correspondences with high Z-scores (>25) in both directions are colored red (top). The set of cell type correspondences with high bi-directional mappings shown in red in top panel are highlighted in yellow (bottom). (C) Gene expression across cell types and tissues in Durante et al. (top) and Deprez et al. (bottom). Each gene is normalized to its maximum value across all tissues. Gene expression from Durante et al. was normalized to that in Deprez et al. to enable comparisons (see Methods and Figure S3). The tissues correspond to the indicated positions along the airway from nasal to distal lung. ACE2 expression in olfactory HBC and sustentacular cells is comparable to that observed in other cell types in the lower respiratory tract.

To further explore the distribution of CoV-2 cell entry genes in the olfactory system we turned to the mouse, which enables interrogative experiments not possible in humans. To evaluate whether mouse expression patterns correspond to those observed in the human OE, we examined published datasets in which RNA-Seq was independently performed on mouse WOM and on purified populations of mature OSNs (46-48). The CoV-2 receptor Ace2 and the protease Tmprss2 were expressed in WOM, as were the cathepsins Ctsb and Ctsl (Figs. 4A and S4A) (46). However, expression of these genes (with the exception of Ctsb) was much lower and Ace2 expression was nearly absent in purified OSN samples (Figs. 4A and S4A, see Legend for counts). Genes used for cell entry by other CoVs (except St3gal4) were also expressed in WOM, and de-enriched in purified OSNs. The de-enrichment of Ace2 and Tmprss2 in OSNs relative to WOM was also observed in two other mouse RNA-Seq datasets (47, 48) (Figure S4B). These data demonstrate that, as in humans, Ace2 and other CoV-2 entry-related genes are expressed in the mouse olfactory epithelium.

ACE2 is expressed in the mouse nasal epithelium but not in mature OSNs. (A) Log2-fold change (FC) in mean across-replicate gene expression between olfactory sensory neurons (OSNs) and whole olfactory mucosa (WOM) for coronavirus (CoV)-related genes and cell type markers (HBC = horizontal basal cells, SUS = sustentacular cells), data from Saraiva et al. (46). (B) UMAP representation of scSeq data from the WOM, colored by cell types (mOSN: mature OSN, iOSN: immature OSN, INP: immediate neural precursor, GBC: globose basal cell, MV: microvillar cell, Resp.: respiratory). (C) Percent of cells expressing Ace2 and Tmprss2 in olfactory and respiratory cell types in the WOM (Drop-seq) dataset. Detection was considered positive if any transcripts (UMIs) were expressed for a given gene. Sustentacular cells (SUS) from dorsal and ventral zones are quantified separately. Ace2 is detected in dorsal sustentacular, Bowmans gland, HBCs, as well as respiratory cell types. (D) UMAP representation of sustentacular cells, with expression of CoV-2 related genes Ace2 and Tmprss2, as well as marker genes for SUS (both pan-SUS marker Cbr2 and dorsal specific marker Sult1c1) indicated. The color represents the normalized expression level for each gene (number of UMIs for a given gene divided by the total number of UMIs for each cell; in this plot Ace2 expression is binarized for visualization purposes). Ace2-positive sustentacular cells are found within the dorsal Sult1c1-positive subset. UMAP plots for other cell types are shown in Figure S4.

The presence of Ace2 and Tmprss2 transcripts in mouse WOM and their (near total) absence in purified OSNs suggest that the molecular components that enable CoV-2 entry into cells are expressed in non-neuronal cell types in the mouse nasal epithelium. To identify the specific cell types that express Ace2 and Tmprss2, we performed scSeq (via Drop-seq, see Methods) on mouse WOM (Fig. 4B). These results were consistent with observations made in the human epithelium: Ace2 and Tmprss2 were expressed in a fraction of sustentacular and Bowmans gland cells, and a very small fraction of stem cells, but not in OSNs (zero of 17,666 identified mature OSNs, Figs. 4C and S4C-D). Of note, only dorsally-located sustentacular cells, which express the markers Sult1c1 and Acsm4, were positive for Ace2 (Figs. 4D and S4D-E). Indeed, reanalysis of the ACE2-positive subset of human sustentacular cells revealed that all positive cells expressed genetic markers associated with the dorsal epithelium (Figure S1D). An independent mouse scSeq data set (obtained using the 10x Chromium platform, see Methods) confirmed that olfactory sensory neurons did not express Ace2 (2 of 28,769 mature OSNs were positive for Ace2), while expression was observed in a fraction of Bowmans gland cells and HBCs (Figure S5, see Methods). Expression in sustentacular cells was not observed in this dataset, which included relatively few dorsal sustentacular cells (a possible consequence of the specific cell isolation procedure associated with the 10x platform; compare Figures S5C and 4D).

Staining of the mouse WOM with anti-ACE2 antibodies confirmed that ACE2 protein is expressed in sustentacular cells and is specifically localized to the sustentacular cell microvilli (Fig. 5). ACE2-positive sustentacular cells were identified exclusively within the dorsal subregion of the OE; critically, within that region many (and possibly all) sustentacular cells expressed ACE2 (Fig. 5B-E). Staining was also observed in Bowmans gland cells but not in OSNs, and in subsets of RE cells (Fig. 5F-G). Taken together, these data demonstrate that ACE2 is expressed by sustentacular cells that specifically reside in the dorsal epithelium in both mouse and human.

ACE2 protein is detected in the mouse olfactory and respiratory epithelium. (A) ACE2 immunostaining of mouse main olfactory epithelium. As shown in this coronal section, ACE2 protein is detected in the dorsal zone and respiratory epithelium. The punctate Ace2 staining beneath the epithelial layer is likely associated with vasculature. Bar = 500 m. Arrowheads depict the edges of ACE2 expression, corresponding to the presumptive dorsal zone (confirmed in G). Dashed boxes indicate the areas shown in B and G (left). (B) ACE2 protein is detected in the dorsal zone of the olfactory epithelium. Bar = 50 m. (C) Dorsal zone-specific expression of ACE2 in the olfactory epithelium was confirmed by co-staining with NQO1, a protein expressed in dorsal-zone OSNs. Bar = 50 m. (D) ACE2 signal in dorsal olfactory epithelium does not overlap with the cilia of olfactory sensory neurons, as visualized by CNGA2. Bar = 50 m. (E) High magnification image of the apical end of the olfactory epithelium reveals that ACE2 signal is localized at the tip of villi of sustentacular cells, visualized by Phalloidin (F-Actin), but does not overlap with cilia of olfactory sensory neurons, as visualized by Acetylated Tubulin (Ac. Tubulin). Bar = 10 m. (F) Bowmans glands, which span from the lamina propria to the apical surface (arrowheads), were positive for ACE2 staining. Bar = 50 m. (G) ACE2 expression in the respiratory epithelium was confirmed by co-staining with TUBB4. Bar = 50 m.

Viral injury can lead to broad changes in OE physiology that are accompanied by recruitment of stem cell populations tasked with regenerating the epithelium (35, 37, 49). To characterize the distribution of Ace2 expression under similar circumstances, we injured the OE by treating mice with methimazole (which ablates both support cells and OSNs), and then employed a previously established lineage tracing protocol to perform scSeq on HBCs and their descendants during subsequent regeneration (see Methods) (36). This analysis revealed that after injury Ace2 and Tmprss2 are expressed in subsets of sustentacular cells and HBCs, as well as in the activated HBCs that serve to regenerate the epithelium (Figs. 6A-C and S6A; note that activated HBCs express Ace2 at higher levels than resting HBCs). Analysis of the Ace2-positive sustentacular cell population revealed expression of dorsal epithelial markers (Fig. 6D). To validate these results, we re-analyzed a similar lineage tracing dataset in which identified HBCs and their progeny were subject to Smart-seq2-based deep sequencing, which is more sensitive than droplet-based scSeq methods (36). In this dataset, Ace2 was detected in more than 0.7% of GBCs, nearly 2% of activated HBCs and nearly 3% of sustentacular cells but was not detected in OSNs (Figures S6B). Immunostaining with anti-ACE2 antibodies confirmed that ACE2 protein was present in activated stem cells under these regeneration conditions (Fig. 6E). These results demonstrate that activated stem cells recruited during injury express ACE2 and do so at higher levels than those in resting stem cells.

ACE2 is expressed in the mouse nasal epithelium in an injury model. (A) UMAP representation of data from an scSeq HBC lineage dataset, which includes several timepoints after epithelial injury induced by methimazole (mOSN: mature OSN, iOSN: immature OSN, INP: immediate neural precursor, SUS: sustentacular cell, GBC: globose basal cell, HBC: horizontal basal cell, HBC*: activated or cycling HBCs. MV: microvillar cell, Resp.: respiratory). (B) UMAP representation of CoV-2 related genes Ace2 and Tmprss2, as well as marker genes for the HBC-derived cell types. The color represents normalized expression (number of UMIs for a given gene divided by the total number of UMIs for each cell). (C) Percent of cells expressing Ace2 and Tmprss2. Ace2 is detected in sustentacular cells, HBC, activated/cycling HBC and respiratory cells. (D) UMAP representation of all sustentacular cells, indicating the normalized expression of CoV-2 related genes Ace2 and Tmprss2, as well as sustentacular (Ermn) cell markers. Ace2-positive sustentacular cells are largely a subset of dorsal SUS cells, as identified via the expression of Sult1c1. Sult1c1-positive sustentacular cells have higher levels of Ace2 (p=1.87E-03, Mann-Whitney test) and Ace2-positive sustentacular cells have higher levels of Sult1c1 (p=8.06E-07, Mann-Whitney test). (E) ACE2 immunostaining of mouse nasal epithelium after methimazole treatment, together with cycling cell marker Ki67 and HBC marker KRT5. At 48 hours after treatment, ACE2 signal is detected in Ki67+/KRT5+ activated HBCs (top). At 96 hours after treatment, ACE2 signal is observed at the apical surface of Ki67+ cells (bottom). Some ACE2-positive cells express low levels of the HBC marker KRT5 and have immunostaining patterns similar to that of dorsal sustentacular cells, suggesting that they are sustentacular cells in the process of differentiating from their HBC precursors. Bar = 25 m.

Given the potential for the RE and OE in the nasal cavity to be directly infected with CoV-2, we assessed the expression of Ace2 and other CoV entry genes in the mouse olfactory bulb (OB), which is directly connected to OSNs via cranial nerve I (CN I); in principle, alterations in OB function could cause anosmia independently of functional changes in the OE. To do so, we performed scSeq (using Drop-seq, see Methods) on the mouse OB, and merged these data with a previously published OB scSeq analysis, yielding a dataset with nearly 50,000 single cells (50) (see Methods). This analysis revealed that Ace2 expression was absent from OB neurons and instead was observed only in vascular cells, predominantly in pericytes, which are involved in blood pressure regulation, maintenance of the blood-brain barrier, and inflammatory responses (51) (Figs. 7A-D and S7-8). Although other potential CoV proteases were expressed in the OB, Tmprss2 was not expressed.

Expression of coronavirus entry genes in mouse olfactory bulb. (A) UMAP visualization of OB scSeq highlighting the main cell classes and subtypes from two integrated scSeq datasets (see Methods). VIP, vasoactive intestinal peptide-expressing neurons; ETCs, external tufted cells; OPCs, oligodendrocyte precursor cells; IPCs, intermediate precursor cells; OECs, olfactory ensheathing cells. Cluster information is summarized in Figures S7-8. (B) UMAP representation of the vascular cell cluster showing expression of CoV-2 entry genes (Ace2 and Tmprss2) and Kcnj8, a pericyte marker. Color scale depicts log-normalized UMI counts. (C) Normalized gene expression of coronavirus entry genes and cell class markers in mouse olfactory bulb. Color scale shows scaled mean expression level per cell type, normalized by their maximum expression across cell types. Ace2 is specifically expressed in vascular cells. (D) Percent of cells expressing Ace2. Other vascular denotes all vascular cells excluding pericytes. Ace2 expression was only detected in vascular cell types. (E) Log2-normalized expression (Log2(TPM+1)) of coronavirus entry genes and dopaminergic neuron markers in manually sorted and deeply-sequenced single olfactory bulb dopaminergic neurons. (F) ACE2 immunostaining of the mouse main olfactory bulb. Left, section of olfactory bulb containing the glomerular layer (with example glomeruli circled), mitral cell layer (MCL) and granule cell layer (GCL). ACE2 protein is present in vascular mural cells but not in OB neurons or OSN axons. Boxes i and ii indicate the locations of enlarged images. Bar = 100 m. (i) enlarged image of glomerular layer (middle). ACE2 protein staining was restricted to vascular cells. Bar = 50 m. (ii) enlarged image of MCL (dashed line) and GCL showing the lack of ACE2 (right). Bar = 50 m. (G) An olfactory bulb section showing ACE2 protein is detected in PDGFRB-positive mural cells, including smooth muscle cells (SMC) and pericytes. Bar = 25 m.

We also performed Smart-seq2-based deep sequencing of single OB dopaminergic juxtaglomerular neurons, a population of local interneurons in the OB glomerular layer that (like tufted cells) can receive direct monosynaptic input from nasal OSNs (Figs. 7E and S9, see Methods); these experiments confirmed the virtual absence of Ace2 and Tmprss2 expression in this cell type. Immunostaining in the OB revealed that blood vessels expressed high levels of ACE2 protein, particularly in pericytes; indeed nearly all pericytes exhibited some degree of staining with ACE2 antibodies. Consistent with the scSeq results, staining was not observed in any neuronal cell type (Fig. 7F-G). These observations may also hold true for at least some other brain regions, as re-analysis of 10 deeply sequenced SMART-Seq2-based scSeq datasets from different regions of the nervous system demonstrated that Ace2 and Tmprss2 expression is almost completely absent from neurons, consistent with prior immunostaining results (52, 53) (Figure S10). Given the extensive similarities detailed above in expression patterns for ACE2 and TMPRSS2 in the mouse and human, these findings (from mouse experiments) suggest that OB neurons are likely not a primary site of infection, but that vascular pericytes may be sensitive to CoV-2

Here we show that subsets of OE sustentacular cells, HBCs, and Bowmans gland cells in both mouse and human samples express the CoV-2 receptor ACE2 and the spike protein protease TMPRSS2. Human OE sustentacular cells express these genes at levels comparable to those observed in lung cells. In contrast, we failed to detect ACE2 expression in mature OSNs at either the transcript or protein levels. Similarly, mouse vascular pericytes in the OB express ACE2, while we did not detect ACE2 in OB neurons. Thus primary infection of non-neuronal cell types rather than sensory or bulb neurons may be responsible for anosmia and related disturbances in odor perception in COVID-19 patients.

The identification of non-neuronal cell types in the OE and OB susceptible to CoV-2 infection suggests four possible, non-mutually-exclusive mechanisms for the acute loss of smell reported in COVID-19 patients. First, local infection of support and vascular cells in the nose and bulb could cause significant inflammatory responses (including cytokine release) whose downstream effects could block effective odor conduction, or alter the function of OSNs or OB neurons (14, 54). Second, damage to support cells (which are responsible for local water and ion balance) could indirectly influence signaling from OSNs to the brain (55). Third, damage to sustentacular cells and Bowmans gland cells in mouse models can lead to OSN death, which in turn could abrogate smell perception (56). Finally, vascular damage could lead to hypoperfusion and inflammation leading to changes in OB function.

Although scSeq revealed ACE2 transcripts in only a subset of OE cells, this low level of observed expression matches or exceeds that observed in respiratory cells types that are infected by CoV-2 in COVID-19 patients (39) (Fig. 3). Critically, immunostaining in the mouse suggests that ACE2 protein is (nearly) ubiquitously expressed in sustentacular cells in the dorsal OE, despite sparse detection of Ace2 transcripts using scSeq. Similarly, nearly all vascular pericytes also expressed ACE2 protein, although only a fraction of OB pericytes were positive for Ace2 transcripts when assessed using scSeq. Although Ace2 transcripts were more rarely detected than protein, there was a clear concordance at the cell type level: expression of Ace2 mRNA in a particular cell type accurately predicted the presence of ACE2 protein, while Ace2 transcript-negative cell types (including OSNs) did not express ACE2 protein. These observations are consistent with recent findings in the respiratory epithelium suggesting that scSeq substantially underestimates the fraction of a given cell type that expresses the Ace2 transcript, but that new Ace2-expressing cell types are not discovered with more sensitive forms of analysis (40). If our findings in the mouse OE translate to the human (a reasonable possibility given the precise match in olfactory cell types that express CoV-2 cell entry genes between the two species), then ACE2 protein is likely to be expressed in a significant subset of human sustentacular cells. Thus, there may be many olfactory support cells available for CoV-2 infection in the human epithelium, which in turn could recruit a diffuse inflammatory process. However, it remains possible that damage to the OE could be caused by more limited cell infection. For example, infection of subsets of sustentacular cells by the SDAV coronavirus in rats ultimately leads to disruption of the global architecture of the OE, suggesting that focal coronavirus infection may be sufficient to cause diffuse epithelial damage (56).

We observe that activated HBCs, which are recruited after injury, express Ace2 at higher levels than those apparent in resting stem cells. The natural history of CoV2-induced anosmia is only now being defined; while recovery of smell on timescales of weeks in many patients has been reported, it remains unclear whether in a subset of patients smell disturbances will be long-lasting or permanent (8-12, 57). While on its own it is unlikely that infection of stem cells would cause acute smell deficits, the capacity of CoV-2 to infect stem cells may play an important role in those cases in which COVID-19-associated anosmia is persistent, a context in which infection of stem cells could inhibit OE regeneration and repair over time.

Two anosmic COVID-19 patients have presented with fMRI-identified hyperintensity in both OBs that reverted to normal after resolution of the anosmia (58, 59), consistent with central involvement in at least some cases. Many viruses, including coronaviruses, have been shown to propagate from the nasal epithelium past the cribriform plate to infect the OB; this form of central infection has been suggested to mediate olfactory deficits, even in the absence of lasting OE damage (60-65). The rodent coronavirus MHV passes from the nose to the bulb, even though rodent OSNs do not express Ceacam1, the main MHV receptor (61, 66) (Figures S4C, S5E, S6A), suggesting that CoVs in the nasal mucosa can reach the brain through mechanisms independent of axonal transport by sensory nerves; interestingly, OB dopaminergic juxtaglomerular cells express Ceacam1 (Fig. 7E), which likely supports the ability of MHV to target the bulb and change odor perception. Although SARS-CoV has been shown to infect the OB in a transgenic mouse model that ectopically expresses human ACE2 (65), it is unclear to what extent similar results will be observed for CoV-2 in these and in recently-developed mouse models expressing human ACE2 that better recapitulate native expression patterns (67-69). One speculative possibility is that local seeding of the OE with CoV-2-infected cells can result in OSN-independent transfer of virions from the nose to the bulb, perhaps via the vascular supply shared between the OB and the OSN axons that comprise CN I. Although CN I was not directly queried in our datasets, it is reasonable to infer that vascular pericytes in CN I also express ACE2, which suggests a possible route of entry for CoV-2 from the nose into the brain. Given the absence of ACE2 in mouse OB neurons and the near-ubiquity of ACE2 expression in OB pericytes we speculate that any central olfactory dysfunction in COVID-19 is the secondary consequence of inflammation arising locally from pericytes, or in response to diffusable factors arising from more distant sources (51).

Multiple immunostaining studies reveal that ACE2 protein in the human brain is predominantly or exclusively expressed in vasculature (and specifically expressed within pericytes) (52, 53, 70), and many neurological symptoms associated with CoV-2 infection like stroke or altered consciousness are consistent with an underlying vasculopathy (71-76). In addition, human CSF samples have failed thus far to reveal CoV-2 RNA (73, 77), and autopsies from human patients have found that the brain contains the lowest levels of CoV-2 across organs sampled (78). On the other hand, multiple other studies have suggested that ACE2 may be expressed in human neurons and glia (79-82). Additionally, two recent studies in mouse models expressing human ACE2 have found CoV-2 in the brain after intranasal inoculation (67, 68), although neither specifically queried the OB; this work stands in contrast to results in a non-human primate model of COVID-19, in which nasal infection did not lead to the presence of identifiable CoV-2 antigens in the brain (83). Further work will be required to resolve these inconsistencies, and to definitively characterize the distribution of ACE2 protein and ultimately CoV2-infected cells in the human OB and brain.

We note several caveats that temper our conclusions. Although current data suggest that ACE2 is the most likely receptor for CoV-2 in vivo, it is possible (although it has not yet been demonstrated) that other molecules such as BSG may enable CoV-2 entry independently of ACE2 (84, 85) (Figures S1E, S4C, S5E, S6A). In addition, it has recently been reported that low level expression of ACE2 can support CoV-2 cell entry (86); it is possible, therefore, that ACE2 expression beneath the level of detection in our assays may yet enable CoV-2 infection of apparently ACE2 negative cell types. We also propose that damage to the olfactory system is either due to primary infection or secondary inflammation; it is possible (although has not yet been demonstrated) that cells infected with CoV-2 can form syncytia with cells that do not express ACE2. Such a mechanism could damage neurons adjacent to infected cells. Finally, it has recently been reported that inflammation can induce expression of ACE2 in human cells (87, 88). It is therefore possible that our survey of ACE2 expression, and other recent reports demonstrating expression of ACE2 in OE support and stem cells but not neurons (81, 89, 90), might under-represent the cell types that express ACE2 under conditions of CoV-2 infection.

Any reasonable pathophysiological mechanism for COVID-19-associated anosmia must account for the high penetrance of smell disorders relative to endemic viruses (12, 91, 92), the apparent suddenness of smell loss that can precede the development of other symptoms (11, 13), and the transient nature of dysfunction in many patients (11, 17, 18); definitive identification of the disease mechanisms underlying COVID-19-mediated anosmia will require additional research. Nonetheless, our identification of cells in the OE and OB expressing molecules known to be involved in CoV-2 entry illuminates a path forward for future studies.

Human scSeq data from Durante et al. (38) was downloaded from the GEO at accession GSE139522. 10x Genomics mtx files were filtered to remove any cells with fewer than 500 total counts. Additional preprocessing was performed as described above, including total counts normalization and filtering for highly variable genes using the SPRING gene filtering function filter_genes with parameters (90, 3, 10). The resulting data were visualized in SPRING and partitioned using Louvain clustering on the SPRING k-nearest-neighbor graph. Four clusters were removed for quality control, including two with low total counts (likely background) and two with high mitochondrial counts (likely stressed or dying cells). Putative doublets were also identified using Scrublet and removed (7% of cells). The remaining cells were projected to 40 dimensions using PCA. PCA-batch-correction was performed using Patient 4 as a reference, as previously described (93). The filtered data were then re-partitioned using Louvain clustering on the SPRING graph and each cluster was annotated using known marker genes, as described in (38). For example, immature and mature OSNs were identified via their expression of GNG8 and GNG13, respectively. HBCs were identified via the expression of KRT5 and TP63 and olfactory HBCs were distinguished from respiratory HBCs via the expression of CXCL14 and MEG3. Identification of SUS cells (CYP2A13, CYP2J2), Bowmans gland (SOX9, GPX3), and MV ionocytes-like cells (ASCL3, CFTR, FOXI1) was also performed using known marker genes. For visualization, the top 40 principal components were reduced to two dimensions using UMAP with parameters (n_neighbors=15, min_dist=0.4).

The filtered human scSeq dataset contained 33358 cells. Each of the samples contained cells from both the olfactory and respiratory epithelium, although the frequency of OSNs and respiratory cells varied across patients, as previously described (38). 295 cells expressed ACE2 and 4953 cells expressed TMPRSS2. Of the 865 identified OSNs, including both immature and mature cells, none of the cells express ACE2 and only 2 (0.23%) expressed TMPRSS2. In contrast, ACE2 was reliably detected in at least 2% and TMPRSS2 was expressed in close to 50% of multiple respiratory epithelial subtypes. The expression of both known cell type markers and known CoV-related genes was also examined across respiratory and olfactory epithelial cell types. For these gene sets, the mean expression in each cell type was calculated and normalized by the maximum across cell types.

Data from Deprez et al. (41) were downloaded from the Human Cell Atlas website (https://www.genomique.eu/cellbrowser/HCA/; Single-cell atlas of the airway epithelium (Grch38 human genome)). A subset of these data was combined with a subset of the Durante data for mapping between cell types. For the Deprez data, the subset consisted of samples from the nasal RE that belonged to a cell type with >20 cells, including Basal, Cycling Basal, Suprabasal, Secretory, Mucous Multiciliated cells, Multiciliated, SMS Goblet and Ionocyte. We observed two distinct subpopulations of Basal cells, with one of the two populations distinguished by expression of Cxcl14. The cells in this population were manually identified using SPRING and defined for downstream analysis as a separate cell type annotation called Basal (Cxcl14+). For the Durante data, the subset consisted of cells from cell types that had some putative similarity to cells in the Deprez dataset, including Olfactory HBC, Cycling respiratory HBC, Respiratory HBC, Early respiratory secretory cells, Respiratory secretory cells, Sustentacular cells, Bowmans gland, Olfactory microvillar cells.

To establish a cell type mapping:

1) Durante et al. (38) and Deprez et al. (41) data were combined and gene expression values were linearly scaled so that all cells across datasets had the same total counts. PCA was then performed using highly variable genes (n=1477 genes) and PCA-batch-correction (93) with the Durante et al. data as a reference set.

2) Mapping was then performed bidirectionally between the two datasets. Each cell from Dataset 1 voted for the 5 most similar cells in the Dataset 2, using distance in PCA space as the measure of similarity. A table T counting votes across cell types was then computed, where for cell type i in the Dataset 1 and cell type j in the Dataset 2,

Tij = {number of votes cast from cells of type i to cells of type j}Thus, if Dataset 1 has N cells, then T would count 5*N votes (Tij=5N)

3) The table of votes T was Z-scored against a null distribution, generated by repeating the procedure above 1000 times with shuffled cell type labels.

The resulting Z-scores were similar between the two possible mapping directions (Durante -> Deprez vs. Deprez -> Durante; R=0.87 Pearson correlation of mapping Z-scores). The mapping Z-scores were also highly robust upon varying the number of votes-cast per cell (R>0.98 correlation of mapping Z-scores upon changing the vote numbers to 1 or 50 as opposed to 5). Only cell-type correspondences with a high Z-score in both mapping directions (Z-score > 25) were used for downstream analysis.

To establish a common scale of gene expression between datasets, we restricted to cell type correspondences that were supported both by bioinformatic mapping and shared a nominal cell type designation based on marker genes. These included: Basal/suprabasal cells = respiratory HBCs from Durante et al., and basal and suprabasal cells from Deprez et al. Secretory cells = rly respiratory secretory cells and respiratory secretory cells from Durante et al., and secretory cells from Deprez et al. Multiciliated cells = respiratory ciliated cells from Durante et al., and multiciliated cells from Deprez et al.

We next sought a transformation of the Durante et al. data so that it would agree with the Deprez et al. data within the corresponding cell types identified above To account for differing normalization strategies applied to each dataset prior to download (log normalization and rescaling with cell-specific factors for Deprez et al. but not for Durante et al.), we used the following ansatz for the transformation, where the pseudocount p is a global latent parameter and the rescaling factors fi are fit to each gene separately. In the equation below, T denotes the transformation and eij represents a gene expression value for cell i and gene j in the Durante data:

The parameter p was fit by maximizing the correlation of average gene expression across all genes between each of the cell type correspondences listed above. The rescaling factors fi. were then fitted separately for each gene by taking the quotient of average gene expression between the Deprez et al. data and the log-transformed Durante et al. data, again across the cell type correspondences above.

Normalized gene expression tables were obtained from previous published datasets (43, 46-48) (Table 1). For the mouse data sets, the means of the replicates from WOM or OSN were used to calculate Log2 fold changes. For the mouse data from Saraiva et al. and the primate data sets (43, 46), the normalized counts of the genes of interest from individual replicates were plotted.

Three different mouse bulk RNA-seq datasets were used, each with replicates from WOM or purified OSNs. An additional dataset contained bulk RNA-seq data from humans and non-human primates.

Tissue dissection and single-cell dissociation for nasal epithelium. A new dataset of whole olfactory mucosa scSeq was generated from adult male mice (812 weeks-old). All mouse husbandry and experiments were performed following institutional and federal guidelines and approved by Harvard Medical Schools Institutional Animal Care and Use Committee (IACUC). Briefly, dissected main olfactory epithelium were cleaned up in 750 l of EBSS (Worthington) and epithelium tissues were isolated in 750 L of Papain (20 U/mL in EBSS) and 50 L of DNase I (2000 U/mL). Tissue pieces were transferred to a 5 mL round-bottom tube (BD) and 1.75 mL of Papain and 450 L of DNase I were added. After 11.5 hour incubation with rocking at 37C, the suspension was triturated with a 5 mL pipette 15 times and passed through 40 m cell strainer (BD) and strainer was washed with 1 mL of DMEM + 10% FBS (Invitrogen). The cell suspension was centrifuged at 300 g for 5 min. Cells were resuspended with 4 mL of DMEM + 10% FBS and centrifuged at 300 g for 5 min. Cells were suspended with PBS + 0.01% BSA and concentration was measured by hemocytometer.

Drop-seq experiments. Drop-seq experiments were performed as previously described (94). Microfluidics devices were obtained from FlowJEM and barcode beads were obtained from chemgenes. 8 of 15 min Drop-seq runs were collected in total, which were obtained from 5 mice.

Sequencing of Drop-seq samples. 8 replicates of Drop-seq samples were sequenced across 5 runs on an Illumina NextSeq 500 platform. Paired end reads from the fastq files were trimmed, aligned, and tagged via the Drop-seq tools (v1.13) pipeline, using STAR (v2.5.4a) with genomic indices from Ensembl Release 93. The digital gene expression matrix was generated for 4,000 cells for 0126_2, 5,000 cells for 0105, 0126_1, 051916_DS11, 051916_DS12, 051916_DS22, 5,500 cells for 051916_DS21, and 9,500 cells for 0106.

Preprocessing of Drop-seq samples. Processing of the WOM Drop-seq samples was performed in Seurat (v2.3.1). Cells with less than 500 UMIs or more than 15,000 UMIs, or higher than 5% mitochondrial genes were removed. Potential doublets were removed using Scrublet. Cells were initially preprocessed using the Seurat pipeline. Variable genes FindVariableGenes (y.cutoff = 0.6) were scaled (regressing out effects due to nUMI, the percent of mitochondrial genes, and replicate ids) and the data was clustered using 50 PCs with the Louvain algorithm (resolution=0.8). In a fraction of sustentacular cells, we observed co-expression of markers for sustentacular cells and other cell types (e.g., OSNs). Re-clustering of sustentacular cells alone separately out these presumed doublets from the rest of the sustentacular cells, and the presumed doublets were removed for the analyses described below.

Processing of Drop-seq samples. The filtered cells from the preprocessing steps were reanalyzed in python using Scanpy and SPRING. In brief, the raw gene counts in each cell were total counts normalized and variable genes were identified using the SPRING gene filtering function filter_genes with parameters (85, 3, 3); mitochondrial and olfactory receptor genes were excluded from the variable gene lists. The resulting 2083 variable genes were z-scored and the dimensionality of the data was reduced to 35 via principal component analysis. The k-nearest neighbor graph (n_neighbors=15) of these 35 PCs was clustered using the leiden algorithm (resolution=1.2) and was reduced to two dimensions for visualization via the UMAP method (min_dist=0.42). Clusters were manually annotated on the basis of known marker genes and those sharing markers (e.g., olfactory sensory neurons) were merged.

The mouse WOM Drop-seq dataset contained 29585 cells that passed the above filtering. Each of the 16 clusters identified contained cells from all 8 replicates in roughly equal proportions. Of the 17666 mature OSNs and the 4674 immature OSNs, none of the cells express Ace2. In contrast, in the olfactory epithelial cells, Ace2 expression was observed in the Bowmans gland, olfactory HBCs, dorsal sustentacular cells.

Mouse olfactory epithelium tissue processing. Mice were sacrificed with a lethal dose of xylazine and nasal epithelium with attached olfactory bulbs were dissected and fixed in 4% paraformaldehyde (Electron Microscope Sciences, 19202) in phosphate-buffered saline (PBS) for overnight at 4C or for 2 hours at room temperature. Tissues were washed in PBS for 3 times (5 min each) and incubated in 0.45M EDTA in PBS overnight at 4C. The following day, tissues were rinsed by PBS and incubated in 30% Sucrose in PBS for at least 30 min, transferred to Tissue Freezing Medium (VWR, 15146-025) for at least 45 min and frozen on crushed dry ice and stored at -80C until sectioning. Tissue sections (20 m thick for the olfactory bulb and 12 m thick for nasal epithelium) were collected on Superfrost Plus glass slides (VWR, 48311703) and stored at -80C until immunostaining.

For methimazole treated samples, Adult C57BL/6J mice (6-12 weeks old, JAX stock No. 000664) were given intraperitoneal injections with Methimazole (Sigma M8506) at 50 g/g body weight and sacrificed at 24, 48, and 96-hour timepoints.

Immunostaining for mouse tissue. Sections were permeabilized with 0.1% Triton X-100 in PBS for 20 min then rinsed 3 times in PBS. Sections were then incubated for 45-60 min in blocking solution that consisted of PBS containing 3% Bovine Serum Albumin (Jackson Immunoresearch, 001-000-162) and 3% Donkey Serum (Jackson ImmunoResearch, 017-000-121) at room temperature, followed by overnight incubation at 4C with primary antibodies diluted in the same blocking solution. Primary antibodies used are as follows. Goat anti-ACE2 (Thermo Fisher, PA5-47488, 1:40), mouse anti-TUBB4 (Sigma, T7941, 1:4000), rabbit anti-KRT5 (abcam, ab52635, 1:200), goat anti-NQO1 (abcam, ab2346, 1:200), mouse anti-acetylated Tubulin (abcam, ab24610, 1:500), rabbit anti-CNGA2 (abcam, ab79261, 1:100), rat anti-CD140b/PDGFRB (Thermo Fisher, 14-1402-82, 1:100).

On the following day, sections were rinsed once and washed three times for 5-10 min in PBS, then incubated for 45 min with secondary antibodies diluted in blocking solution at 1:300 ratios and/or Alexa 555-conjugated Phalloidin (1:400). Secondary antibodies used were as follows: Donkey anti-Goat IgG Alexa 488 (Jackson ImmunoResearch, 705-546-147), donkey anti-Goat IgG Alexa 555, (Invitrogen, A21432), donkey anti-Rabbit IgG Alexa 555 (Invitrogen, A31572), donkey anti-Rabbit IgG Alexa 647 (Jackson ImmunoResearch, 711-605-152), donkey anti-Mouse IgG Alexa 555 (Invitrogen, A31570), donkey anti-Mouse IgG Alexa 647 (Invitrogen, A31571), and donkey anti-Rat IgG Alexa 488 (Invitrogen, A21208).

After secondary antibody incubation, sections were washed twice for 5-10 min in PBS, incubated with 300 nM DAPI in PBS for 10 min and then rinsed with PBS. Slides were mounted with glass coverslips using Vectashield Mounting Medium (Vector Laboratories, H-1000) or ProLong Diamond Antifade Mountant (Invitrogen, P36961).

For co-staining of ACE2 and NQO1, slides were first stained with ACE2 primary antibody and donkey anti-Goat IgG Alexa 488 secondary. After 3 washes of secondary antibody, tissues were incubated with unconjugated donkey anti-Goat IgG Fab fragments (Jackson ImmunoResearch, 705-007-003) at 30 g/mL diluted in blocking solution for 1 hour at room temperature. Tissues were washed twice with PBS, once in blocking solution, and incubated in blocking solution for 30-40 min at room temperature, followed by a second round of staining with the NQO1 primary antibody and donkey anti-Goat IgG Alexa 555 secondary antibody.

Confocal images were acquired using a Leica SPE microscope (Harvard Medical School Neurobiology Imaging Facility) with 405 nm, 488 nm, 561 nm, and 635 nm laser lines. Multi-slice z-stack images were acquired, and their maximal intensity projections are shown. For Fig. 5A, tiled images were acquired and stitched by the Leica LAS X software. Images were processed using Fiji ImageJ software (95), and noisy images were median-smoothed using the Remove Outliers function built into Fiji.

Fluorescent in situ hybridization for mouse tissue. Sult1c1 RNA was detected by fluorescent RNAscope assay (Advanced Cell Diagnostics, kit 320851) using probe 539921-C2, following the manufacturers protocol (RNAscope Fluorescent Multiplex Kit User Manual, 320293-UM Date 03142017) for paraformaldehyde-fixed tissue. Prior to initiating the hybridization protocol, the tissue was pre-treated with two successive incubations (first 30 min, then 15 min long) in RNAscope Protease III (Advanced Cell Diagnostics, 322337) at 40C, then washed in distilled water. At the end of protocol, the tissue was washed in PBS and subjected to the 2-day immunostaining protocol described above.

Immunostaining of human nasal tissue. Human olfactory mucosa biopsies were obtained via IRB-approved protocol at Duke University School of Medicine, from nasal septum or superior turbinate during endoscopic sinus surgery. Tissue was fixed with 4% paraformaldehyde and cryosectioned at 10 m and sections were processed for immunostaining, as previously described (38).

Sections from a 28-year old female nasal septum biopsy were stained for ACE2 (Fig. 2E) using the same Goat anti-ACE2 (Thermo Fisher, PA5-47488, 1:40) and the protocol described above for mouse tissue. The human sections were co-stained with Rabbit anti-keratin 5 (Abcam, ab24647; AB_448212, 1:1000) and were detected with AlexaFluor 488 Donkey anti-goat (Jackson ImmunoResearch, 705-545-147) and AlexaFluor 594 Donkey anti-rabbit (Jackson ImmunoResearch, 711-585-152) secondary antibodies (1:300).

As further validation of ACE2 expression and to confirm the lack of ACE2 expression in human olfactory sensory neurons (Figure S2), sections were stained with a rabbit anti-ACE2 (Abcam, ab15348; RRID:AB_301861, used at 1:100) antibody immunogenized against human ACE2 and a mouse Tuj1 antibody against neuron-specific tubulin (BioLegend, 801201; RRID:AB_2313773). Anti-ACE2 was raised against a C-terminal synthetic peptide for human ACE2 and was validated by the manufacturer to not cross-react with ACE1 for immunohistochemical labeling of ACE2 in fruit bat nasal tissue as well as in human lower airway. Recombinant human ACE2 abolished labeling with this antibody in a previous study in human tissue, further demonstrating its specificity (53). The Tuj1 antibody was validated, as previously described (38). Biotinylated secondary antibodies (Vector Labs), avidin-biotinylated horseradish peroxidase kit (Vector) followed by fluorescein tyramide signal amplification (Perkin Elmer) were applied per manufacturers instructions. For dual staining, Tuj1 was visualized using AlexaFluor 594 Goat anti-Mouse (Jackson ImmunoResearch, 115-585-146; RRID: AB_2338881).

Human sections were counterstained with 4,6-diamidino-2-phenylindole (DAPI) and coverslips were mounted using Prolong Gold (Invitrogen) for imaging, using a Leica DMi8 microscope system. Images were processed using Fiji ImageJ software (NIH). Scale bars were applied directly from the Leica acquisition software metadata in ImageJ Tools. Unsharp Mask was applied in ImageJ, and brightness/contrast was adjusted globally.

Mice. 2 month-old and 18 month-old wild type C57BL/6J mice were obtained from the National Institute on Aging Aged Rodent Colony and used for the WOM experiments; each experimental condition consisted of one male and one female mouse to aid doublet detection. Mice containing the transgenic Krt5-CreER(T2) driver (96) and Rosa26-YFP reporter allele (97) were used for the HBC lineage tracing dataset. All mice were assumed to be of normal immune status. Animals were maintained and treated according to federal guidelines under IACUC oversight at the University of California, Berkeley.

Single-Cell RNA Sequencing. The olfactory epithelium was surgically removed, and the dorsal, sensory portion was dissected and dissociated, as previously described (36). For WOM experiments, dissociated cells were subjected to fluorescence-activated cell sorting (FACS) using propidium iodide to identify and select against dead or dying cells; 100,000 cells/sample were collected in 10% FBS. For the HBC lineage tracing experiments Krt5-CreER; Rosa26YFP/YFP mice were injected once with tamoxifen (0.25 mg tamoxifen/g body weight) at P21-23 days of age and sacrificed at 24 hours, 48 hours, 96 hours, 7 days and 14 days post-injury, as previously described (36, 98). For each experimental time point, YFP+ cells were isolated by FACS based on YFP expression and negative for propidium iodide, a vital dye.

Cells isolated by FACS were subjected to single-cell RNA-seq. Three replicates (defined here as a FACS collection run) per age were analyzed for the WOM experiment; at least two biological replicates were collected for each experimental condition for the HBC lineage tracing experiment. Single cell cDNA libraries from the isolated cells were prepared using the Chromium Single Cell 3 System according to the manufacturers instructions. The WOM preparation employed v3 chemistry with the following modification: the cell suspension was directly added to the reverse transcription master mix, along with the appropriate volume of water to achieve the approximate cell capture target. The HBC lineage tracing experiments were performed using v2 chemistry. The 0.04% weight/volume BSA washing step was omitted to minimize cell loss. Completed libraries were sequenced on Illumina HiSeq4000 to produce paired-end 100nt reads.

Sequence data were processed with the 10x Genomics Cell Ranger pipeline (2.0.0 for v2 chemistry), resulting in the initial starting number before filtering of 60,408 WOM cells and 25,469 HBC lineage traced cells. The scone R/Bioconductor package (99) was used to filter out lowly-expressed genes (fewer than 2 UMIs in fewer than 5 cells) and low-quality libraries (using the metric_sample_filter function with arguments hard_nreads = 2000, zcut = 4).

Preliminary Filtering. Cells with co-expression of male (Ddx3y, Eif2s3y, Kdm5d, and Uty) and female marker genes (Xist) were removed as potential doublets from the WOM dataset. For both datasets, doublet cell detection was performed per sample using DoubletFinder (100) and Scrublet (101). Genes with at least 3 UMIs in at least 5 cells were used for downstream clustering and cell type identification. For the HBC lineage tracing dataset, the Bioconductor package scone was used to pick the top normalization (none,fq,ruv_k=1,no_bio,batch), corresponding to full quantile normalization, batch correction and removing one factor of unwanted variation using RUV (102). A range of cluster labels were created by clustering using the partitioning around medoids (PAM) algorithm and hierarchical clustering in the clusterExperiment Bioconductor package (103), with parameters k0s= (10, 13, 16, 19, 22, 25) and alpha=(NA,0.1,0.2,0.3). Clusters that did not show differential expression were merged (using the function mergeClusters with arguments mergeMethod = adjP, cutoff = 0.01, and DEMethod = limma for the lineage-traced dataset). Initial clustering identified one Macrophage (Msr1+) cluster consisting of 252 cells; upon its removal and restarting from the normalization step a subsequent set of 15 clusters was obtained. These clusters were used to filter out 1515 cells for which no stable clustering could be found (i.e., unassigned cells), and four clusters respectively consisting of 31, 29 and 23 and 305 cells. Doublets were identified using DoubletFinder and 271 putative doublets were removed. Inspection of the data in a three-dimensional UMAP embedding identified two groups of cells whose experimentally sampled timepoint did not match their position along the HBC differentiation trajectory, and these additional 219 cells were also removed from subsequent analyses.

Analysis of CoV-related genes in WOM and HBC lineage 10x datasets. Analysis of WOM scSeq data were performed in python using the open-source Scanpy software starting from the raw UMI count matrix of the 40179 cells passing the initial filtering and QC criteria described above. UMIs were total-count normalized and scaled by 10,000 (TPT, tag per ten-thousands) and then log-normalized. For each gene, the residuals from linear regression models using the total number of UMIs per cell as predictors were then scaled via z-scoring. PCA was then performed on a set of highly-variable genes (excluding OR genes) calculated using the highly_variable_genes function with parameters: min_mean=0.01, max_mean=10, min_disp=0.5. A batch corrected neighborhood graph was constructed by the bbknn function with 42 PCs with the parameters: local_connectivity=1.5, and embedding two-dimensions using the UMAP function with default parameters (min_dist = 0.5). Cells were clustered using the neighborhood graph via the Leiden algorithm (resolution = 1.2). Identified clusters were manually merged and annotated based on known marker gene expression. We removed 281 cells containing mixtures of marker genes with no clear gene expression signature. The identified cell types and the number of each of the remaining 39898 cells detected were as follows. 28,769 mOSN: mature OSN; 2,607 iOSN: immature OSN; 859 INP: Immediate Neural Precursor; 623 GBC: Globose Basal Cell; HBC: Horizontal Basal Cell (1,083 Olfactory and 626 Respiratory); 480 SUS: sustentacular cell; 331 BG: Bowmans gland; MV: Microvillar cell (563 Brush-like and 1,530 Ionocyte-like); 92 OEC: Olfactory Ensheathing Cell; 76 Resp. Secretory cells; 227 Resp. unspecified cells; 172 atypical OSN; 1,757 various immune cells, 103 RBC: Red Blood Cell. TPT gene expression levels were visualized in two-dimensional UMAP plots.

The filtered HBC lineage dataset containing 21722 cells was analyzing in python and processed for visualization using pipelines in SPRING and Scanpy (104, 105). In brief, total counts were normalized to the median total counts for each cell and highly variable genes were selected using the SPRING gene filtering function (filter_genes) using parameters (90, 3, 3). The dimensionality of the data was reduced to 20 using principal components analysis (PCA) and visualized in two-dimensions using the UMAP method with parameters (n_neighbors=20, min_dist=0.5). Clustering was performed using the Leiden algorithm (resolution=1.45) and clusters were merged manually using known marker genes. The identified cell types and number of each type were: 929 mOSN: mature OSN; 2073 iOSN: immature OSN; 786 INP: Immediate Neural Precursor; 755 GBC: Globose Basal Cell; HBC: Horizontal Basal Cell (7782 Olfactory, 5418 Regenerating, and 964 Respiratory); 2666 SUS: sustentacular cell; and 176 Ionocyte-like Microvillar (MV) cell.

Expression of candidate CoV-2-related genes was defined if at least one transcript (UMI) was detected in that cell, and the percent of cells expressing candidate genes was calculated for each cell type. In the WOM dataset Ace2 was only detected in 2 out of 28,769 mature OSNs (0.007%), and in the HBC lineage dataset, Ace2 was not detected in any OSNs. Furthermore, Ace2 was not detected in immature sensory neurons (GBCs, INPs, or iOSNs) in either dataset.

Single-cell RNA-seq data from HBC-derived cells from Fletcher et al. and Gadye et al. (36, 98), labeled via Krt5-CreER driver mice, were downloaded from GEO at accession GSE99251 using the file GSE95601_oeHBCdiff_Cufflinks_eSet_counts_table.txt.gz. Processing was performed as described above, including total counts normalization and filtering for highly variable genes using the SPRING gene filtering function filter_genes with parameters (75, 20, 10). The resulting data were visualized in SPRING and a subset of cells were removed for quality control, including a cluster of cells with low total counts and another with predominantly reads from ERCC spike-in controls. Putative doublets were also identified using Scrublet and removed (6% of cells) (101). The resulting data were visualized in SPRING and partitioned using Louvain clustering on the SPRING k-nearest-neighbor graph using the top 40 principal components. Cell type annotation was performed manually using the same set of markers genes listed above. Three clusters were removed for quality control, including one with low total counts and one with predominantly reads from ERCC spike-in controls (likely background), and one with high mitochondrial counts (likely stressed cells). For visualization, and clustering the remaining cells were projected to 15 dimensions using PCA and visualized with UMAP with parameters (n_neighbors=15, min_dist=0.4, alpha=0.5, maxiter=500). Clustering was performed using the Leiden algorithm (resolution=0.4) and cell types were manually annotated using known marker genes.

The filtered dataset of mouse HBC-derived cells contained 1450 cells. The percent of cells expressing each marker gene was calculated as described above. Of the 51 OSNs identified, none of them expressed Ace2, and only 1 out of 194 INPs and iOSNs expressed Ace2. In contrast, Ace2 and Tmprss2 were both detected in HBCs and SUS cells.

Juvenile mouse data. Single-cell RNAseq data from whole mouse olfactory bulb (50) were downloaded from mousebrain.org/loomfiles_level_L1.html in loom format (l1 olfactory.loom) and converted to a Seurat object. Samples were obtained from juvenile mice (age postnatal day 26-29). This dataset comprises 20514 cells passing cell quality filters, excluding 122 cells identified as potential doublets.

Tissue dissection and single-cell dissociation. A new dataset of whole olfactory bulb scSeq was generated from adult male mice (812 weeks-old). All mouse husbandry and experiments were performed following institutional and federal guidelines and approved by Harvard Medical Schools Institutional Animal Care and Use Committee (IACUC). Briefly, dissected olfactory bulbs (including the accessory olfactory bulb and fractions of the anterior olfactory nucleus) were dissociated in 750 l of dissociation media (DM: HBSS containing 10mM HEPES, 1 mM MgCl2, 33 mM D-glucose) with 28 U/mL Papain and 386 U/mL DNase I (Worthington). Minced tissue pieces were transferred to a 5 mL round-bottom tube (BD). DM was added to a final volume of 3.3 mL and the tissue was mechanically triturated 5 times with a P1000 pipette tip. After 1-hour incubation with rocking at 37C, the suspension was triturated with a 10 mL pipette 10 times and 2.3 mL was passed through 40 m cell strainer (BD). The suspension was then mechanically triturated with a P1000 pipette tip 10 times and 800 L were filtered on the same strainer. The cell suspension was further triturated with a P200 pipette tip 10 times and filtered. 1 mL of Quench buffer (22 mL of DM, 2.5 mL of protease inhibitor prepared by resuspending 1 vial of protease inhibitor with 32 mL of DM, and 2000U of DNase I) was added to the suspension and centrifuged at 300 g for 5 min. Cells were resuspended with 3 mL of Quench buffer and overlaid gently on top of 5 mL of protease inhibitor, then spun down at 70 g for 10min. The pellet was resuspended using DM supplemented with 0.04% BSA and spun down at 300 g for 5 min. Cells were suspended in 400 L of DM with 0.04% BSA.

Olfactory bulb Drop-seq experiments. Drop-seq experiments were performed as previously described (94). Microfluidics devices were obtained from FlowJEM and barcode beads were obtained from chemgenes. Two 15 min Drop-seq runs were collected from a single dissociation preparation obtained from 2 mice. Two such dissociations were performed, giving 4 total replicates.

Sequencing of Drop-seq samples. 4 replicates of Drop-seq samples were pooled and sequenced across 3 runs on an Illumina NextSeq 500 platform. Paired end reads from the fastq files were trimmed, aligned, and tagged via the Drop-seq tools (1-2.0) pipeline, using STAR (2.4.2a) with genomic indices from Ensembl Release 82. The digital gene expression matrix was generated for 8,000 cells per replicate.

Preprocessing of Drop-seq samples. Cells with low numbers of genes (500), low numbers of UMIs (700) or high numbers of UMIs (>10000) were removed (6% of cells). Potential doublets were identified via Scrublet and removed (3.5% of cells). Overall, this new dataset comprised 27004 cells.

Integration of whole olfactory bulb scRNAseq datasets. Raw UMI counts from juvenile and adult whole olfactory bulb samples were integrated in Seurat (106). Integrating the datasets ensured that clusters with rare cell types could be identified and that corresponding cell types could be accurately matched. As described below (see Figure S7), although some cell types were observed with different frequencies, the integration procedure yielded stable clusters with cells from both datasets. Briefly, raw counts were log-normalised separately and the 10000 most variable genes identified by variance stabilizing transformation for each dataset. The 4529 variable genes present in both datasets and the first 30 principal components (PCs) were used as features for identifying the integration anchors. The integrated expression matrix was scaled and dimensionality reduced using PCA. Based on their percentage of explained variance, the first 28 PCs were chosen for UMAP visualization and clustering.

Graph-based clustering was performed using the Louvain algorithm following the standard Seurat workflow. Cluster stability was analyzed with Clustree on a range of resolution values (0.4 to 1.4), with 0.6 yielding the most stable set of clusters (107). Overall, 26 clusters were identified, the smallest of which contained only 43 cells with gene expression patterns consistent with blood cells, which were excluded from further visualization plots. Clustering the two datasets separately yielded similar results. Moreover, the distribution of cells from each dataset across clusters was homogenous (Figure S7) and the clusters corresponded previous cell class and subtype annotations (50). As previously reported, a small cluster of excitatory neurons (cluster 13) contained neurons from the anterior olfactory nucleus. UMAP visualizations of expression level for cell class and cell type markers, and for genes coding for coronavirus entry proteins, depict log-normalized UMI counts. The heatmap in Fig. 7C shows the mean expression level for each cell class, normalised to the maximum mean value. The percentage of cells per cell class expressing Ace2 was defined as the percentage of cells with at least one UMI. In cells from both datasets, Ace2 was enriched in pericytes but was not detected in neurons.

Tissue dissociation and manual cell sorting. Acute olfactory bulb 300 m slices were obtained from Dat-Cre/Flox-tdTomato (B6.SJL-Slc6a3tm1.1(cre) Bkmn/J, Jax stock 006660 / B6.Cg Gt(ROSA)26Sortm9(CAG-tdTomato)Hze, Jax stock 007909) P28 mice as previously described (108). As part of a wider study, at P27 these mice had undergone brief 24 hours unilateral naris occlusion via a plastic plug insert (N = 5 mice) or were subjected to a sham control manipulation (N = 5 mice); all observed effects here were independent of these treatment groups. Single cell suspensions were generated using the Neural Tissue Dissociation Kit Postnatal Neurons (Miltenyi Biotec. Cat no. 130-094-802), following manufacturers instructions for manual dissociation, using 3 fired-polished Pasteur pipettes of progressively smaller diameter. After enzymatic and mechanical dissociations, cells were filtered through a 30 m cell strainer, centrifuged for 10 min at 4C, resuspended in 500 l of ACSF (in mM: 140 NaCl, 1.25 KCl, 1.25 NaH2PO4, 10 HEPES, 25 Glucose, 3 MgCl2, 1 CaCl2) with channel blockers (0.1 M TTX, 20 M CNQX, 50 M D-APV) and kept on ice to minimise excitotoxicity and cell death.

For manual sorting of fluorescently labeled dopaminergic neurons we adapted a previously described protocol (109). 50 l of single cell suspension was dispersed on 3.5mm petri dishes (with a Sylgard-covered base) containing 2 ml of ACSF + channel blockers. Dishes were left undisturbed for 15 min to allow the cells to sink and settle. Throughout, dishes were kept on a metal plate on top of ice. tdTomato-positive cells were identified by their red fluorescence under a stereoscope. Using a pulled glass capillary pipette attached to a mouthpiece, individual cells were aspirated and transferred to a clean, empty dish containing 2 ml ACSF + channel blockers. The same cell was then transferred to a third clean plate, changing pipettes for every plate change. Finally, each individual cell was transferred to a 0.2 ml PCR tube containing 2 l of lysis buffer (RLT Plus - Qiagen). The tube was immediately placed on a metal plate sitting on top of dry ice for flash-freezing. Collected cells were stored at -80C until further processing. Positive (more than 10 cells) and negative (sample collection procedure without picking a cell) controls were collected for each sorting session. In total, we collected samples from 10 mice, averaging 50 tdTomato-positive cells collected per session. Overall, less than 2.5 hours elapsed between mouse sacrifice and collection of the last cell in any session.

Preparation and amplification of full-length cDNA and sequencing libraries. Samples were processing using a modified version of the Smart-Seq2 protocol(110). Briefly, 1 l of a 1:2,000,000 dilution of ERCC spike-ins (Invitrogen. Cat. no. 4456740) was added to each sample and mRNA was captured using modified oligo-dT biotinylated beads (Dynabeads, Invitrogen). PCR amplification was performed for 22 cycles. Amplified cDNA was cleaned with a 0.8:1 ratio of Ampure-XP beads (Beckman Coulter). cDNAs were quantified on Qubit using HS DNA reagents (Invitrogen) and selected samples were run on a Bioanalyzer HS DNA chip (Agilent) to evaluate size distribution.

For generating the sequencing libraries, individual cDNA samples were normalised to 0.2ng/l and 1l was used for one-quarter standard-sized Nextera XT (Illumina) tagmentation reactions, with 12 amplification cycles. Sample indexing was performed using index sets A and D (Illumina). At this point, individual samples were pooled according to their index set. Pooled libraries were cleaned using a 0.6:1 ratio of Ampure beads and quantified on Qubit using HS DNA reagents and with the KAPA Library Quantification Kits for Illumina (Roche). Samples were sequenced on two separate rapid-runs on HiSeq2500 (Illumina), generating 100bp paired-end reads. An additional 5 samples were sequenced on MiSeq (Illumina).

Full-length cDNA sequencing data processing and analysis. Paired-end read fastq files were demultiplexed, quality controlled using FastQC (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and trimmed using Trim Galore (https://www.bioinformatics.babraham.ac.uk/projects/trim_galore/). Reads were pseudoaligned and quantified using kallisto (111) against a reference transcriptome from Ensembl Release 89 (Gencode Release M17 GRCm38.p6) with sequences corresponding to the ERCC spike-ins and the Cre recombinase and tdT genes added to the index. Transcripts were collapsed into genes using the sumAcrossFeatures function in scater.

Cell level quality control and cell filtering was performed in scater (112). Cells with <1000 genes, <100,000 reads, >75% reads mapping to ERCC spike-ins, >10% reads mapping to mitochondrial genes or low library complexity were discarded (14% samples). The population of olfactory bulb cells labeled in DAT-tdTomato mice is known to include a minor non-dopaminergic calretinin-positive subgroup (113), so calretinin-expressing cells were excluded from all analyses. The scTransform function in Seurat was used to remove technical batch effects.

An analysis of single-cell gene expression data from 10 studies was performed to investigate the expression of genes coding for coronavirus entry proteins in neurons from a range of brain regions and sensory systems. Processed gene expression data tables were obtained from scSeq studies that evaluated gene expression in retina (GSE81905) (114) inner ear sensory epithelium (GSE115934) (115, 116) and spiral ganglion (GSE114997) (117), ventral midbrain (GSE76381) (118), hippocampus (GSE100449) (119), cortex (GSE107632) (120), hypothalamus (GSE74672) (121), visceral motor neurons (GSE78845) (122), dorsal root ganglia (GSE59739) (123) and spinal cord dorsal horn (GSE103840) (124). Smart-Seq2 sequencing data from Vsx2-GFP positive cells was used from the retina dataset. A subset of the expression matrix that corresponds to day 0 (i.e., control, undisturbed neurons) was used from the layer VI somatosensory cortex dataset. A subset of the data containing neurons from untreated (control) mice was used from the hypothalamic neuron dataset. From the ventral midbrain dopaminergic neuron dataset, a subset comprising DAT-Cre/tdTomato positive neurons from P28 mice was used. A subset comprising Type I neurons from wild type mice was used from the spiral ganglion dataset. The unclassified neurons were excluded from the visceral motor neuron dataset. A subset containing neurons that were collected at room temperature was used from the dorsal root ganglia dataset. Expression data from dorsal horn neurons obtained from C57/BL6 wild type mice, vGat-cre-tdTomato and vGlut2-eGFP mouse lines was used from the spinal cord dataset. Inspection of all datasets for batch effects was performed using the scater package (version 1.10.1) (112). Publicly available raw count expression matrices were used for the retina, hippocampus, hypothalamus, midbrain, visceral motor neurons and spinal cord datasets, whereas the normalized expression data was used from the inner ear hair cell datasets. For datasets containing raw counts, normalization was performed for each dataset separately by computing pool-based size factors that are subsequently deconvolved to obtain cell-based size factors using the scran package (version 1.10.2) (125). Violin plots were generated in scater.

S. H. R. Bagheri, A. M. Asghari, M. Farhadi, A. R. S. Shamshiri, A. Kabir, S.K. Kamrava, M. Jalessi, A. Mohebbi, R. Alizadeh, A.A.Honormand, Coincidence of COVID-19 epidemic and olfactory dysfunction outbreak. medRxiv 20041889, 27 March 2020. .doi:10.1101/2020.03.23.20041889

V. Parma et al., More than smell. COVID-19 is associated with severe impairment of smell, taste, and chemesthesis. medRxiv, 20090902 24 May 2020.

H. J. Hedrich, The Laboratory Mouse. (Academic Press, 2012), pp. 845.

M. Deprez et al., A single-cell atlas of the human healthy airways. bioRxiv 884759 23 December 2019.

W. Sungnak, N. Huang, C. Bcavin, M. Berg, H. C. A. L. B. Network, SARS-CoV-2 entry genes are most highly expressed in nasal goblet and ciliated cells within human airways. arXiv preprint arXiv:2003.06122, (2020).

L. S. Politi, E. Salsano, M. Grimaldi, Magnetic Resonance Imaging Alteration of the Brain in a Patient With Coronavirus Disease 2019 (COVID-19) and Anosmia. JAMA Neurology, (2020).

L. He et al., Pericyte-specific vascular expression of SARS-CoV-2 receptor ACE2 implications for microvascular inflammation and hypercoagulopathy in COVID-19 patients. bioRxiv,088500 (2020). doi:10.1101/2020.05.11.088500

T. Coolen et al., Early postmortem brain MRI findings in COVID-19 non-survivors. medRxiv, 20090316 8 May 2020.

R. Ueha et al., Background mechanisms of olfactory dysfunction in COVID-19: expression of ACE2, TMPRSS2, and Furin in the nose and olfactory bulb in human and mice. bioRxiv, 097352 15 May 2020.

R. Chen et al., The spatial and cell-type distribution of SARS-CoV-2 receptor ACE2 in human and mouse brain. bioRxiv, 030650 9 April 2020.

K. Wang et al., SARS-CoV-2 invades host cells via a novel route: CD147-spike protein. bioRxiv 988345. 14 March 2020.

S. P. Sajuthi et al., Type 2 and interferon inflammation strongly regulate SARS-CoV-2 related gene expression in the airway epithelium. bioRxiv, 034454 10 April 2020.

L. Fodoulian et al., SARS-CoV-2 receptor and entry genes are expressed by sustentacular cells in the human olfactory neuroepithelium. bioRxiv 013268 2 April 2020.

M. Chen et al., Elevated ACE2 expression in the olfactory neuroepithelium: implications for anosmia and upper respiratory SARS-CoV-2 entry and replication. bioRxiv.084996 9 May 2020.

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Non-neuronal expression of SARS-CoV-2 entry genes in the olfactory system suggests mechanisms underlying COVID-19-associated anosmia - Science...

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Stem Cell Rev Rep. 2020 Aug 3. doi: 10.1007/s12015-020-10012-x. Online ahead of print.

ABSTRACT

Severe acute respiratory syndrome corona virus 2 (SARS-CoV-2) is a single stranded RNA virus and responsible for infecting human being. In many cases the individual may remain asymptomatic. Some recently reported studies revealed that individuals of elderly age group and with pre-existing medical conditions such as hypertension, diabetes mellitus had severe consequences, even may lead to death. However, it is not clearly delineated whether hypertension itself or associated comorbidities or antihypertensive therapy contributes to the grave prognosis of COVID-19 infections. This review is aimed to decipher the exact mechanisms involved at molecular level from existing evidence and as reported. It has been reported that SARS-CoV-2 enters into the host cell through interaction between conserved residues of viral spike protein and angiotensin converting enzyme 2 (ACE2) receptor which is highly expressed in hosts cardiac and pulmonary cells and finally transmembrane protease, serine-2 (TMPRSS2), helps in priming of the surface protein. Subsequently, symptom related to multi organ involvement is primarily contributed by cytokine storm. Although various clinical trials are being conducted on renin- angiotensin- system inhibitor, till to date there is no standard treatment protocol approved for critically ill COVID-19 positive cases with pre-existing hypertension. Recently, several studies are carried out to document the safety and efficacy outcome of mesenchymal stem cell transplantation based on its immunomodulatory and regenerative properties. Therefore, identification of future novel therapeutics in the form of mesenchymal stem cell either alone or in combination with pharmacological approach could be recommended for combating SARS-CoV-2 which might be dreadful to debilitating elderly people. Graphical Abstract.

PMID:32748331 | DOI:10.1007/s12015-020-10012-x

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Physiological Relevance of Angiotensin Converting Enzyme 2 As a Metabolic Linker and Therapeutic Implication of Mesenchymal Stem Cells in COVID-19 and...