Archive for the ‘Cardiac Stem Cells’ Category

Abstract

The control of stem and progenitor cell fate is emerging as a compelling urgency for regenerative medicine. Here, we propose a innovative strategy to gain optical control of endothelial colony-forming cell fate, which represents the only known truly endothelial precursor showing robust in vitro proliferation and overwhelming vessel formation in vivo. We combine conjugated polymers, used as photo-actuators, with the advantages offered by optical stimulation over current electromechanical and chemical stimulation approaches. Light modulation provides unprecedented spatial and temporal resolution, permitting at the same time lower invasiveness and higher selectivity. We demonstrate that polymer-mediated optical excitation induces a robust enhancement of proliferation and lumen formation in vitro. We identify the underlying biophysical pathway as due to light-induced activation of TRPV1 channel. Altogether, our results represent an effective way to induce angiogenesis in vitro, which represents the proof of principle to improve the outcome of autologous cell-based therapy in vivo.

In recent years, organic semiconductors have emerged as highly promising materials in biotechnology, thanks to several key-enabling features. Differently from silicon-based electronics, they support both electronic and ionic charge transport (1); they can be easily functionalized with specific excitation and sensing capabilities (24); and they are solution processable, soft, and conformable (5). They are highly biocompatible, being suitable for in vivo implantation and long-term operation, as recently reported for many different applications, including electrocorticography, precise delivery of neurotransmitters, electrocardiography, deep brain stimulation, and spinal cord injury (69). An important, distinctive feature of organic semiconductors is their sensitivity to the visible and near-infrared light. Recently, our and other groups have exploited it for optical modulation of cell electrophysiological activity, by using conjugated polymers and organic molecules as exogenous light-sensitive actuators (1012). Interesting applications have been reported in the field of artificial visual prosthesis (5), photothermal excitation or inhibition of cellular activity (13, 14), and modulation of animal behavior (15).

In this framework, the opportunity to use polymer-based phototransduction mechanisms to regulate the very early stages of living cell development has been very scarcely considered (16, 17). The possibility to selectively and precisely regulate a number of cell processes, such as adhesion, differentiation, proliferation, and migration, would be key to regenerative medicine and drug screening. The presently dominant approaches to reliably regulate stem and progenitor cell fate for regenerative purposes mainly rely on the use of chemical cues. However, irreversibility and lack of spatial selectivity represent important limitations of these methods. Whenever targeting in vivo applications, one must face the major, unsolved problem of diffusion of neurotrophic molecules by the conventional intravenous or oral routes. In addition, the therapeutic outcome of autologous cell-based therapy is often impaired by low engraftment, survival, and poor integration of stem cells within the environment of the targeted tissue. Other stimuli, mainly consisting of mechanical and electrical cues, were recently reported to have some notable effects, and recent advances in nanotechnology and material science enabled versatile, robust, and larger-scale modulation of the cell fate. In particular, carbon-based materials and conjugated polymers led to interesting results (18). However, their distinctive visible light absorption was never exploited in optically driven techniques.

Use of light actuation has been proposed either by viral transfer of light-sensitive proteins, by optogenetics tools, or by absorption of endogenously expressed light-sensitive moieties, based on low lightlevel therapies (1921). In the first case, interesting results were obtained (22); however, this approach bears all the drawbacks related to the need for viral gene transfer. Photobiomodulation led to interesting outputs as well, but overall efficiency is hampered by the limited absorption of light-responsive molecules endogenously expressed in living cells.

In this work, we propose to couple the use of conjugated polymers with visible light excitation to gain optical control of cell fate. We focus our attention on endothelial progenitor cells (EPCs) and, in particular, on endothelial colony-forming cells (ECFCs), which are currently considered the bona fide best surrogate of EPCs (23). ECFCs are mobilized from the bone marrow and vascular stem cell niche to reconstruct the vascular network destroyed by an ischemic insult and to restore local blood perfusion (24). ECFCs may be easily harvested from peripheral blood, display robust clonogenic potential, exhibit tube-forming capacity in vitro, and generate vessel-like structures in vivo (24, 25), thereby representing a promising candidate for autologous cell-based therapy of ischemic disorders (24). Manipulating the signaling pathways that drive ECFC proliferation, migration, differentiation, and tubulogenesis could represent a reliable strategy to improve the regenerative outcome of therapeutic angiogenesis in the harsh microenvironment of an ischemic tissue, such as the infarcted heart (24, 25). Intracellular Ca2+ signals play a crucial role in stimulating ECFC proliferation and tubulogenesis by promoting the nuclear translocation of the Ca2+-sensitive nuclear transcription factor B (NF-B) (2628). It has, therefore, been suggested that intracellular Ca2+ signaling could be targeted to boost the regenerative potential of autologous ECFCs for regenerative purposes (29). For the above-mentioned reasons, ECFCs represent a valuable test bed model for assessing the possibility to exploit the visible light sensitivity of conjugated polymers to gain touchless, optical modulation of cell proliferation and function.

In this framework, we demonstrate that polymer-mediated optical excitation during the first steps of ECFC growth leads to a robust enhancement of both proliferation and tubulogenesis through the optical modulation of the Ca2+-permeable transient receptor potential vanilloid 1 (TRPV1) channel and NF-Bmediated gene expression. Our results represent, to the best of our knowledge, the first report on the use of polymer photoexcitation for the in vitro modulation of ECFC fate and function, thereby representing the proof of principle to obtain direct control of progenitor cell fate.

Figure 1A shows a sketch of the bio/polymer interface developed for obtaining optical control of ECFC proliferation and network formation, together with the polymer chemical structure and the optical absorption spectrum. The material of choice for light absorption and phototransduction is a workhorse organic semiconductor, widely used in photovoltaic and photodetection applications, namely, regioregular poly(3-hexyl-thiophene) (P3HT) (6). It is characterized by a broad optical absorption spectrum, in the blue-green visible region, peaking at 520 nm. P3HT outstanding biocompatibility properties have been reported in a number of different systems, both in vitro and in vivo, including astrocytes (30), primary neurons and brain slices (14), and invertebrate models of Hydra vulgaris (15). Chronical implantation of P3HT-based devices in the rat subretinal space did not show substantial inflammatory reactions up to 6 months in vivo (10). Here, polymer thin films (approximate thickness, 150 nm) have been deposited by spin coating on top of polished glass substrates, as detailed in Materials and Methods. Both polymer-coated and glass substrates have been thermally sterilized (120C, 2 hours), coated with fibronectin, and, lastly, used as light-sensitive and control cell culturing substrates, respectively. ECFCs have been isolated from peripheral blood samples of human volunteers and seeded on top of polymer and glass substrates.

(A) P3HT polymer optical absorption spectrum. Insets show the chemical structure of the conjugated polymer and a sketch of the polymer device used for cell optical activation. ECFCs are cultured on top of P3HT thin films, deposited on glass substrates. (B) ECFC viability at fixed time points after plating (24, 48, and 72 hours). Cell cultures were kept in dark conditions at controlled temperature (37C) and fixed CO2 levels (5%). No statistically significant difference was observed between the glass and polymer substrates at any fixed time point (unpaired Students t test). (C) Experimental setup and optical excitation protocol for evaluation of polymer-mediated cell photoexcitation effects on cell fate. Polymer and control samples are positioned within a sterilized, home-designed petri holder. Light scattering effects are completely screened. The geometry and the photoexcitation protocol have been implemented to minimize overheating effects and to keep the overall extracellular bath temperature fairly unaltered. Thirty-millisecond-long green light pulses are followed by 70 ms in dark condition.

ECFC proliferation on polymer substrates has been primarily assessed in dark conditions at three different time points, namely, 24, 48, and 72 hours after plating (Fig. 1B). Polymer-coated samples, while showing from the very beginning a slightly lower number of cells as compared with control substrates, exhibit a proliferation rate fully similar to cells plated on glass substrates (slope of the linear fitting is 0.034 0.003, R2 = 0.99 and 0.034 0.005, R2 = 0.96 for control and P3HT polymer samples, respectively).

Once assessed that the P3HT polymer surface represents a nicely biocompatible substrate for ECFC seeding and proliferation in the dark, we moved to investigate the effect of polymer photoexcitation. In more detail, to evaluate the effect of optical stimulation on cell proliferation and network formation, we continuously shined light for the whole temporal window required for cell growth, and we realized an ad hoc system suitable for operation within the cell incubator. The experimental configuration and the excitation protocol are schematically represented in Fig. 1C. Optical excitation is provided by a light-emitting diode (LED) source, with maximum emission wavelength at 525 nm, incident from the substrate side. The choice of the protocol, continuously administered to the cell cultures during early seeding and proliferation stages, has been mainly dictated by the need to avoid overheating effects, with possible negative outcomes on the overall cell culture viability. On the basis of these considerations, we opted for a protocol based on 30-ms excitation pulses, followed by a 70-ms dark condition, at a photoexcitation density of 40 mW/cm2. The whole protocol is continuously repeated for a minimum of 4 up to 36 hours, depending on the type of functional assay, at controlled temperature (37C) and CO2 levels (5%).

The temporally precise and spatially localized measurement of the temperature variation upon polymer photoexcitation at the polymer/cell interface (i.e., within the cell cleft) is not straightforward because it requires the use of localized, submicrometer probes with a fast response time. However, according to the heat diffusion equation, we expect that dissipation occurs within a few milliseconds, following exponential decrease dynamics (14). Moreover, we used the well-known method of the calibrated pipette (31) to characterize the temperature variation dynamics within the extracellular bath volume, defined by the cylinder with the base area corresponding to the light spot size and the height of about 1 m. This choice is a good approximation of the overall volume occupied by a single ECFC cell; thus, it provides a realistic estimation of the average heating experienced by the cell (fig. S1A). We observe that temperature variation closely follows short optical pulse dynamics, reaching a maximum temperature at the end of the 30-ms illumination period, quickly followed by an almost complete thermal relaxation to the basal temperature during the 70-ms-long dark period. We conclude that our polymer-based system provides a highly spatially and temporally resolved method for optical excitation, making it possible, in perspective, to selectively target single cells and even cell subcompartments. Upon prolonged illumination (hours), one should also consider possible overheating effects of the whole extracellular medium volume. The average temperature of the bath for the entire duration of the long-term experiment was measured by a thermocouple immersed in the medium. Data show that an equilibrium situation is established after 5 hours and that the absolute temperature of the bath is increased by about 1.5 (fig. S1B). The adopted prolonged excitation protocol does not negatively affect overall cell culture viability (see below).

Figure 2 reports specific effects mediated by P3HT substrates and visible light stimulation on ECFC proliferation. ECFCs were plated in the presence of EGM-2 medium to facilitate the adhesion to the substrate. After 12 hours, the medium was switched to EBM-2 supplemented with 2% fetal bovine serum, and the cells were subjected to the long-term lighting protocol for 36 hours at controlled temperature (37C) and CO2 levels (5%). Under these conditions, ECFCs seeded on P3HT and subjected to light stimulation undergo a significant increase in proliferation rate, as compared with the control condition, i.e., to cells also seeded on P3HT polymer substrates but kept in dark conditions for the whole duration of the experiment (+158% versus P3HT dark; P < 0.05). No statistically significant difference in proliferation was observed among cells seeded on glass, whether they were subjected to optical excitation or not (Fig. 2A).

(A) Relative variation of the proliferation rate of ECFCs subjected to long-term optical excitation seeded on both bare glass and P3HT thin films, together with corresponding control samples kept in dark conditions. Cell proliferation was measured after 36 hours of culture in the presence of EBM-2 supplemented with 2% fetal calf serum. (B) Relative variation of the proliferation rate of ECFCs subjected to long-term optical excitation seeded on P3HT in the absence (CTRL) and presence of 10 M capsazepine (CPZ), 10 M ruthenium red (RR), 20 M RN-1734 (RN-1734), and 30 M BAPTA-AM (BAPTA). The results are represented as the means standard error of the mean (SEM) of three different experiments conducted on cells harvested from three different donors. The significance of differences was evaluated with one-way analysis of variance (ANOVA) coupled with Tukey (A) or Dunnetts (B) post hoc test. *P < 0.05.

Recent evidence demonstrated an interesting correlation between processes key to ECFC vascular regeneration, including proliferation and network formation, and activation of TRPV1 channels, which are expected to be endogenously expressed in ECFCs (32). In addition, we recently reported that polymer photoexcitation leads to selective TRPV1 activation in transfected human embryonic kidney (HEK) cell models (33). Therefore, we were prompted to evaluate whether the increase in cell proliferation is distinctively determined by a polymer-mediated photoactivation of the TRPV1 channel. To this goal, we preliminarily checked the actual expression of the TRPV1 channel in the ECFC models by carrying out electrophysiology experiments in patch-clamp configuration. Methods and results are extensively discussed in the Supplementary Materials (fig. S2 and related description). Briefly, the expression of the TRPV1 channel was confirmed, as well as the capability to selectively excite its activity through localized polymer excitation at high optical power density. To establish whether the TRPV1 channel also has a role in the observed increase in cell proliferation upon polymer excitation, we performed the experiments under light illumination upon administration of a highly specific TRPV1 antagonist [capsazepine (CPZ), 10 M], an aspecific TRPV channel inhibitor [ruthenium red (RR), 10 M], and a selective antagonist of a different temperature-sensitive channel, TRPV4, which is also endogenously expressed in ECFCs (RN-1734, 20 M) (34) (Fig. 2B). TRPV1 inactivation by CPZ and RR results in a relative, strong reduction in cell proliferation by 51 and 30%, respectively, as compared with untreated cells. Conversely, in the case of RN-1734 treatment, the proliferation increase due to polymer photoexcitation is completely unaltered.

As mentioned earlier, intracellular Ca2+ signaling has been reported to drive ECFC proliferation (26, 28). To further investigate whether TRPV1-mediated extracellular Ca2+ entry mediates the proangiogenic response to light illumination, we pretreated ECFCs with [1,2-Bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid tetrakis(acetoxymethyl ester) BAPTA-AM] (30 M), a membrane-permeable buffer of intracellular Ca2+ levels (26, 28). BAPTA-AM is widely used to prevent the increase in intracellular Ca2+ concentration ([Ca2+]i) induced by extracellular stimuli and inhibits the downstream Ca2+-dependent processes. For instance, BAPTA-AM represents the most suitable tool to prevent the activation of Ca2+-sensitive decoders residing within tens of nanometers from the inner pore of plasmalemmal Ca2+ channels (35). It was recently reported that, in the absence of Ca2+-mobilizing growth factors, it does not impair the low rate of ECFC growth (27). Here, however, BAPTA-AM clearly reduced the light-driven proliferation increase, thus confirming that TRPV1 stimulates ECFCs through an increase in [Ca2+]i (Fig. 2B).

We further examined the physiological outcome of chronic light stimulation by carrying out a tube formation assay within an extracellular matrix protein-based scaffold, which is a surrogate of the basement membrane extracellular matrix. This assay recapitulates many steps of the angiogenic process, including adhesion, migration, protease activity, and tubule formation (27, 28). ECFCs were plated in the presence of EBM-2 medium supplemented with 2% fetal calf serum and subjected to the long-term lighting protocol for 8 hours at controlled levels of temperature and CO2. Control experiments carried out in dark conditions, either onto glass (see Fig. 3A for a representative optical image) or onto polymer substrates (Fig. 3C), as well as control experiments carried out upon photoexcitation of cells seeded on glass substrates (Fig. 3B), do not show remarkable differences. Conversely, ECFC cultures subjected to polymer-mediated optical excitation clearly tend to assemble into an extended bidimensional capillary-like network (Fig. 3D). Cell cultures were monitored up to 24 hours after illumination onset, but results were comparable to observations reported here, after 8 hours of illumination. This qualitative observation is fully confirmed by quantitative morphological analysis (27). As depicted in the sketch of Fig. 3E, we quantitatively evaluated the main features typical of the capillary-like network formation and, in particular, the number of master segments (Fig. 3F), master junctions (Fig. 3G), and meshes (Fig. 3H). In all cases, a notable, statistically relevant difference is observed between cells subjected to polymer-mediated optical excitation and controls. Within the same considered temporal window, the combined use of polymer substrates and visible light stimuli does not lead to sizable toxicity effects or delays in cell proliferation. Conversely, it leads to enhanced cell proliferation (Fig. 2) and allows the achievement of the formation of a more extended and mature tubular network (Fig. 3).

(A to D) Representative images of in vitro tubular networks of ECFCs subjected to long-term optical excitation seeded on both bare glass and P3HT, as well as on corresponding control samples in dark conditions. Cultures were observed up to 24 hours, but their appearance did not substantially change after pictures were taken after 8-hour culture. Scale bars, 250 m. (E) Sketch representing the main features typical of the capillary-like network that were considered for the topologic analysis. Number of master segments (F), master junctions (G), and meshes (H) analyzed in the different conditions. The results are represented as the means SEM of three different experiments conducted on cells harvested from three different donors. The significance of differences was evaluated with one-way ANOVA coupled with Tukey post hoc test. **P < 0.01 and ***P < 0.001.

As evidenced for the proliferation rate, the TRPV1 channel activation emerges to play also a fundamental role in tubulogenesis (Fig. 4). The TRPV1 pharmacological blockade with the specific inhibitor CPZ deterministically leads to a marked reduction in network formation (Fig. 4A). Upon CPZ administration, a statistically significant decrease in the relative variation of the number of master segments (Fig. 4E), master junctions (Fig. 4F), and meshes (Fig. 4G) is observed. In line with the results shown in Figs. 2 and 3, RR administration resulted in a less marked but still sizable reduction in the tubular network (Fig. 4, B and E to G), probably due to the minor specificity toward TRPV1, while the protubular effect of light remained fully unaltered in the presence of the TRPV4 inhibitor RN-1734 (Fig. 4, C and E to G). Notably, the treatment with BAPTA-AM (30 M), which affected ECFC proliferation, was able to prevent also in vitro tubulogenesis, thus corroborating the key role of intracellular Ca2+ signaling in the proangiogenic response to light illumination (Fig. 4, D and E to G). Control measurements carried out in dark conditions on polymer substrates upon the considered pharmacological treatments do not show any relevant effect (fig. S4, A to C). Overall, this evidence supports the notion that TRPV1 stimulates ECFC proliferation and network formation and demonstrates that optical excitation, properly mediated by biocompatible polymer substrates, positively affects ECFC fate by spatially and temporally selective activation of the TRPV1 channel.

(A to D) Representative optical images of in vitro tubular network of ECFCs subjected to long-term optical excitation seeded either on bare glass or on P3HT thin films and treated respectively with CPZ (A), RR (B), RN-1734 (C), and BAPTA-AM (D). Scale bars, 250 m. (E to G) Relative variation of number of master segments (E), master junctions (F), and meshes (G) of ECFCs subjected to long-term optical excitation seeded on P3HT in the absence [control (CTRL)] and presence of 10 M CPZ, 10 M RR, 20 M RN-1734 (RN-1734), and 30 M BAPTA-AM (BAPTA). The results are represented as the means SEM of three different experiments conducted on cells harvested from three different donors. The significance of differences was evaluated with one-way ANOVA coupled with Dunnetts post hoc test. *P < 0.05 and **P < 0.01.

We now turn our attention to elucidating the possible mechanisms leading to optically enhanced tubulogenesis, through TRPV1 channel activation, upon prolonged polymer excitation.

Reliable optical modulation of the cell activity mediated by polymer photoexcitation has been reported in several, previous reports, both in vitro, at the level of single cells, and in vivo, at the level of the whole animal, as evidenced by behavioral studies on both invertebrate and vertebrate models. Three different photostimulation mechanisms, active at the polymer/cell interface, have been proposed so far. These include (i) the creation of an interface capacitance, i.e., of a localized electric field, possibly affecting the cell membrane potential (11); (ii) photothermal processes, establishing a localized temperature increase upon polymer photoexcitation (13, 36); and (iii) photoelectrochemical reactions, mainly oxygen reduction processes, leading to a local variation of extracellular and/or intracellular pH (33) and sizable production of reactive oxygen species (ROS), at a nontoxic concentration, and intracellular calcium modulation (37).

In electrophysiological experiments, carried out at a photoexcitation density higher than the one used in chronic stimulation by about two orders of magnitude, we clearly observe TRPV1 excitation, corresponding however to a small variation of the cell membrane potential, in the order of a few millivolts (Supplementary Materials). Thus, upon much lower light intensity, the effects of either direct photothermal channel activation and of photocapacitive charging are expected to be negligible. To further corroborate this hypothesis, we carry out control experiments aimed at disentangling photoelectrical from photothermal transduction processes.

First, we use a different material as a cell-seeding substrate, characterized by optical absorption and heat conductivity similar to the ones typical of P3HT (13) but fully electrically inert (i.e., unable to sustain electronic charge generation upon photoexcitation). The material of choice is a photoresist (MicroPosit S1813). S1813 thin films are realized by spin coating, and deposition parameters are optimized to obtain optical absorbance values similar to the semiconducting polymer samples at the considered excitation wavelength. The capability of photoresist substrates to sustain ECFC proliferation was successfully assessed in a control measurement, obtaining fully comparable results with respect to the P3HT substrates (Fig. 5A). The functional effect eventually driven by photoresist optical excitation on tubulogenesis was then investigated by using the same experimental conditions and analysis technique previously adopted for polymer and glass substrates (Fig. 5B). Data show that long-term photoresist excitation does not lead to sizable enhancement of the cellular network formation, thus pointing out that a purely photothermal effect does not play a major role in boosting the tubulogenesis process at variance with semiconducting polymer substrates. In a complementary experiment, we directly assessed the occurrence of photoelectrochemical reactions at the polymer/extracellular bath interface by measuring ROS production. We previously demonstrated that P3HT polymer thin films exposed to saline electrolytes sustain efficient light-triggered charge generation and charge transfer processes, giving rise to photoelectrochemical reactions (38, 39). Moreover, we also reported that P3HT nanoparticles are efficiently internalized within the cytosol of secondary line cell models (HEK-293) and that their photoexcitation leads to the production of ROS and subsequent intracellular calcium modulation (15, 37). However, the actual capability to sustain photoelectrochemical reactions in the specific experimental conditions used in this work (polymer film deposition conditions, sterilization process, prolonged exposure to specific cellular growth medium in an incubating environment, prolonged exposure to a light excitation protocol, light wavelength, pulses duty cycle, and power density) was never assessed. In particular, direct measurement of intracellular ROS was never carried out in the presence of polymer thin films. To this goal, we realized ECFC cultures on top of polymer and glass control substrates, and we exposed them to the same optical stimulation protocol previously used in the tubulogenesis assay. ROS production was then evaluated by means of a fluorescence experiment based on the use of the well-known ROS probe 2,7-dichlorodihydrofluorescein diacetate (H2DCF-DA) (Fig. 5C). Results show that light induces an increase in ROS production both on glass and polymer substrates. Relative percentage variation amounts to +34 and +200%, respectively, thus pointing out that polymer surface photocatalytic activity plays a major role in the phototransduction phenomenon.

(A) An electrically insulating, thermally conducting material (photoresist) is successfully used as an ECFC seeding substrate. (B) Photoresist long-term photoexcitation does not lead to sizable enhancement in tubulogenesis parameters. (C) Evaluation of intracellular ROS production following long-term photoexcitation protocol of ECFC cultures on polymer and glass substrates (glass dark, n = 629; glass light, n = 656; P3HT dark, n = 686; and P3HT light, n = 583). For each panel, the results are represented as the means SEM of three different experiments conducted on cells harvested from three different donors. The significance of differences was evaluated with unpaired Students t test (A and B) or one-way ANOVA coupled with Tukey post hoc test (C). ***P < 0.001.

Altogether, data in Fig. 5 indicate that photoelectrochemical reactions induced by light at the interface between the organic semiconducting polymer and the extracellular bath play a key role in triggering the observed enhancement in cell network formation through indirect activation of the TRPV1 channel. The occurrence of faradaic phenomena at the polymer/bath interface may give rise to material degradation effects. The photostability of the polymer substrates was carefully checked by optical absorption, photoluminescence, and Raman spectra measurements. By treating the samples with the same experimental protocol used for cell tubulogenesis assays (photoexcitation density, pulses frequency, overall exposure duration, temperature, and humidity levels), no sign of irreversible polymer degradation was observed, as compared with nonilluminated samples (fig. S5).

The Ca2+-sensitive transcription factor NF-B might provide the missing link between the influx of Ca2+ through TRPV1 and the increase in proliferation and tubulogenesis observed in ECFCs upon photostimulation (26). We therefore monitored the nuclear translocation of the cytoplasmic p65 NF-B subunit via immunofluorescence staining and mRNA levels of a number of genes induced during tubulogenesis in an NF-Bdependent manner (26, 40) (Fig. 6). Our data indicate that ECFCs seeded on polymer and subjected to light stimulation have a significantly enhanced p65 NF-B nuclear translocation compared with the control conditions consisting of cells also seeded on P3HT but kept in dark conditions (+35% versus P3HT dark; P < 0.05; Fig. 6, A and B), and seeded on bare glass (+28% versus glass dark; P < 0.05; Fig. 6B). No differences were observed between samples seeded on glass, whether they were subjected to optical excitation or not (fig. S6).

ECFCs seeded on P3HT samples and glass controls are subjected to long-term photostimulation protocol. Corresponding control samples are kept in dark conditions. After photostimulation, p65 NF-B nuclear translocation (A and B) and mRNA levels of tubulogenic/angiogenic genes that have been shown to be activated downstream of NF-B (C) are evaluated. (A) Representative images of immunofluorescence staining showing p65 NF-B (green) nuclear translocation. Cell nuclei are detected by 4,6-diamidino-2-phenylindole (DAPI) (blue). Scale bars, 50 m. (B) Quantitative evaluation of p65 NF-B nuclear translocation, as evidenced by colocalization experiments. Results are expressed as means SEM of the relative percentage of p65 nucleipositively stained cells to the total number of cells (glass dark, n = 151; glass light, n = 125; P3HT dark, n = 147; and P3HT light, n = 159). Ten fields per condition are analyzed. Data are obtained from two different experiments conducted on cells harvested from two different donors. (C) mRNA levels of intercellular adhesion molecule 1 (ICAM1), selectin E (SELE), and matrix metalloproteinases (MMP1, MMP2, and MMP9) are quantified by real-time polymerase chain reaction (PCR). Data are expressed as means SEM of percentage variation with respect to cells grown in the dark (n = 6). The significance of differences was evaluated with unpaired Students t test (C) or one-way ANOVA coupled with Tukey post hoc test (B). *P < 0.05 and **P < 0.01.

In addition, we have checked the expression of nine genes whose expression is known to be induced in endothelial cells during tubulogenesis/angiogenesis in an NF-Bdependent manner. We considered intercellular adhesion molecule 1 (ICAM1); vascular adhesion molecule 1 (VCAM1); selectin E (SELE), matrix metalloproteinases (MMPs) 1, 2, and 9; vascular endothelial growth factor A (VEGFA); cyclooxygenase 2 (COX2, PTGS2); and cyclin D1 (CCND1) (40). Of these, five are significantly up-regulated by light exposure in cells grown on P3HT substrates, namely, ICAM1 (+90% versus P3HT dark; P < 0.05), SELE (+1119%; P < 0.01), MMP1 (+242%; P < 0.01), MMP2 (+467%; P < 0.05), and MMP9 (+458%; P < 0.05) (Fig. 6C). Conversely, VCAM1, VEGFA, PTGS2, and CCND1 do not show relevant variation upon light stimulation (fig. S7A). Light excitation on cells grown on bare glass substrates does not show any significant effect as compared with control samples in dark conditions (fig. S7B).

Therapeutic angiogenesis via autologous EPC transplantation represents a promising strategy to preserve or, at least, partially restore cardiac function after myocardial infarction (24, 41). Nevertheless, the regenerative outcome of EPC-based therapies in preclinical studies was rather disappointing and did not lead to sufficient neovascularization of the ischemic heart (41). This led to the proposal to boost their angiogenic activity by using emerging technologies, including tissue engineering of vascular niches, pharmacological preconditioning, or genetic and epigenetic reprogramming (42). ECFCs are regarded among the most suitable EPC subtypes to induce therapeutic angiogenesis and cardiac regeneration due to their high clonal proliferative potential and ability to assemble into capillary-like structures (23, 24). In addition, they can be easily isolated and expanded from the peripheral blood of patients and healthy donors. It has recently been suggested that their angiogenic activity could be boosted by targeting the intracellular Ca2+ toolkit (29). Here, we target ECFCs by adopting a fully different approach, i.e., by exploiting visible light as a modulation trigger and by the use of a thiophene-based conjugated polymer as the exogenous, light-responsive actuator. We demonstrate that photoexcitation of the organic material deterministically leads to robustly enhanced proliferation and tubulogenesis. Pharmacological assays, supported by electrophysiology experiments, allow the identification of TRPV1 selective excitation as a key player in the molecular pathway leading to macroscopic outcomes, as observed by quantitative analysis of the angiogenic response.

All data unambiguously show that polymer photoexcitation leads to selective activation of the TRPV1 channel, which has recently been shown to be expressed and drive angiogenesis in human ECFCs (32). TRPV1 is a polymodal Ca2+-permeable channel that integrates multiple chemical and physical cues to sense major changes in the local microenvironment of most mammalian cells (43). TRPV1 is activated by either noxious heat (>42C) and acidic solutions (pH < 6.5), whereas mild acidification (pH 6.3) of the extracellular milieus sensitizes TRPV1 to heat stimulation and results in channel activation at temperature thresholds (30 to 32C) well below the normal one (43). ROS production is also expected to further contribute to TRPV1 activation, as previously reported in mouse coronary endothelial cells (44), in which hydrogen peroxide elicits a depolarizing inward current at negative holding potentials. Likewise, ROS may stimulate TRPV1 to depolarize the membrane potential, thereby triggering trains of action potentials in airway C fibers (45, 46).

On the basis of measurements carried out in cells seeded on the photoresist substrate, as well as on direct evaluation of a limited, local temperature increase upon light stimuli during the long-term photoexcitation protocol, we infer that the excitation of the TRPV1 channel through direct photothermal transduction is not the predominant process leading to enhanced tubulogenesis.

We have previously demonstrated that polymer photoexcitation leads to generation of faradaic current, to electron transfer reactions at the polymer/electrolyte interface, and to sizable intracellular enhancement of ROS (37, 38). Briefly, optical excitation of P3HT polymer thin films leads to photoexcited species (Eq. 1), namely, singlets and charge states, which react with the oxygen dissolved in the cell medium, thus reducing oxygen (Eq. 2)P3HT+hP3HT*(1)P3HT*+O2P3HT++O2(2)

The superoxide further evolves, leading to the generation of different ROS and, lastly, ending up with hydrogen peroxide production. It has been reported that extracellular H2O2 can cross the plasma membrane through aquaporin AQP3, thereby triggering intracellular ROS signaling (47, 48). In line with our previous results, we have demonstrated here that intracellular ROS enhancement does occur in ECFCs upon photoexcitation of polymer thin films, thus contributing to TRPV1 activation.

Altogether, the evidence supports the hypothesis of a transduction mechanism mainly governed by photoelectrochemical reactions. Moreover, these same observations could explain why TRPV4, which is also expressed in ECFCs (34), is not sensitive to optical modulation. Although TRPV4 is activated by moderate heat (24 to 38C), it is supposed to be inhibited by local pH variation, although this is still a matter of debate (49, 50).

On the one hand, the role attributed in the phototransduction mechanism to the capability of the polymer to generate and transport electronic charges, as well as to its photocatalytic activity in an aqueous environment, clearly implies the need for a biocompatible, visible lightresponsive, semiconducting material. This excludes any possible implementation of the reported technique by using a thermally conducting, electrically insulating plastic substrate. Suitable cell-seeding materials have to be selected and developed within the wide arena of organic semiconducting polymers. On the other hand, the key role played by ROS raises additional issues about material photostability, cell viability, and overall safety and reliability of the technique. We extensively verified that the main polymer optoelectronic properties are not substantially altered by the exposure to light and to incubating conditions. From the biological point of view, it is very well known that high ROS levels can induce highly toxic effects and, finally, lead to cell death. We notice, however, that the established photoactivation protocol (illuminator geometry and air flow, light photoexcitation density, duty cycle, and repetition rate) has been implemented to avoid any detrimental effect. Accordingly, no toxicity effects were detected for the overall duration of the experiments, as proven by the robust increase in ECFC proliferation and tubulogenesis exposed to light. This observation is consistent with the emerging notion that appropriate ROS levels can exert a signaling role and control angiogenesis in endothelial cells (51).

The biophysical mechanisms whereby the photoactivation of TRPV1 stimulates in vitro angiogenesis in ECFCs deserve a more detailed discussion as well. Earlier work showed that TRPV1 stimulates proliferation and tube formation in vascular endothelial cells by mediating extracellular Ca2+ entry. The following increase in intracellular Ca2+ concentration ([Ca2+]i) leads to the recruitment of several downstream Ca2+-dependent decoders, such as endothelial nitric oxide synthase and Ca2+/calmodulin-dependent protein kinase II (CaMKII) (52). Recently, TRPV1 was found to induce also proliferation and tube formation in ECFCs by mediating the uptake of the endocannabinoid anandamide (32). This study, however, did not investigate whether TRPV1 activation was per se able to stimulate ECFCs by engaging Ca2+-dependent pathways. Intracellular Ca2+ signaling is a crucial determinant of ECFC fate and behavior (2628). Accordingly, light-induced ECFC proliferation and tube formation were markedly reduced by the pharmacological blockade of TRPV1-mediated Ca2+ entry with CPZ and RR and by preventing the subsequent increase in [Ca2+]i with BAPTA-AM. This finding endorses the view that optical excitation stimulates ECFCs through TRPV1-mediated extracellular Ca2+ entry, and we suggest here that this occurs via downstream activation of transcriptional factor NF-B. NF-B has previously been shown to stimulate cell proliferation and tubulogenesis in endothelial cells (53, 54) and in hepatocytes (55). Our group has shown that NF-B triggers the transcriptional program underlying the angiogenic response to extracellular Ca2+ entry in ECFCs (26). Moreover, NF-B activation in response to extracellular stimulation and Ca2+ entry through TRPV1 has also been demonstrated (56, 57). Under resting conditions, NF-B is retained in the cytoplasm by the complex with the inhibitory protein IB. An increase in [Ca2+]i results in IB degradation by ubiquitination, which is triggered upon the Ca2+-dependent phosphorylation of IB. As a consequence, the p65 NF-B subunit is released from IB inhibition and translocates into the nucleus (58) where it induces the expression of multiple proangiogenic genes (40). Consistently, we found that optical excitation significantly boosted the nuclear translocation of p65 in ECFCs cultured on the conjugated polymer compared with those not exposed to light. Robust up-regulation of several angiogenic genes, such as ICAM, SELE, MMP1, MMP2, and MMP9, which are under NF-Bdependent transcriptional control, was also consequently observed. Intriguingly, NF-B also mediates VEGFA-induced gene expression and angiogenesis in vascular endothelial cells (59, 60) through an increase in [Ca2+]i (61). These observations strongly hint at NF-B as the Ca2+-sensitive decoder that translates optical excitation into an angiogenic response in human ECFCs interfaced with the light-sensitive conjugated polymer.

Overall, our findings represent the proof of principle that optical modulation may be successfully exploited to directly control the fate of a progenitor cell population, i.e., ECFCs, which has been shown to support revascularization of ischemic tissues. The in vitro activation of ECFC angiogenic activity is made possible by the use of a biocompatible, light-sensitive polymer as the phototransduction element.

The combined use of optical excitation and organic polymer technology can open interesting perspectives for several different reasons. First, the use of light modulation allows unprecedented spatial and temporal resolution to be achieved in a fully reversible way. Light temporal and spatial patterns can be specifically designed and adapted to different in vitro cell models, allowing ideally endless combinations of possibilities, to finely tune overall output in cell proliferation and network formation. The demonstrated technology is minimally invasive, allows for massive parallelization of experiments, and can be virtually implemented in any cell therapy model in a straightforward way. In addition, the use of different polymers, with lower energy gap and in the form of nanobeads, may pave the way to the optical enhancement of therapeutic angiogenesis in vivo. Further work is needed to understand whether the pattern and/or intensity of the illumination protocol may be adjusted to further boost the angiogenic response. For instance, the optical excitation protocol consisted of 30-ms-long light pulses that were delivered at 1 Hz for 4 (tubulogenesis) up to 36 (proliferation) hours. This is likely to result in oscillations in [Ca2+]i, which are known to deliver the most instructive signal for ECFCs to undergo angiogenesis by inducing the nuclear translocation of the p65 NF-B subunit (26). As the frequency of intracellular Ca2+ oscillations can be artificially manipulated to regulate NF-Bdependent gene expression in virtually any cell type (62), we envisage an additional layer of specificity and control that could be exploited to further improve the angiogenic response to optical excitation. Future work will also be devoted to assess the outcome of optical modulation on patient-derived ECFCs. One of the main hurdles associated to autologous cell-based therapy is the impairment of the angiogenic activity of EPCs, including ECFCs harvested from cardiovascular patients (29). The therapeutic translation of our findings will require the demonstration that light-induced TRPV1 activation boosts angiogenesis also in ECFCs derived from individuals affected by severe cardiovascular disorders, such as hypertension, atherosclerosis, and heart failure. In this view, the combination of organic semiconductors and genetic manipulation to increase endogenous TRPV1 expression could be sufficient to restore the reparative phenotype of autologous ECFCs from cardiovascular patients.

Regioregular P3HT (99.995% purity; Mn 54,000 to 75,000 molecular weight) was purchased from Sigma-Aldrich and used without any further purification. The samples for cell cultures were prepared by spin coating on a square 18 mm by 18 mm glass (VWR International) substrates carefully rinsed in subsequent ultrasonic baths of ultrapure water, acetone, and isopropanol. P3HT solution was prepared in chlorobenzene at a final P3HT concentration of 20 g/liter and spin coated on the cleaned substrates with a two-step recipe: (i) 3 s at 800 rpm and (ii) 60 s at 1600 rpm. Polymer film thickness is about 150 nm.

Microposit S1813 photoresist was purchased from Shipley and used without any further purification. Photoresist thin films were prepared by spin coating on cleaned substrates with a two-step recipe: (i) 3 min at 300 rpm and (ii) 30 s at 2600 rpm. Parameters were adjusted to obtain homogeneous films and similar optical absorbance to the one of the polymer thin films, at the same excitation wavelength used in the long-term stimulation protocol (see below). All films were thermally treated in an oven at 120C for 2 hours for annealing and sterilization. To promote adhesion, samples were coated with fibronectin (from bovine plasma; Sigma-Aldrich) at a concentration of 2 mg/ml in phosphate-buffered saline (PBS) for at least 30 min at 37C and then rinsed with PBS.

ECFCs were isolated from peripheral blood and expanded as shown elsewhere (26). Blood samples (40 ml) collected in EDTA-containing tubes were obtained from healthy male human volunteers aged from 28 to 38 years. The Institutional Review Board at Istituto di Ricovero e Cura a Carattere Scientifico Policlinico San Matteo Foundation in Pavia approved all protocols and specifically approved this study. Informed written consent was obtained according to the Declaration of Helsinki of 1975 as revised in 2008. We focused on the so-called ECFCs, a subgroup of EPCs that are found in the CD34+ CD45 fraction of circulating mononuclear cells (MNCs), exhibit robust proliferative potential, and form capillary-like structures in vitro (23). To isolate ECFCs, MNCs were obtained from peripheral blood by density gradient centrifugation on lymphocyte separation medium for 30 min at 400g and washed twice in EBM-2 with 2% fetal calf serum. A median of 36 106 MNCs (range, 18 to 66) was plated on fibronectin-coated culture dishes (BD Biosciences) in the presence of the endothelial cell growth medium EGM-2 MV (Lonza) containing endothelial basal medium (EBM-2), 5% fetal bovine serum (FBS), recombinant human (rh) EGF, rhVEGF, recombinant human Fibroblast Growth Factor-Basic (rhFGF-B), recombinant human Insulin-like Growth Factor-1 (rhIGF-1), ascorbic acid, and heparin and maintained at 37C in 5% CO2 and humidified atmosphere. Nonadherent cells were discarded after 2 days, and thereafter, medium was changed three times a week. The outgrowth of ECFCs from adherent MNCs was characterized by the formation of a cluster of cobblestone-shaped cells. That ECFC-derived colonies belonged to the endothelial lineage was confirmed by staining with anti-CD31, anti-CD105, anti-CD144, anti-CD146, antivon Willebrand factor, anti-CD45, and anti-CD14 monoclonal antibodies and by assessment of capillary-like network formation in the in vitro tube formation assay.

For our experiments, we have mainly used endothelial cells obtained from early-passage ECFCs (P2-4, which roughly encompasses a 15- to 18-day period) with the purpose to avoid, or maximally reduce, any potential bias due to cell differentiation. However, to make sure that the phenotype of the cells did not change throughout the experiments, in the preliminary experiments, we tested the immunophenotype of ECFCs at different passages, and we found no differences. We also tested whether functional differences occurred when early (P2) and late (P6)passage ECFCs were used by testing the in vitro capacity of capillary network formation in a Cultrex assay and found no differences between early- and late-passage ECFC-derived cells (data not shown).

Electrophysiological recordings were performed using a patch-clamp setup (Axopatch 200B; Axon Instruments) coupled to an inverted microscope (Nikon Eclipse Ti). ECFCs were measured in whole-cell configuration with freshly pulled glass pipettes (3 to 6 M), filled with the following intracellular solution: 12 mM KCl, 125 mM K-gluconate, 1 mM MgCl2, 0.1 mM CaCl2, 10 mM EGTA, 10 mM Hepes, and 10 mM ATP (adenosine 5-triphosphate)Na2. The extracellular solution contained the following: 135 mM NaCl, 5.4 mM KCl, 5 mM Hepes, 10 mM glucose, 1.8 mM CaCl2, 1 mM MgCl2. Only single cells were selected for recordings. Acquisition was performed with the pCLAMP 10 software (Axon Instruments). Membrane currents were low pass filtered at 2 kHz and digitized with a sampling rate of 10 kHz (Digidata 1440 A; Molecular Devices). Data were analyzed with Clampfit (Axon Instruments) and Origin 8.0 (OriginLab Corporation).

For optical excitation of the polymer, a homemade petri cell culture illuminator, compatible with the use within the cell incubator, was designed and implemented. Its design included a black spacer made by fused filament fabrication, both to minimize overheating effects in the extracellular bath and to avoid unwanted light scattering/diffusion effects and cross-talk between different specimens. Optical excitation was provided by a green LED system, whose duty cycle, repetition rate, and intensity were set through a custom-made control circuit, comprising a microcontroller, a digital-to-analog converter, and an analog LED driver. The driver was connected to five green LEDs (SMB1N-525V-02; Roithner LaserTechnik GmbH, Vienna, Austria), with maximum emission wavelength at 525 nm, each carrying a collimator lens reducing the emission angle to 22. This way, up to five 3.5-cm petri dishes can be simultaneously treated with a homogeneous photoexcitation density of 40 mW/cm2. The long-term optical excitation protocol adopted for cell fate modulation consists of 30-ms-long pulses, followed by 70-ms-long dark conditions, continuously repeated for a minimum of 4 up to 36 hours in the case of tubulogenesis and proliferation assays, respectively.

Growth dynamics were evaluated by plating a total of 5 103 ECFC-derived cells into 10-mm fibronectin-treated cloning cylinders (5 104/cm2) in the presence of EGM-2 MV medium to facilitate the adhesion. After 12 hours, the medium was switched to EBM-2 supplemented with 2% fetal calf serum. For the pharmacological treatment, one of compounds was added to the medium: BAPTA (30 M), CPZ (10 M), RN-1734 (20 M), or RR (10 M). Cultures were incubated at 37C (in 5% CO2 and humidified atmosphere), and cell growth was assessed after 36 hours since the beginning of the long-term illumination protocol. At this point, cells were recovered by trypsinization from all the dishes, and the cell number was assessed by counting in a hemocytometer. Preliminary experiments showed no unspecific or toxic effect for each agent when used at these concentrations. Each assay was repeated in triplicate.

ECFC-derived cells from early-passage (P2 to P4) cultures were obtained by trypsinization and resuspended in EBM-2 supplemented with 2% FBS. EPC-derived cells (10 103) per well were plated in Cultrex basement membrane extract (Trevigen Inc., Gaithersburg, MD, USA) 10-mm fibronectin-treated cloning cylinders. Plates were then incubated at 37C, 5% CO2, and capillary network formation was assessed starting from 4 to 24 hours later. At least three different sets of cultures were performed every experimental point. Quantification of tubular structures was performed after 8 hours of incubation by measuring the total length of structures per field with the aid of the ImageJ software (National Institutes of Health, USA; http://rsbweb.nih.gov/ij/). To evaluate the role of TRPV1, the same protocol was repeated in the presence of the following drugs: BAPTA (30 M), CPZ (10 M), RN-1734 (20 M), or RR (10 M).

H2DCF-DA (Sigma-Aldrich) was used for the intracellular detection of ROS. ECFCs were seeded onto polymer and control substrates and subjected to the same photoexcitation protocol used for the in vitro tube formation assay. Immediately after the end of the protocol, cell cultures were incubated with the ROS probe for 30 min. After careful washout of the excess probe from the extracellular medium, the fluorescence of the probe was recorded (excitation/emission wavelengths, 490/520 nm; integration time, 70 ms for H2DCF-DA) with an inverted microscope (Nikon Eclipse Ti) equipped with an Analog-WDM Camera (CoolSNAP MYO, Teledyne Photometrics). To minimize the effects of the spectral overlap between the polymer absorption and emission spectra, and the probe emission, samples were turned upside down by using a homemade chamber with a 500-m-thick channel filled with extracellular medium. Variation of fluorescence intensity was evaluated over regions of interest covering single-cell areas, and reported values represent the average over multiple cells. See figure captions for additional details about statistical analysis. Image processing was carried out with ImageJ and subsequently analyzed with Origin 8.0.

Two sets of P3HT thin films (n = 12) were prepared as described above. The optical absorbance, the emission, and the Raman spectrum were measured immediately after fabrication. Then, all samples were exposed to ECFC growth medium (EBM-2 supplemented with 2% FBS) and incubated at 37C, 5% CO2 for 24 hours. The first set was taken in dark conditions (n = 6), and the second one was treated with the same optical excitation protocol used in the tubulogenesis assays (n = 6). After incubation, absorption, emission, and Raman spectrum were measured again in the same conditions as before. Absorption spectra were recorded by using a spectrophotometer (PerkinElmer Lambda 1040) in transmission mode. Photoluminescence spectra were acquired by using a Jobin-Yvon spectrofluorometer; the excitation wavelength was set at the polymer absorption peak wavelength (530 nm). Resonant Raman spectra were recorded by using visible light excitation at 532 nm (HORIBA Jobin-Yvon HR800 micro-Raman spectrometer system). Laser power intensity on the sample was kept at values lower than 0.03 mW to avoid laser-induced sample degradation. Spectra were typically recorded in the region 600 to 2000 cm1 and were calibrated against the 520.5 cm1 line of an internal silicon wafer. The signal-to-noise ratio was enhanced by repeated acquisitions (100). The measurements were conducted at room temperature (RT), and the resulting spectral resolution was 0.4 cm1.

To examine NF-B p65 subunit translocation into the nucleus in the individual ECFCs, the coverslips were fixed with 4% formaldehyde in PBS (20 min at RT) and permeabilized with 0.1% Triton X-100 in PBS (7 min at RT). Primary rabbit polyclonal anti-p65 antibody (Santa Cruz Biotechnology, catalog no. Sc-372) was applied at a final dilution of 1:100 for 1 hour at 37C in a humidified chamber. After three washes with PBS, secondary chicken anti-rabbit Alexa(488)-conjugated antibody (1:200; Invitrogen, catalog no. A-21441) was applied for 1 hour at RT. After washing (three times in PBS), nuclei were counterstained with 4,6-diamidino-2-phenylindole, dihydrochloride (DAPI; 1:5000 dilution in PBS; 20 min at RT; Invitrogen, catalog no. D1306). Last, the coverslips with cells were mounted on microscope glass slides using Fluoroshield mount medium (Sigma, catalog no. F6182). Fluorescence images were taken with the same fluorescence microscope used for the electrophysiology experiments, using standard DAPI and fluorescein isothiocyanate filters set for the acquisition of DAPI and Alexa(488) fluorescence emission, respectively.

Cells were lysed in 0.5 ml of TRI Reagent (Sigma, catalog no. T9424), and total RNA was extracted according to the manufacturers protocol. One microgram of total RNA was retrotranscribed using SensiFAST cDNA Synthesis Kit (Bioline, London, UK, catalog no. BIO-65054). Real-time polymerase chain reaction (PCR) was performed using iTaq qPCR master mix according to the manufacturers instructions (Bio-Rad, Segrate, Italy, catalog no. 1725124) on a SFX96 Real-Time System (Bio-Rad). As a control, S18 ribosomal subunit was used, whose expression did not change across the conditions. For each gene, Ct was calculated by using the formula Ct = 2^(Ct(gene) Ct(S18)). The data are expressed as a percentage variation between P3HT light and glass light conditions and P3HT dark and glass dark samples, respectively. Sequences of oligonucleotide primers are listed in table S1.

The significance of differences was evaluated with unpaired Students t test or one-way analysis of variance (ANOVA) coupled with Tukey or Dunnetts post hoc test, as appropriate. Data are represented as means standard error of the mean (SEM). P < 0.05 was considered statistically significant. Statistical analysis was performed using the GraphPad Prism 7 software (GraphPad Software Inc., La Jolla, CA).

Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/9/eaav4620/DC1

Fig. S1. Local and global evaluation of the extracellular bath temperature.

Fig. S2. TRPV1 is endogenously expressed in ECFCs, and it is efficiently activated by polymer photostimulation.

Fig. S3. Current clamp measurements in HEK-293 cells.

Fig. S4. Pharmacological study on ECFCs seeded on polymer substrates in the darkEvaluation of effect on tubulogenesis.

Fig. S5. Polymer photostability.

Fig. S6. p65 NF-B nuclear translocation is unaltered in ECFCs seeded on glass subjected to light-induced photostimulation.

Fig. S7. mRNA levels of proangiogenic genes downstream of NF-B signaling.

Table S1. List of oligonucleotide primers used for real-time PCR.

This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial license, which permits use, distribution, and reproduction in any medium, so long as the resultant use is not for commercial advantage and provided the original work is properly cited.

Acknowledgments: We gratefully thank I. Abdel Aziz for the characterization of the homemade petri cell culture illuminator used for long-term optical excitation and P. Falvo for the constructive criticism of the manuscript and the helpful scientific discussions. Funding: This work was jointly supported by the European Research Council (ERC) under the European Unions Horizon 2020 research and innovation program LINCE, grant agreement no. 803621 (M.R.A.), the EU Horizon 2020 FETOPEN-2018-2020 Programme LION-HEARTED, grant agreement no. 828984 (F.L., F.M., and M.R.A.), the Italian Ministry of Education, University and Research (MIUR): Dipartimenti di Eccellenza Program (20182022)Department of Biology and Biotechnology L. Spallanzani, University of Pavia (F.M.), and Fondo Ricerca Giovani from the University of Pavia (F.M.). Author contributions: F.L., F.M., and M.R.A. planned the experiments. F.L. carried out the experimental measurements (electrophysiology, short- and long-term photoexcitation, evaluation of effects on proliferation, tubulogenesis, and ROS production). V.R. provided the ECFC models, took care of the cell cultures, and contributed to the tubulogenesis and proliferation experiments. G.T. prepared the polymer samples. A.D. designed, realized, and optimized the experimental setup for the long-term photoexcitation. L.T. and D.L. carried out the immunofluorescence and real-time PCR assays. P.C. contributed to the methodological discussion about gene expression. F.L. and M.R.A. wrote the main manuscript, with help from F.M. All authors contributed to the data interpretation and approved the final 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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Conjugated polymers optically regulate the fate of endothelial colony-forming cells - Science Advances

Stem cells are biological cells which have the ability to distinguish into specialized cells, which are capable of cell division through mitosis. Amniotic fluid stem cells are a collective mixture of stem cells obtained from amniotic tissues and fluid. Amniotic fluid is clear, slightly yellowish liquid which surrounds the fetus during pregnancy and is discarded as medical waste during caesarean section deliveries. Amniotic fluid is a source of valuable biological material which includes stem cells which can be potentially used in cell therapy and regenerative therapies. Amniotic fluid stem cells can be developed into a different type of tissues such as cartilage, skin, cardiac nerves, bone, and muscles. Amniotic fluid stem cells are able to find the damaged joint caused by rheumatoid arthritis and differentiate tissues which are damaged. Medical conditions where no drug is able to lessen the symptoms and begin the healing process are the major target for amniotic fluid stem cell therapy. Amniotic fluid stem cells therapy is a solution to those patients who do not want to undergo surgery. Amniotic fluid has a high concentration of stem cells, cytokines, proteins and other important components. Amniotic fluid stem cell therapy is safe and effective treatment which contain growth factor helps to stimulate tissue growth, naturally reduce inflammation. Amniotic fluid also contains hyaluronic acid which acts as a lubricant and promotes cartilage growth.

With increasing technological advancement in the healthcare, amniotic fluid stem cell therapy has more advantage over the other therapy. Amniotic fluid stem cell therapy eliminates the chances of surgery and organs are regenerated, without causing any damage. These are some of the factors driving the growth of amniotic fluid stem cell therapy market over the forecast period. Increasing prevalence of chronic diseases which can be treated with the amniotic fluid stem cell therapy propel the market growth for amniotic fluid stem cell therapy, globally. Increasing funding by the government in research and development of stem cell therapy may drive the amniotic fluid stem cell therapy market growth. But, high procedure cost, difficulties in collecting the amniotic fluid and lack of reimbursement policies hinder the growth of amniotic fluid stem cell therapy market.

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The global amniotic fluid stem cell therapy market is segmented on basis of treatment, application, end user and geography: Segmentation by Treatment Allogeneic Amniotic Fluid stem cell therapy Autologous Amniotic Fluid stem cell therapy Segmentation by Application Regenerative medicines Skin Orthopedics Oncology Fetal tissue reconstruction Kidney regeneration Regeneration of neural tissue Cardiac regeneration Lung epithelial regeneration Others Drug research and development Segmentation by End User Hospital Ambulatory Surgical Centers Specialty Clinics Academic and Research Institutes Segmentation by Geography North America Latin America Europe Asia-Pacific Excluding China China Middle East & Africa

Rapid technological advancement in healthcare, and favorable results of the amniotic fluid stem cells therapy will increase the market for amniotic fluid stem cell therapy over the forecast period. Increasing public-private investment for stem cells in managing disease and improving healthcare infrastructure are expected to propel the growth of the amniotic fluid stem cell therapy market.

However, on the basis of geography, global Amniotic Fluid Stem Cell Therapy Market is segmented into six key regions viz. North America, Latin America, Europe, Asia Pacific Excluding China, China and Middle East & Africa. North America captured the largest shares in global Amniotic Fluid Stem Cell Therapy Market and is projected to continue over the forecast period owing to technological advancement in the healthcare and growing awareness among the population towards the new research and development in the stem cell therapy. Europe is expected to account for the second largest revenue share in the amniotic fluid stem cell therapy market. The Asia Pacific is anticipated to have rapid growth in near future owing to increasing healthcare set up and improving healthcare expenditure. Latin America and the Middle East and Africa account for slow growth in the market of amniotic fluid stem cell therapy due to lack of medical facilities and technical knowledge.

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Some of the key players operating in global amniotic fluid stem cell therapy market are Stem Shot, Provia Laboratories LLC, Thermo Fisher Scientific Inc. Mesoblast Ltd., Roslin Cells, Regeneus Ltd. etc. among others.

The report covers exhaustive analysis on: Amniotic Fluid Stem Cell Therapy Market Segments Amniotic Fluid Stem Cell Therapy Market Dynamics Historical Actual Market Size, 2012 2016 Amniotic Fluid Stem Cell Therapy Market Size & Forecast 2016 to 2024 Amniotic Fluid Stem Cell Therapy Market Current Trends/Issues/Challenges Competition & Companies involved Amniotic Fluid Stem Cell Therapy Market Drivers and Restraints

Regional analysis includes North America Latin America Europe Asia Pacific Excluding China China The Middle East & Africa

Report Highlights: Shifting Industry dynamics In-depth market segmentation Historical, current and projected industry size Recent industry trends Key Competition landscape Strategies of key players and product offerings Potential and niche segments/regions exhibiting promising growth A neutral perspective towards market performance

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Amniotic Fluid Stem Cell Therapy Market to Rear Excessive Growth During 2018 2026 - Herald Space

Heart cells derived from patients stem cells and grown in a lab dish can reveal important clues about the development of heart ailments associated with Fabry disease.

The study, A Human Stem Cell Model of Fabry Disease Implicates LIMP-2 Accumulation in Cardiomyocyte Pathology, was published in Stem Cell Reports.

Fabry is a rare genetic disorder caused by mutations in the GLA gene. Located on the X chromosome, the gene provides instructions for the production of an enzyme called alpha-galactosidase A (alpha-GAL A).

These mutations typically affect the activity of alpha-GAL A, leading to the accumulation of a type of fat called globotriaosylceramide (GL-3) in different tissues and organs, including the heart, kidneys and nervous system, gradually compromising their normal function.

For this reason, most Fabry patients develop heart disease over the course of their lives, which may progress to heart failure, the most common cause of death among people living with the disorder.

A major obstacle for advancing therapy for patients with [Fabry disease] is the knowledge gap between the direct molecular consequences of alpha-GAL A deficiency in CMs [cardiomyocytes, or heart cells] and the cascade of events driving disease in the heart; the inaccessibility of CMs from patients precludes adequate investigation of these events, especially at early stages, the investigators wrote.

In a previous study, researchers describe the generation of induced pluripotent stem cells (iPSCs) from Fabry patients carrying nonsense mutations in the GLA gene. This gave them the possibility, for the first time, to study the impact of alpha-GAL A deficiency on heart cells derived from patients iPSCs grown in a lab dish.

(iPSCs are fully matured cells that are reprogrammed back to a stem cell state, where they are able to grow into any type of cell. A nonsense mutation is a mutation in which the alteration of a single nucleotide (the building blocks of DNA) makes proteins shorter.)

Investigators from Sanofi, in collaboration with researchers at the University of Manchester, further investigated the properties of heart cells derived from patients iPSCs. Their aim was to discover more clues about the molecular mechanisms involved in the development of heart disease linked to Fabry.

Functional and structural characterization experiments revealed that heart cells from Fabry patients had higher levels of GL-3, and showed a series of abnormalities in the way they responded to electrical stimuli and in how they regulated their calcium usage, compared to heart cells from healthy people serving as controls. Calcium is essential to coordinate the hearts function by contributing to the electrical signals involved in heart muscle contraction.

When researchers analyzed the protein contents of heart cells grown in a lab dish, they found these cells produced more than 5,500 different proteins. This analysis also showed that compared to controls, heart cells from Fabry patients produced large amounts of lysosomal membrane protein 2 (LIMP-2) and heat shock-related 70 kDa protein 2 (HSPA2/HSP70-2).

(LIMP-2 is a protein normally found on the membrane of lysosomes small structures within cells that accumulate, digest, and recycle materials that regulates their transport within cells; HSPA2/HSP70-2 is a protein involved in cellular quality control, participating in the folding of other proteins and targeting abnormal proteins for degradation.)

Heart cells from Fabry patients released high amounts of cathepsin F, a protein that helps breakdown materials being transported inside lysosomes, as well as HSPA2/HSP70-2. As expected, when researchers corrected the genetic mutation associated with Fabry in heart cells derived from patients iPSCs, all these defects were reversed.

To confirm the validity of these proteins as Fabry biomarkers, researchers then forced healthy heart cells to produce high amounts of LIMP-2. They discovered this also triggered the release of large amounts of cathepsin F and HSPA2/HSP70-2, resulting in a massive accumulation of vacuoles (enclosed compartments filled with water and other substances) inside cells.

In summary, our study has shown the power of the iPSC model to reveal early functional changes and the development of a distinctive biomarker expression profile in [Fabry disease] CMs. These biomarkers may be of utility in drug screening and in elucidating the earliest pathological events and cascades in [Fabry disease] cells. Quantification in patient plasma and urine samples will be an important next step toward validating their relevance in patients, the researchers wrote.

A better understanding of these mechanisms will no doubt accelerate the development of more effective and increasingly personalized therapies for patients, they added.

Joana is currently completing her PhD in Biomedicine and Clinical Research at Universidade de Lisboa. She also holds a BSc in Biology and an MSc in Evolutionary and Developmental Biology from Universidade de Lisboa. Her work has been focused on the impact of non-canonical Wnt signaling in the collective behavior of endothelial cells cells that make up the lining of blood vessels found in the umbilical cord of newborns.

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Fabry Heart Cells Grown in Lab Dish Give Hints to Cardiac Complications - Fabry Disease News

Artificial intelligence has been embraced for its ability to offer insight from big data. By applying the technology to genetics, a research team led by Queen Mary University of London has found clues that they say could aid the development of new drugs for heart failure and identify people at risk of the disease.

Based on an AI analysis of heart MRI images from 17,000 volunteers in UK Biobank, the researchers linked genetic factors to 22% to 39% of abnormalities in the size and function of the hearts left ventricle, which pumps blood into the aorta. They published the findings in the journal Circulation.

The team identified or confirmed 14 regions in the human genome that play a part in determining the size and function of the left ventricle, becausethey contain genes that regulate the early development of heart chambers and the contraction of heart muscle. Enlargement of left ventricle is a condition that can hamper the heart muscles ability to contract and pump blood, putting the patient at high risk of heart attack.

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This study has shown that several genes known to be important in heart failure also appear to regulate the heart size and function in healthy people, said study co-author Steffen Petersen of Queen Mary in a statement. That understanding of the genetic basis of heart structure and function in the general population improves our knowledge of how heart failure evolves.

RELATED:Bayer teams up with AI firm Sensyne Health to mine NHS data for its heart disease pipeline

There is a growing interest in using AI to gain insights into cardiovascular disease. Bayer recently partnered up with Sensyne Health, which uses AI to mine patient data from the U.K. National Health Service, including genomic sequencing data and real-world evidence, to help design clinical studies and accelerate drug discovery.

Many research teams having been looking at different ways to treat heart disease, including using immune therapies and regenerative approaches. Scientists at the University of Pennsylvania, for example,developed genetically modified T cells to attack and remove cardiac fibroblasts, which can lead to cardiac fibrosis. Vanderbilt University researchers identified Roches SYN0012, originally designed to treat rheumatoid arthritis, as a promising candidate that could dampen inflammation of heart tissue after a heart attack. Such inflammation can progress to acute episodes andchronic heart failure.

To help repair damaged cardiac tissue after a heart attack, scientists at the University of Cambridge in the United Kingdom and the University of Washington combined two types of cells derived from human stem cellsheart muscle cells and supportive epicardial cells that help the muscle cells live longer. A team at the the Morgridge Institute for Research previously added a drug called RepSox to stem cells to build better smooth muscle cells that can grow into functional arterial cells.

The Queen Mary researchers believe the 14 regions of the genome they fingered in their new study could be just the beginning of a larger story about genes and heart disease. Our academic and commercial partners are further developing these AI algorithms to analyze other aspects of cardiac structure and function,lead researcher Nay Aung said in the statement.

Aung and colleagues argue the genetic markers theyve already uncovered could help identify those at high risk of developing heart disease or open up new avenues for targeted treatments. The genetic risk scores established from this study could be tested in future studies to create an integrated and personalized risk assessment tool for heart failure, Aung said.

Link:
AI uncovers genes linked to heart failure - FierceBiotech

All living organisms function under specific conditions, the so-called laws of nature. But with the expansion of scientific knowledge in the past centuries, humans havent always just been helplessly swayed by the powers of nature.

While far from controlling nature, we have invented substances and came up with strategies that allow us to stretch the boundaries of science, from altering cell processes to becoming fully immune to diseases.

One of the most promising innovations of our times nanotechnology is taking it even further. Sometimes nicknamed the next industrial revolution, it pervades virtually every field from manufacturing to the food and drink industry.

By operating on the atomic and molecular scale, nanotechnology carries out the most precise interventions. What new possibilities does it bring to industries dealing with biology?

In 2018, global healthcare nanotechnology reached $160 billion and this number is likely to grow to over $300 billion by the end of 2025. Theres no wonder why healthcare is the true harvest field of nanotechnology with diverse opportunities, mostly in diagnostics, treatment, and prevention of diseases itself.

Experts have developed nanosized diagnostic devices that can be deployed throughout the human body to monitor levels of toxins or other substances. This allows for constant and real-time monitoring of an individual on a very detailed level something that was hardly imaginable just a few years ago.

Read More:Challenges in achieving precision medicine, personalized healthcare

And whats more! due to their size, these tiny sensors can enter spaces that are normally difficult to examine, including the brain.

Regarding treatment, there have been experiments with nanosized robots that can travel through bodily fluids. They can work to deliver active substances in a highly effective way.

Likewise, they could bring implants that destroy old cells and inject healing substances to promote the growth of new ones or the recovery of existing ones.

In this respect. In fact, researchers at North Carolina State University are developing a method to deliver cardiac stem cells to damaged heart tissue.

This highly-targeted approach also means a revolution in cancer treatment as we know it: nanotechnology could eliminate the adversary side effects of the conventional methods that affect the whole system.

Read More:Doctors will navigate this passive pill cam like they were playing Xbox

And with closer oversight and enhanced possibilities for direct intervention, cancerous cells can be destroyed even before a major breakout occurs.

Scientists at the Worcester Polytechnic Institute are working on such non-invasive preventive strategies. They have developed a chip made of carbon nanotubes that can capture circulating tumor cells of all sizes.

These can be analyzed easily to help identify any early-stage tumors and monitor treatment progress.

Nanotechnology has also seen a big boom in the cosmetics industry. In recent years, we have seen a rise in the usage of various nano-substances, including peptides, proteomics, stem cells, and epigenetics. These could directly intervene against the sources of any dermatological phenomenon, be it wrinkles, pigment spots, or acne.

The potential is immense, which is demonstrated by the industrys rising investment and the fact that major cosmetic producers, including LOral, P&G, Dior, and Johnson & Johnson, publish several nanotechnology-related patents every year.

LOral specifically designated a web page to nanoparticles to educate their consumers about the power of these substances in many aspects; from intensifying the shade of mascara to providing a matte finish effect on the skin.

The uses are truly diverse. For example, we can find the adoption of nanoemulsions that encapsulate active ingredients to be carried deeper into hair shafts, or nanosilvers and nanogolds that are known for their antibacterial effects and are used in deodorant or toothpaste.

Nanotechnology is practically used in all everyday products, including moisturizers, haircare, or sunscreens.

In fact, the usage of nanoparticles in sunscreens has perhaps earned the most attention. These SPF creams contain zinc oxide and titanium dioxide as their main compounds. Such products can reflect UV rays, in contrast to the traditional chemical sunscreens that absorb the rays.

Thats why nano-powered sunscreens appear transparent, instead of leaving a white layer on the skin. Yet, this method has been associated with safety concerns, arising from the risk of the particles penetrating tissue and entering the human organism.

While there is still research to be done, an Australian study from last year disproved this notion and asserted that nano-powered sunscreens are unlikely to be harmful.

But its not just healthcare and beauty. Nanotechnology also brings opportunities to conservation and preservation. By being able to disrupt biological processes at the most detailed level, scientists are working to delay wilting and enhance desired processes, such as fostering an environment unfriendly to bacteria.

Specifically, there has been a lot of progress done in the field of food storage and preservation. For example, the encapsulation of nutraceuticals through nanotechnology is a step towards greater food safety and bioavailability, allowing us to benefit from food to its full nutritional potential.

However, similar applications could boost a plethora of other industries, including design and art, education, and science. Laboratories could find easier ways to preserve biological samples, while impressive natural artworks could be exhibited in museums for decades, bringing awe to many generations.

One of the innovations already being put to practice is the NanoFreeze technology, which can directly battle the sources of flower decay. This preservation strategy relies on a uniquely set up freezing process that can halt decomposition.

It kills present microorganisms, stops enzymatic reactions, and establishes a protected environment that prevents the occurrence of parasites. This way, NanoFreeze technology succeeds in maintaining the bloom looking fresh even years after it was cut off, bringing unprecedented possibilities to the floriculture industry and beyond.

From live-saving innovations to more mundane consumer upgrades, nanotechnology presents many opportunities for the future.

While predicted to grow significantly in the upcoming years, its crucial to understand that the innovation still hasnt reached its peak and is yet to experience its full bloom.

Disclosure: This article is brought to you by a client of an Espacio portfolio company

Originally posted here:
How nanotechnology is sizing up healthcare, beauty and conservation - The Sociable

ORLANDO, Fla.--(BUSINESS WIRE)--

- Grant is part of the Helping to End Addiction Long-Term Initiative (NIH HEAL Initiative) launched in 2018 -

Hesperos, Inc., (www.hesperosinc.com) announced today the University of Central Florida (UCF) and Hesperos have received the first phase of a $3.8 million milestone-based National Institutes of Health (NIH) grant for research involving the companys human-on-a-chip system. The goal of the research is to better understand how overdosing on opiates works, their impact on multiple organs and the effect of drugs used to treat those overdose episodes including their potential toxicity to organs. James Hickman, Ph.D., Chief Scientist at Hesperos and Professor at UCF, is the principal investigator for the research.

Hesperos, Inc., (www.hesperosinc.com) announced today the University of Central Florida (UCF) received the first phase of a $3.8 million milestone-based National Institutes of Health (NIH) grant for research involving the companys human-on-a-chip system.

"We are grateful to have funding to support research in an area that represents such a large and growing need," said Dr. Hickman. "Our interconnected human-on-a-chip system provides a non-invasive way to emulate the response of compounds among all 'organ' compartments, and to concurrently predict potential toxicity and efficacy of drugs, including opioids and opioid antagonists such as Narcan."

The funding comes from the NIHs Helping to End Addiction Long-term Initiative, or the NIH HEAL Initiative. The initiative aims to improve treatments for chronic pain, curb the rates of opioid use disorder and overdose and achieve long-term recovery from opioid addiction. The National Institutes of Health launched the Helping to End Addiction Long-term Initiative, or NIH HEAL Initiative, in April 2018 to improve prevention and treatment strategies for opioid misuse and addiction and enhance pain management. More information about the grants awarded by the NIH HEAL Initiative can be found here.

Its clear that a multi-pronged scientific approach is needed to reduce the risks of opioids, accelerate development of effective non-opioid therapies for pain and provide more flexible and effective options for treating addiction to opioids, said NIH Director Francis S. Collins, M.D., Ph.D., who launched the initiative in early 2018. This unprecedented investment in the NIH HEAL Initiative demonstrates the commitment to reversing this devastating crisis.

Under this program, Hesperos will build overdose models in a multi-organ system and evaluate the acute and chronic effects of overdose treatments, such as Narcan, on overdose recovery and efficacy. The research will provide insight into the impact of both opiate overdoses and treatment drugs on the kidneys, heart, muscles and liver, as well as explore how these drugs impact the part of the brain that controls breathing to reproduce overdose conditions.

Hesperos seeks to radically change established practice in drug discovery and testing by bypassing animal experiments and extensive clinical trials to provide treatments for diseases and clinical conditions such as overdose. Dr. Hickman developed the human-on-a-chip system at UCF in collaboration with Michael Shuler, President and CEO at Hesperos and Professor Emeritus, Cornell University. UCF licensed the technology to Hesperos, which was co-founded by Dr. Hickman and Dr. Shuler.

Over the past few years we have formed multiple collaborations with companies and nonprofit organizations seeking more efficient and effective alternatives to preclinical evaluation of drugs or toxicity tests on chemicals without lengthy, expensive animal studies, Dr. Shuler said. We recently published results in Nature Scientific Reports and Science Translational Medicine supporting the ability of our system to truly revolutionize the drug discovery process.

About Hesperos:

Hesperos, Inc. is a leader in efforts to characterize an individuals biology with human-on-a-chip microfluidic systems. Founders Michael L. Shuler and James J. Hickman have been at the forefront of every major scientific discovery in this realm, from individual organ-on-a-chip constructs to fully functional, interconnected multi-organ systems. With a mission to revolutionize toxicology testing as well as efficacy evaluation for drug discovery, the company has created pumpless platforms with serum-free cellular mediums that allow multi-organ system communication and integrated computational PKPD modeling of live physiological responses utilizing functional readouts from neurons, cardiac, muscle, barrier tissues and neuromuscular junctions as well as responses from liver, pancreas and barrier tissues. Created from human stem cells, the fully human systems are the first in vitro solutions that accurately utilize in vitro systems to predict in vivo functions without the use of animal models. More information is available at http://www.hesperosinc.com

View source version on businesswire.com: https://www.businesswire.com/news/home/20190926005748/en/

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Hesperos Human-on-a-Chip Technology Awarded First Phase of $3.8 Million Milestone-based NIH Grant to Study Opiate Overdoses in Collaboration With UCF...

CAIRO 22 September 2019: Renowned Professor of Cardiothoracic surgery Magdi Yacoub said that the foundation stone of a heart center in Cairo will be laid soon. The center will provide cardiac care.

In an interview with Egypt Today, the Egyptian-British cardiothoracic surgeonsaid that an inauguration ceremony of the Cairo center will beheldin January 2020, and will beattended by a large number of parliamentarians, senior doctors and statesmen to support the center and urge Egyptians to donate.

The MagdiYacoub Global Heart Foundation launched a campaign in May to raise fund for the new center.

A set of remarkable scientists and public figures took part in the campaign such as Professor MagdyIshak, and Egyptian Ambassador to the United States Yasser Reda, among others.

The MagdiYacoub Global Heart Foundation supports Aswan heart centre in Upper Egypt and is raising funds for the future MagdiYacoubglobal heart centre in Cairo.

Besides providing urgently needed cardiac care, the centers impact the region and continent by advancing scientific understanding through research and building human health capacities with training programs.

The new center will cost an estimate of $150 million and will include 300 beds, hence expected to upgrade network care capacity from 33,000 to 140,000 outpatients and from 4,000 to 17,000 inpatients annually.

Moreover, the training capacity will grow from 550 to over 2300, dramatically increasing the sectors workforce.

Yacoub was among the first three surgeons to perform an open heart surgery in Nigeria in 1974. In 1986, he was part of the team that developed the techniques of the heart-lung transplantation at the National Heart and Lung Institute.

He also led a British research team at Harefield hospital in 2007, aiming to grow a part of the human heart using stem cells. These efforts were all exerted in order to overcome the shortage of heart transplant donations.

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Cairo heart center to be inaugurated January: Magdi Yacoub - Egypttoday

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Autologous Stem Cell Based Therapies Market Report with Depth Analysis 2019 | Regeneus, Mesoblast - Tech Platform

Overview of current stem cell-based approaches to treat heart disease

Since heart failure after heart attacks results from death of heart muscle cells, researchers have been developing strategies to remuscularize the damaged heart wall in efforts to improve its function. Researchers are transplanting different types of stem cell and progenitor cells (see above) into patients to repair the damaged heart muscle. These strategies have mainly used either adult stem cells (found in bone marrow, fat, or the heart itself) or pluripotent (ES or iPS) cells.

Preliminary results from experiments with adult stem cells showed that they appeared to improve cardiac function even though they died shortly after transplantation. This led to the idea that these cells can release signals that can improve function without replacing the lost muscle. Clinical trials began in the early 2000s transplanting adult stem cells from the bone marrow and then from the heart. These trials demonstrated that transplanting cells into damaged hearts is feasible and generally safe for patients. However, larger trials that were randomized, blinded, and placebo-controlled, showed fewer indications of improved function. The consensus now is that adult stem cells have modest, if any, benefit to cardiac function.

Research shows that pluripotent stem cell-derived cardiomyocytes can form beating human heart muscle cells that both release the necessary signals and replace muscle lost to heart attack. Transplantation of pluripotent stem cell-derived cardiac cells have demonstrated substantial benefits to cardiac function in animal models of heart disease, from mice to monkeys. Recently, pluripotent stem cell-derived interventions were used in clinical trials for the first time. Patches of human heart muscle cells derived from the stem cells were transplanted onto the surface of failing hearts. Early results suggest that this approach is feasible and safe, but it is too early to know whether there are functional benefits.Research is ongoing to test cellular therapies to treat heart attacks by combining different types of stem cells, repeating transplantations, or improving stem cell patches. Clinical trials using these improved methods are currently targeted to begin around 2020.Unfortunately, many unscrupulous clinics are making unsubstantiated claims about the efficacy of stem cell therapies for heart failure, creating confusion about the current state of cellular approaches for heart failure. To learn more about warning signs of these unproven interventions, please visit Nine Things to Know About Stem Cell Treatments.

More here:
Heart Disease A Closer Look at Stem Cells

How do Stem Cellsfunction?Stem cells have the capacity to migrate to injured tissues, a phenomenon calledhoming. This occurs by injury or disease signals that are released from the distressed cells and tissue. Once stem cells arrive,they dock on adjacent cells to commence performing their job to repair the problem.

Stem cells serve as a cell replacementwhere they change into the required cell type such as a muscle cell, bone orcartilage. This is ideal for traumaticinjuries and many orthopedic indications.

They do not express specific human leukocyte antigens (HLAs) which helpthem avoid the immune system. Stemcells dock on adjacent cells and release proteins called growth factors, cytokines and chemokines. These factors help control many aspects of the healing and repairprocess systemically.

Stem cells control the immune system and regulate inflammation which is a keymediator of disease, aging, and is ahallmark of autoimmune diseases such as rheumatoid arthritis and multiple sclerosis.

They help to increase new blood vesselformation so that tissues receive proper blood flow and the correct nutrients needed to heal as in stroke, peripheral arterydisease and heart disease.

Stem cells provide trophic support forsurrounding tissues and help hostendogenous repair. This works wellwhen used for orthopedics. In case ofdiabetes, it may help the remaining beta cells in the pancreas to reproduce orfunction optimally.

As CSN research evolves, the field ofregenerative medicine and stem cells offers the greatest hope for those suffering from degenerative diseases, conditions for which there is currently no effective treatment or conditions that have failed conventional medical therapy.

Stem cell treatment is a complex process allowing us to harvest the bodys own repair mechanism to fight against degeneration, inflammation and general tissue damage. Stem cells are cells that can differentiate into other types of tissue to restore function and reduce pain.

Adult stem cells are found in abundance in adipose (fat) tissue, where more than 5million stem cells reside in every gram. These stem cells are called adult mesenchymal stem cells.

Our medical doctors extract stromal vascular fraction (SVF) from your own body to provide treatment using your very own cells. This process is calledautologous mesenchymal stem celltherapy. Our multi-specialty team deploys SVF under an institutional review board (IRB). This is an approved protocol that governs investigational work and the focus is to maintain safety of autologous use of SVF for various degenerative conditions.

How do we perform the stem cell treatment?Our procedure is very safe and completed in a single visit to our clinic. On the day of treatment, our physicians inject a localanaesthetic and harvest approximately 60 cc (2 oz.) of stromalvascular fraction (SVF) from under the skin of your flanks or abdomen. The extracted SVF is then refined in a closed system using strictCSN protocols to produce pure stromalvascular fraction (SVF). SVF containsregenerative cells including mesenchymal and hematopoietic stem cells, macrophages, endothelial cells, immune regulatory cells, and important growth factors that facilitate your stem cell activity. CSN technology allows us to isolate high numbers of viable stem cells that we can immediately deploy directly into a joint, trigger point, and/or byintravascular infusion. Specific deployment methods have been developed that are unique for each condition being treated.

During the refinement process, thesubcutaneous harvested cells andtheir connecting collagen matrix willbe separated, leaving purified free stem cells. About half of the SVF will be pure stem cells, while the remainder will be acombination of other regenerative cellsand growth factors. Before the SVF isre-injected into your body during the final part of the process we perform a qualityand quantity test which will examine the cell count and viability.

Perfecting the stem cell treatmentOur team records cell numbers and viability so that we can gain a better understanding of what constitutes a successful treatment. Although it is not yet possible to predict what number of cells that will be recovered in a harvest, it is very important that we know the total cell count and cell viability. It is only with this data that we will beginto understand why treatments are verysuccessful, only slightly successful orunsuccessful.

While vigilant about patient safety, we are also learning and sharing with the CSN data bank about which diseases respond best and which deployment methods are most effective with over 80 other clinics.

This data collection from all over the world makes the Cell Surgical Network the worlds largest regenerative medicineclinical research organization.

Network physicians have the opportunity to share their data, as well as their clinical experiences, thus helping one anotherto achieve higher levels of scientificunderstanding and optimizing medical protocols.

Injecting into thevascular system and/ora jointWe will administer the stem cell treatment with two methods:

The belief is that for many degenerative joint conditions IV and intra-articulardeployment is superior because each of these conditions have a local pathology and a central pathology. The local resident stem cell population has been working very hard to repair the damage and over the course of time these stem cells have become worn out, depleted and slowly die. This essentially causes a state of stem cell depletion. When we inject our mix of stem cells, cytokines and growth factors (known as SVF)inflammation is decreased and theregenerative process improved.

The stem cells that we have injected will then bring the level of stem cells closer to the normal level, thus restoring the natural balance and allow the body to heal itself.

Caplan et al, The MSC: An Injury Drugstore, DOI 10.1016/j .stem.2011.06.008

How long does it last?Many studies have shown the healing and regenerative ability of stem cells. Forexample, a study in World Journal of Plastic Surgery (Volume 5[2]; May 2016) followed a woman with knee arthritis. Before and after analysis of MRI images confirmed new growth of cartilage tissue. Unlike steroids, lubricants, and other injectable treatments, stem cells actually repair damaged tissue.

As published in Experimental andTherapeutic Medicine (Volume 12[2]; August 2016), numerous studies with hundreds of patients showed continuous improvement of arthritis for two years. Patients showed improvement three months after a single treatment and they continued to show improvement for two full years. This is why stem cells are often referred to as regenerative medicine.

No one can guarantee results for this or any other treatment. Outcomes will vary from patient to patient. Each potential patient must be assessed individually to determine the potential for optimum results from this regenerative therapy. To learn more about stem cell therapy, please contact us by clicking here or calling our clinic at 604-708-CELL (604-708-2355).

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Vancouver Stem Cell Treatment Centre | Stem Cells

There are many ways in which human stem cells can be used in research and the clinic. Studies of human embryonic stem cells will yield information about the complex events that occur during human development. A primary goal of this work is to identify how undifferentiated stem cells become the differentiated cells that form the tissues and organs. Scientists know that turning genes on and off is central to this process. Some of the most serious medical conditions, such as cancer and birth defects, are due to abnormal cell division and differentiation. A more complete understanding of the genetic and molecular controls of these processes may yield information about how such diseases arise and suggest new strategies for therapy. Predictably controlling cell proliferation and differentiation requires additional basic research on the molecular and genetic signals that regulate cell division and specialization. While recent developments with iPS cells suggest some of the specific factors that may be involved, techniques must be devised to introduce these factors safely into the cells and control the processes that are induced by these factors.

Human stem cells are currently being used to test new drugs. New medications are tested for safety on differentiated cells generated from human pluripotent cell lines. Other kinds of cell lines have a long history of being used in this way. Cancer cell lines, for example, are used to screen potential anti-tumor drugs. The availability of pluripotent stem cells would allow drug testing in a wider range of cell types. However, to screen drugs effectively, the conditions must be identical when comparing different drugs. Therefore, scientists must be able to precisely control the differentiation of stem cells into the specific cell type on which drugs will be tested. For some cell types and tissues, current knowledge of the signals controlling differentiation falls short of being able to mimic these conditions precisely to generate pure populations of differentiated cells for each drug being tested.

Perhaps the most important potential application of human stem cells is the generation of cells and tissues that could be used for cell-based therapies. Today, donated organs and tissues are often used to replace ailing or destroyed tissue, but the need for transplantable tissues and organs far outweighs the available supply. Stem cells, directed to differentiate into specific cell types, offer the possibility of a renewable source of replacement cells and tissues to treat diseases including maculardegeneration, spinal cord injury, stroke, burns, heart disease, diabetes, osteoarthritis, and rheumatoid arthritis.

Figure 3. Strategies to repair heart muscle with adult stem cells. Click here for larger image.

2008 Terese Winslow

For example, it may become possible to generate healthy heart muscle cells in the laboratory and then transplant those cells into patients with chronic heart disease. Preliminary research in mice and other animals indicates that bone marrow stromal cells, transplanted into a damaged heart, can have beneficial effects. Whether these cells can generate heart muscle cells or stimulate the growth of new blood vessels that repopulate the heart tissue, or help via some other mechanism is actively under investigation. For example, injected cells may accomplish repair by secreting growth factors, rather than actually incorporating into the heart. Promising results from animal studies have served as the basis for a small number of exploratory studies in humans (for discussion, see call-out box, "Can Stem Cells Mend a Broken Heart?"). Other recent studies in cell culture systems indicate that it may be possible to direct the differentiation of embryonic stem cells or adult bone marrow cells into heart muscle cells (Figure 3).

Cardiovascular disease (CVD), which includes hypertension, coronary heart disease, stroke, and congestive heart failure, has ranked as the number one cause of death in the United States every year since 1900 except 1918, when the nation struggled with an influenza epidemic. Nearly 2,600 Americans die of CVD each day, roughly one person every 34 seconds. Given the aging of the population and the relatively dramatic recent increases in the prevalence of cardiovascular risk factors such as obesity and type 2 diabetes, CVD will be a significant health concern well into the 21st century.

Cardiovascular disease can deprive heart tissue of oxygen, thereby killing cardiac muscle cells (cardiomyocytes). This loss triggers a cascade of detrimental events, including formation of scar tissue, an overload of blood flow and pressure capacity, the overstretching of viable cardiac cells attempting to sustain cardiac output, leading to heart failure, and eventual death. Restoring damaged heart muscle tissue, through repair or regeneration, is therefore a potentially new strategy to treat heart failure.

The use of embryonic and adult-derived stem cells for cardiac repair is an active area of research. A number of stem cell types, including embryonic stem (ES) cells, cardiac stem cells that naturally reside within the heart, myoblasts (muscle stem cells), adult bone marrow-derived cells including mesenchymal cells (bone marrow-derived cells that give rise to tissues such as muscle, bone, tendons, ligaments, and adipose tissue), endothelial progenitor cells (cells that give rise to the endothelium, the interior lining of blood vessels), and umbilical cord blood cells, have been investigated as possible sources for regenerating damaged heart tissue. All have been explored in mouse or rat models, and some have been tested in larger animal models, such as pigs.

A few small studies have also been carried out in humans, usually in patients who are undergoing open-heart surgery. Several of these have demonstrated that stem cells that are injected into the circulation or directly into the injured heart tissue appear to improve cardiac function and/or induce the formation of new capillaries. The mechanism for this repair remains controversial, and the stem cells likely regenerate heart tissue through several pathways. However, the stem cell populations that have been tested in these experiments vary widely, as do the conditions of their purification and application. Although much more research is needed to assess the safety and improve the efficacy of this approach, these preliminary clinical experiments show how stem cells may one day be used to repair damaged heart tissue, thereby reducing the burden of cardiovascular disease.

In people who suffer from type1 diabetes, the cells of the pancreas that normally produce insulin are destroyed by the patient's own immune system. New studies indicate that it may be possible to direct the differentiation of human embryonic stem cells in cell culture to form insulin-producing cells that eventually could be used in transplantation therapy for persons with diabetes.

To realize the promise of novel cell-based therapies for such pervasive and debilitating diseases, scientists must be able to manipulate stem cells so that they possess the necessary characteristics for successful differentiation, transplantation, and engraftment. The following is a list of steps in successful cell-based treatments that scientists will have to learn to control to bring such treatments to the clinic. To be useful for transplant purposes, stem cells must be reproducibly made to:

Also, to avoid the problem of immune rejection, scientists are experimenting with different research strategies to generate tissues that will not be rejected.

To summarize, stem cells offer exciting promise for future therapies, but significant technical hurdles remain that will only be overcome through years of intensive research.

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Stem Cell Basics VII. | stemcells.nih.gov

Adult stem cells are the bodys toolbox, called into action by normal wear and tear on the body, and when serious damage or disease attack. Researchers believe that adult stem cells also have the potential, as yet untapped, to be tools in medicine. Scientists and physicians are working towards being able to treat patients with their own stem cells, or with banked donor stem cells that match them genetically.

Grown in large enough numbers in the lab, then transplanted into the patient, these cells could repair an injury or counter a diseaseproviding more insulin-producing cells for people with type 1 diabetes, for example, or cardiac muscle cells to help people recover from a heart attack. This approach is called regenerative medicine.

A number of challenges must be overcome before the full therapeutic potential of adult stem cells can be realized. Scientists are exploring practical ways of harvesting and maintaining most types of adult stem cells. Right now, scientists do not have the ability to grow the cells in the amounts needed for treatment. More work is also needed to find practical ways to direct the different kinds of cells to where theyre needed in the body, preferably without the need for surgery or other invasive methods.

Research in all aspects of adult stem cells and their potential is underway at Childrens Hospital Boston. Realizing that potential will require years of research, but promising strides are being made.

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Why are Adult Stem Cells Important? Boston Children's ...

By: Ian Murnaghan BSc (hons), MSc - Updated: 12 Sep 2015| *Discuss

One of the biggest hurdles in stem cell research involves the use of embryonic stem cells. While these stem cells have the greatest potential in terms of their ability to differentiate into many different kinds of cells in the human body, they also bring with them enormous ethical controversies. The extraction of embryonic stem cells involves the destruction of an embryo, which upsets and outrages some policy makers and researchers as well as a number of public members. Not only that, but actually obtaining them is a challenge in itself and one that has become more difficult in places such as the United States, where policies have limited the availability of embryonic stem cells for use.

Although researchers have focused on harnessing the power of adult stem cells, there have still been many difficulties in the practical aspects of these potential therapies. In an ideal world, we would be able to use embryonic stem cells without destroying an embyro. Now, however, this ideal hope may actually have some realistic basis. In recent medical news, there has been important progress in the use of embryonic stem cells.

There are still many more tests and research that must be conducted to verify the safety and reliability of the procedure but it is indeed hopeful that funding can now increase for stem cell research. If you are an avid reader of health articles, you will probably be able to stay up-to-date on the latest developments related to this medical news. This newest research into embryonic stem cells holds promise and hope for appeasing the controversy around embryonic stem cell use and allowing for research to finally move forward with fewer challenges and controversies. For those who suffer from one of the many debilitating diseases and conditions that stem cell treatments may help or perhaps cure one day, this is welcome news.

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Creating Embryonic Stem Cells Without Embryo Destruction

Cardiovascular disease, also called heart disease, is a broad medical term used to describe a group of conditions that affect the blood vessels or the heart. It is the most common cause of death worldwide.1

Conditions of cardiovascular disease include:

The Stem Cells Transplant Institutein Costa Rica, uses adult autologous stem cells for the treatment of cardiovascular disease (heart disease). The symptoms of cardiovascular disease will depend on the specific type of heart disease.

Treatment at the Stem Cells Transplant Institute could help improve the symptoms of cardiovascular disease such as:

Heart disease and cardiovascular disease are often used interchangeably. These terms refer to a group of conditions that affect the blood vessels and heart. Valvular heart disease affects how the valves pump blood flow in and out of the heart. Cardiomyopathy affects the contractions of the heart muscle. Heart arrhythmias are disturbances in the electrical conduction making the heart beat irregular. Coronary artery disease is the most common cause of cardiovascular disease and stem cell therapy may be an effective treatment.

Coronary artery disease is caused by atherosclerosis, the buildup of plaque, causing a narrowing or blocking the blood vessels in the coronary arteries. Coronary artery disease is the leading cause of cardiovascular disease. Atherosclerosis can lead to chest pain, heart attack or stroke.

Coronary arteries carry oxygen rich blood to the heart. Plaque is caused by the presence of cholesterol, calcium, fat, and other substances in the blood. When plaque builds up in the blood vessels it narrows the arteries causing them to harden and weaken, reducing the amount of oxygen rich blood to the heart. As a result, the heart cannot pump blood effectively to the rest of the body potentially leading to heart failure and ultimately death.

If the plaque building up in the coronary arteries breaks, a blood clot forms around the plaque. If the clot cuts off the blood flow to the heart muscle completely, the heart muscle is unable to get the necessary oxygen and nutrients causing a part of the heart muscle to die. The result is a heart attack or myocardial infarction,

Coronary artery disease, high blood pressure or a previous heart attack can lead to the onset of heart failure. Heart failure is a chronic, progressive disease typically caused by another heart condition resulting in the heart muscle losing its ability to supply the rest of body with enough blood and oxygen.

Atherosclerosis can also cause peripheral artery disease. Peripheral arterial disease occurs when the narrowed peripheral arteries cannot send enough blood flow to the extremities, usually the legs. The most common symptoms of peripheral artery disease are; cramping, pain, and/or tiredness in the leg or hip muscles during exertion. The most severe symptom of peripheral artery disease is critical limb ischemia, pain at rest due to reduced blood flow to the limb.

Approximately 85% of strokes are ischemic strokes. Atherosclerosis is the most common cause of ischemic stroke. If the arteries become too narrow due to plaque buildup, the blood cells may collect and form a clot. A larger clot can block the artery where it is formed (thrombotic stroke) while a smaller clot may travel until it reaches an artery closer to the brain (embolic stroke). When the arteries to your brain become narrow or blocked, the required blood flow is reduced resulting in stroke. Other causes of ischemic stroke are clots due to an irregular heartbeat or heart attack.

Stem cell therapy at the Stem Cells Transplant Institute may be a good alternative for patients seeking a safe, non-surgical treatment for cardiovascular disease.

Notably, adult stem and progenitor cells including.mesenchymal stem cells have progressed into clinical trials and have shown positive benefits.5

Stem cell transplantation uses healthy cells to promote the repair of damaged cells and regeneration of healthy and functional cells to repair injured tissue.1 The therapeutic effect of stem cell transplantation in patients with cardiovascular disease may be due to the paracrine effect. The theory is transplanted stem cells repair damaged tissue by releasing factors that promote regeneration of healthy stem cells, reduce inflammation, promote the growth of new blood vessels, inhibit cell death, and reduce hypertrophy.1

The results of initial research using mesenchymal stem cell transplantation:

Heart Failure

Adipose derived stem cells improve left ventricular function, promote angiogenesis, lower fibrosis, and decrease inflammation. Several months following treatment, stem cells continue to migrate to the heart muscle regenerating and renewing healthy heart function. Stem cell therapy cannot help all patients with cardiovascular disease but for many patients stem cell therapy combined with lifestyle modification may be a safe, effective, non-surgical alternative treatment.

Lifestyle changes that can help improve cardiovascular disease include:

The Stem Cells Transplant Institute uses autologous mesenchymal stem cells for the treatment of cardiovascular disease. Autologous means the stem cells are collected from the recipient so the risk of rejection is virtually eliminated. Mesenchymal stem cells are one type of adult stem cells that are found in a variety of tissues including; adipose tissue, lung, bone marrow, and blood. Mesenchymal stem cells have several advantages over other types of stem cells; ability to migrate to sites of tissue injury, strong immunosuppressive effect, and better safety after infusion.2,3 Mesenchymal stem cells are a promising treatment for cardiovascular disease. Treatment at the Stem Cells Transplant Institute may improve the symptoms and long-term complications of cardiovascular disease.

A team of stem cell experts developed an FDA approved method and protocol for harvesting and isolating adipose derived stem cells for autologous reimplantation. The collection and use of adult stem cells does not require the destruction of embryos and for this reason, more U.S. federal funding is being spent on stem cell research.

The stem cells are administered intravenously.

Costa Rica has one of the best healthcare systems in world and is ranked among the highest for medical tourism. Using the most advanced technologies, the team of experts at The Stem Cells Transplant Institute believes in the potential of stem cell therapy for the treatment of cardiovascular disease. We are committed to providing personalized service and the highest quality of care to every patient. Contact us to see if stem cell therapy may be a treatment option for you.

1.Sun R.Advances in stem cell therapy for cardiovascular disease (Review). National Journal of Mol. Med. 38: 23-29, 2016. 2 Hare JM, Fishman JE, Gerstenblith G, DiFede Velazquez DL, Zambrano JP, Suncion VY, Tracy M, Ghersin E, Johnston PV, Brinker JA, et al: Comparison of allogeneic vs autologous bone marrow-derived mesenchymal stem cells delivered by transendocardial injection in patients with ischemic cardiomyopathy: the POSEIDON randomized trial. JAMA 308: 2369-2379, 2012.3 Miyahara Y, Nagaya N, Kataoka M, Yanagawa B, Tanaka K, Hao H, Ishino K, Ishida H, Shimizu T, Kangawa K, et al: Monolayered mesenchymal stem cells repair scarred myocardium after myocardial infarction. Nat Med 12: 459-465, 2006. 4 Mazo M, Planat-Bnard V, Abizanda G, Pelacho B, Lobon B, Gavira JJ, Peuelas I, Cemborain A, Pnicaud L, Laharrague P, et al: Transplantation of adipose derived stromal cells is associated with functional improvement in a rat model of chronic myocardial infarction. Eur J Heart Fail 10: 454-462, 2008. 5 Stem cell-based therapies to promote angiogenesis in ischemic cardiovascular disease Luqia Hou,1,2 Am J Physiol Heart Circ Physiol 310: H455H465, 2016. 6 Kinnaird T, Stabile E, Burnett MS, Lee CW, Barr S, Fuchs S, Epstein SE. Marrow-derived stromal cells express genes encoding a broad spectrum of arteriogenic cytokines and promote in vitro and in vivo arteriogenesis through paracrine mechanisms. Circ Res 94: 678685, 2004. 7 Kinnaird T, Stabile E, Burnett MS, Shou M, Lee CW, Barr S, Fuchs S, Epstein SE. Local delivery of marrow-derived stromal cells augments collateral perfusion through paracrine mechanisms. Circulation 109: 15431549, 2004.

8 Hare JM, Fishman JE, Gerstenblith G, DiFede Velazquez DL, Zambrano JP, Suncion VY, Tracy M, Ghersin E, Johnston PV, Brinker JA, Breton E, Davis-Sproul J, Schulman IH, Byrnes J, Mendizabal AM, Lowery MH, Rouy D, Altman P, Wong Po Foo C, Ruiz P, Amador A, Da Silva J, McNiece IK, Heldman AW, George R, Lardo A. Comparison of allogeneic vs autologous bone marrowderived mesenchymal stem cells delivered by transendocardial injection in patients with ischemic cardiomyopathy: the POSEIDON randomized trial. JAMA 308: 23692379, 2012.

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Stem Cell Treatment Cardiovascular Disease, Heart Disease ...

Stem cells in menstrual blood have similar regenerative capabilities as thestem cells in umbilical cord blood and bone marrow. Cryo-Cell's patent-pendingmenstrual stem cell service offers women in their reproductive years the ability to store and preserve these cells for potential use by herself or a family memberfree from ethical or political controversy.

Cryo-Cell is the only stem cell bank in the world that can offer womenthe reassurance and peace of mind that comes with this opportunity.

What are menstrual stem cells?Stem cells in menstrual blood are highly proliferativeandpossess the unique ability to develop into various other types of healthy cells. During a womans menstrual cycle, these valuable stem cells are discarded.

Cryo-Cell'smenstrual stem cell bankingservice captures those self-renewing stem cells, processes and cryopreserves them for emerging cellular therapies that hold the promise of potentially treatinglife-threatening diseases.

How are menstrual stem cells collected, processed and stored?The menstrual blood is collected in a physicians officeusing a medical-grade silicone cup in place of a tampon orsanitary napkin. The sample is shipped to Cryo-Cell via a medical courier and processed in our state-of-the-art ISO Class 7 clean room.

The menstrual stem cells are stored in two cryovials that are overwrapped to safeguard them during storage. The overwrapped vials are cryogenically preserved in a facility that isclosely monitored at all times to ensure that your menstrual stem cells are safe and ready for future use.

What are the benefits of banking menstrual stem cells?Cryo-Cell's innovative menstrual stem cell banking service provides women with the exclusive opportunity to build their own personal healthcare portfolio with stem cells that will be a 100% match for the donor. Menstrual stem cells have demonstrated the capability of differentiating into many other types of stem cells such as cardiac, neural, bone, fat and cartilage.

Bankingmenstrual stem cells now is an investment in your future medical needs. Currently, they are being studied to treat stroke, heart disease, diabetes, neurodegenerative disease, and ischemic wounds in pre-clinical and clinical models.

Cryo-Cells activities for New York State residents are limited to collection, processing, and long-term storage ofmenstrual stem cells. Cryo-Cells possession of a New York State license for such collection, processing, and long-term storage does not indicate approval or endorsement of possible future uses or future suitability of these cells.

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Banking Menstrual Stem Cells | What are Menstrual Stem ...

Stem cells are incredible. Science is only starting to scratch the surface of how these amazing cells can help people suffering from heart failure and other cardiovascular issues. Heres some information on what stem cells are, and how they may help heart attack patients and others who have problems involving their heart tissue.

There are more than 200 kinds of cells in the body, and each type is specifically structured for the job its supposed to do. There are skin cells, nerve cells, and cells that form heart tissue and other tissues in the body.1

Theyre found in bone marrow, blood vessels, the liver, the brain, and other parts of the body. Stem cells are even found in the umbilical cord. These sophisticated cells change over time as the body matures. Some of them disappear shortly after youre born, while others stay with you for a lifetime.2

There are three main types of stem cells tissue-specific (adult stem cells), embryonic stem cells, and induced pluripotent (iPS) stem cells. Heres a quick look at each type:

These typically reside in a specific organ, generating other cells to support the health of that organ. They replace those that are lost through injury, or through everyday living.3

Embryonic stem cells form about three to five days after a sperm fertilizes an egg. These are also known as pluripotent cells. This simply means they can develop into any sort of cell the body needs to develop.4

Embryonic cells have been the source of a massive controversy. The main reason is that harvesting these cells destroys the embryo.5 Scientists are working to develop iPS cells that come from adult stems cells rather than embryonic cells. Early research indicates that these cells may share many of the same characteristics of embryonic cells. But there are differences between the two, and there is more work to be done before scientists know exactly what those differences are.6

Research is ongoing into the potential use of stem cells for heart health. For example, work is being done to see if stem cells can help improve heart attack survival rates. Scientists are also looking into the potential for giving a patient their own cardiac stem cells after a heart attack, or even giving patients non-cardiac stem cells from a donor after an attack takes place.7

The goal of this research is to eventually provide cardiac patients with stem cells that can regenerate heart tissue that has been damaged. Some researchers feel that these advances are imminent, while others believe there is a great deal of work yet to be done.8

Early results from ongoing clinical trials involving stem cells for heart health are extremely promising. In one study, a group of 109 patients suffering from heart failure received either stem cell therapy or a placebo. According to the results, the patients who received stem cells were at significantly lower risk of hospitalization or death due to a sudden worsening of their condition.9

Heart failure affects more than 5 million people in the U.S.10 It occurs when the heart gradually weakens to the point to where it cant pump enough blood to meet the needs of the rest of the body. For those with severe heart failure, the only options are either to have a heart transplant or have a device planted to help the heart continue pumping. And even this is only a temporary measure theyll still need a transplant.11

Another study involved the use of stem cells from the umbilical cord. This trial involved 30 heart failure patients. Like the previous study, one group received stem cells while the other received a placebo. The umbilical cords were donated by healthy mothers whose babies were delivered through cesarean section.12

According to the results, the hearts of patients who received the umbilical cord stem cells pumped better than those of the placebo group. The stem cell patients also showed improved quality of life and day-to-day functioning. In addition, the stem cell group did not report any adverse effects, such as immune system reactions.13

As you can see, the use of stem cells to treat heart patients shows great promise. But this is still an extremely young scientific field, and a great deal more research must be performed. Many questions have to be answered, such as what approaches to stem cell harvesting will work the best and what types of side effects are possible from stem cell treatment.

However, this research does bring hope. And hope is something that is incredibly important to many of those suffering from severe cardiac illnesses.

Learn More:How Cardio Can Change Your Brain (And Why Thats Good News!)NEWS: A Vaccine For Arthritis Is Closer Than You ThinkAre Organ Donors At Risk of Becoming Obsolete?

Sources1.https://askabiologist.asu.edu/questions/human-cell-types2.https://www.medicalnewstoday.com/info/stem_cell3.http://www.closerlookatstemcells.org/learn-about-stem-cells/types-of-stem-cells4.https://stemcells.nih.gov/info/basics/3.htm5.http://www.cnn.com/2013/07/05/health/stem-cells-fast-facts/index.html6.http://www.closerlookatstemcells.org/learn-about-stem-cells/types-of-stem-cells#induced-pluripotent7.https://my.clevelandclinic.org/health/diseases/17508-stem-cell-therapy-for-heart-disease8.https://www.health.harvard.edu/heart-health/repairing-the-heart-with-stem-cells9.https://www.ncbi.nlm.nih.gov/pubmed/2705988710.https://www.cdc.gov/dhdsp/data_statistics/fact_sheets/fs_heart_failure.htm11.http://www.heart.org/HEARTORG/Conditions/HeartFailure/TreatmentOptionsForHeartFailure/Devices-and-Surgical-Procedures-to-Treat-Heart-Failure_UCM_306354_Article.jsp#.WleO-yMrJ3k12.https://www.medicalnewstoday.com/articles/319552.php13.http://circres.ahajournals.org/content/early/2017/09/15/CIRCRESAHA.117.310712

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Stem Cells For Heart Health: What The Current Research ...

Bellin, M., Marchetto, M. C., Gage, F. H. & Mummery, C. L. Induced pluripotent stem cells: the new patient? Nat. Rev. Mol. Cell Biol. 13, 713726 (2012).

Matsa, E., Burridge, P. W. & Wu, J. C. Human stem cells for modeling heart disease and for drug discovery. Sci. Transl. Med. 6, 239 (2014).

Wang, G. et al. Modeling the mitochondrial cardiomyopathy of Barth syndrome with induced pluripotent stem cell and heart-on-chip technologies. Nat. Med. 20, 616623 (2014).

Yazawa, M. et al. Using induced pluripotent stem cells to investigate cardiac phenotypes in Timothy syndrome. Nature 471, 230234 (2011).

Yang, X., Pabon, L. & Murry, C. E. Engineering adolescence: maturation of human pluripotent stem cell-derived cardiomyocytes. Circ. Res. 114, 511523 (2014).

Feric, N. T. & Radisic, M. Maturing human pluripotent stem cell-derived cardiomyocytes in human engineered cardiac tissues. Adv. Drug Deliv. Rev. 96, 110134 (2016).

Domian, I. J. et al. Generation of functional ventricular heart muscle from mouse ventricular progenitor cells. Science 326, 426429 (2009).

Lundy, S. D., Zhu, W. Z., Regnier, M. & Laflamme, M. A. Structural and functional maturation of cardiomyocytes derived from human pluripotent stem cells. Stem Cells Dev. 22, 19912002 (2013).

Nunes, S. S. et al. Biowire: a platform for maturation of human pluripotent stem cell-derived cardiomyocytes. Nat. Methods 10, 781787 (2013).

Mannhardt, I. et al. Human engineered heart tissue: analysis of contractile force. Stem Cell Reports 7, 2942 (2016).

Ribeiro, M. C. et al. Functional maturation of human pluripotent stem cell derived cardiomyocytes in vitrocorrelation between contraction force and electrophysiology. Biomaterials 51, 138150 (2015).

Shadrin, I. Y. et al. Cardiopatch platform enables maturation and scale-up of human pluripotent stem cell-derived engineered heart tissues. Nat. Commun. 8, 1825 (2017).

Brette, F. & Orchard, C. T-tubule function in mammalian cardiac myocytes. Circ. Res. 92, 11821192 (2003).

Wiegerinck, R. F. et al. Force frequency relationship of the human ventricle increases during early postnatal development. Pediatr. Res. 65, 414419 (2009).

Lopaschuk, G. D. & Jaswal, J. S. Energy metabolic phenotype of the cardiomyocyte during development, differentiation, and postnatal maturation. J. Cardiovasc. Pharmacol. 56, 130140 (2010).

Jackman, C. P., Carlson, A. L. & Bursac, N. Dynamic culture yields engineered myocardium with near-adult functional output. Biomaterials 111, 6679 (2016).

Radisic, M. et al. Functional assembly of engineered myocardium by electrical stimulation of cardiac myocytes cultured on scaffolds. Proc. Natl Acad. Sci. USA 101, 1812918134 (2004).

Eng, G. et al. Autonomous beating rate adaptation in human stem cell-derived cardiomyocytes. Nat. Commun. 7, 10312 (2016).

Hasenfuss, G. et al. Energetics of isometric force development in control and volume-overload human myocardium. Comparison with animal species. Circ. Res. 68, 836846 (1991).

Chung, S. et al. Mitochondrial oxidative metabolism is required for the cardiac differentiation of stem cells. Nat. Clin. Pract. Cardiovasc. Med. 4, S60S67 (2007).

Gong, G. et al. Parkin-mediated mitophagy directs perinatal cardiac metabolic maturation in mice. Science 350, aad2459 (2015).

Porter, G. A. Jr et al. Bioenergetics, mitochondria, and cardiac myocyte differentiation. Prog. Pediatr. Cardiol. 31, 7581 (2011).

Vega, R. B., Horton, J. L. & Kelly, D. P. Maintaining ancient organelles: mitochondrial biogenesis and maturation. Circ. Res. 116, 18201834 (2015).

Gottlieb, R. A. & Bernstein, D. Metabolism. Mitochondria shape cardiac metabolism. Science 350, 11621163 (2015).

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Bers, D. M. Cardiac excitationcontraction coupling. Nature 415, 198205 (2002).

Huebsch, N. et al. Miniaturized iPS-cell-derived cardiac muscles for physiologically relevant drug response analyses. Sci. Rep. 6, 24726 (2016).

Tulloch, N. L. et al. Growth of engineered human myocardium with mechanical loading and vascular coculture. Circ. Res. 109, 4759 (2011).

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Advanced maturation of human cardiac tissue grown from ...

To determine why the first stem cell trials were not providing the anticipated therapeutic potential, all variables, such as which stem cells were used, and how they were developed and administered, were open to consideration, says Dr. Epstein.

A key issue was the use of autologous stem cells in all previous studies. Studies demonstrated these old stem cells are functionally defective when compared to stem cells obtained from young healthy individuals. So harvesting a healthy young donors bone marrow and growing the resident stem cells might produce more robust cells.

However, giving a patient allogenic stem cells raised an important issue: whether such cells will be rejected by an immune response. But research showed mesenchymal stem cells (MSCs), a type of adult stem cell, have been designed by nature to be stealth bombers, explainsDr. Epstein. They express molecules on their surface that prevent the body from recognizing the cells as foreign, so the patient does not reject the donated MSCs.

To further explore and refine potential stem cell cardiovascular therapies, MHVI expanded the translational research team to include Michael Lipinski, MD, PhD, an expert in molecular biology and scientific lead for preclinical research at the MedStar Cardiovascular Research Network, and Dror Luger, PhD, an expert in immunology and inflammatory responses. By bringing together these diverse areas of expertise, we forged a team with the potential to produce research that could lead to important breakthroughs in understanding how stem cells might work and thereby provide more successful treatment of patients with cardiac disease, says Dr. Epstein.

CardioCell, a San Diego-based stem cell company focused on stem cell therapy for cardiovascular disease, found that MSCs grew faster and showed improved function when cultured in a reduced oxygen environment. Stem cells typically grow in the body, in bone marrow and other tissues, in a low oxygen environmentonly five percent oxygen, as opposed to room air, which is about 20 percent, explains Dr. Lipinski. All previous stem cell trials used cells exposed to, and grown under, room air oxygen conditions.

Using CardioCells low oxygen-grown MSCs, the MHVI scientists demonstrated biologically important effects occurred, even when the MSCs were administered intravenously. This mode of administration was previously rejected by scientists who thought cells would be trapped in the first capillary bed they traversedthe lungsand never reach the heart.

However, the MHVI team demonstrated a small percentage of these IV administered MSCs did reach the heart, where they could exert beneficial effects. The cells seek out inflamed cardiac tissue after a heart attack because they upregulate receptors that allow them to be attracted to and penetrate inflamed tissue in high numbers, says Dr. Luger.

The investigators also found the cells residing in other tissues could provide other benefits. It has been shown that a heart attack activates the immune and inflammatory systems, including those in the spleen, explains Dr. Luger. The systemic anti-inflammatory effects produced by MSCs in the spleen, lungs and other tissues caused by the molecules secreted by the MSCs could exert positive effects as well. Dr. Epstein added that such anti-inflammatory effects could also benefit the excessive inflammatory activities that exist in many heart failure patients.

For the clinical heart failure trial, MHVI is partnering with CardioCell, which will grow and provide stem cells already used in Phase I and 2a clinical trials and approved by the Food and Drug Administration.

As an extension of their stem cell work, the MHVI investigators are building on the fact that any beneficial effect of adult stem cells will not derive from their transformation into heart muscle, but rather from the molecules they secrete; these, in turn, stimulate pathways favoring tissue healing. The team is investigating the use of liposomes as therapeutic delivery vehicles for these secreted products, which include those with anti-inflammatory and angiogenesis activities.

If successful, using MSCs for anti-inflammatory and immune-modulatory effects could have implicationsfor many different diseases, including arthritis and autoimmune diseases like rheumatoid arthritis. Dr. Epstein cautions that a great deal of research is yet to be done before such applications can be routinely used to treat patients with these conditions. For now, they hope the current studies in heart failure patients will demonstrate effectiveness. If so, Dr. Epstein says, it changes the whole playing field for stem cells.

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Jeff Huffman, MD,is the Director of the Cardiac Psychiatry Research Program (CPRP), Director of Inpatient Psychiatry Research, and an Associate Professor of Psychiatry at Harvard Medical School. He currently serves as principal investigator for over ten projects, and has been awarded grants from the American Heart Association, American Diabetes Association, the Templeton Foundation, American Foundation for Suicide Prevention, and the National Institutes of Health (NHLBI and NIDDK). He has numerous peer-reviewed publications, including 100 first or senior author publications. He has mentored post doctoral psychology fellows, junior psychiatrist and psychologist faculty, medical students, psychiatry residents, research fellows, psychologists, social workers, and he received the 2015 Mass General Psychiatry Outstanding Research Mentor Award. His areas of interest include the impact of psychiatric illness on patients with cardiac disease, and the development and use of positive psychological interventions in a wide range of populations.

Christopher Celano, MD,is an attending psychiatrist at Mass General, an Assistant Professor in Psychiatry at Harvard Medical School, and the Associate Director of the CPRP. He is the recipient of a K23 career development award sponsored by the National Heart, Lung, and Blood Institute to develop a psychological intervention to improve health behaviors in patients with heart failure. He has published over 35 articles with the team, is an active co-investigator on several projects, and serves as the project director of health behavior trials in patients with coronary artery disease and diabetes. His areas of interest include the impact of depression and anxiety on cardiac health as well as the promotion of positive psychological states and health behaviors in patients with mental illness and cardiovascular disease.

Scott Beach, MD,is an Assistant Professor in Psychiatry at Harvard Medical School. He is Program Director for the Mass General/McLean Adult Psychiatry Residency and an attending psychiatrist on the consultation service at Mass General. He is currently PI of a study investigating neuroimaging and gene expression in patients with catatonia prior to and following lysis with lorazepam, and an active co-investigator on multiple projects. He has published over 50 book chapters and peer-reviewed articles on topics including QTc prolongation with psychotropic medications, catatonia, and deception syndromes.

James Januzzi, MD,is an Associate Professor of Medicine in the Division of Cardiology at Harvard Medical School, and the Director of the Cardiac Intensive Care Unit at Mass General. He is a well-established researcher at Mass General with over 300 peer-reviewed research publications, over 100 review articles and chapters, and has edited three text books. He is internationally known as an expert in the study of biomarkers in patients with heart failure and other cardiac illnesses, and has served as a section editor on the recent American College of Cardiology/American Heart Association clinical practice guidelines for heart failure, and was the lead for the heart failure section for the Universal Definition of Myocardial Infarction Global Task Force. He has served as the primary cardiologist on projects for the CPRP for the past nine years, including collaborative care depression and anxiety management trials in hospitalized cardiac patients, and studies of positive psychological states in persons with heart disease.

Laura Duque, MD, is a research fellow at the CPRP. Her areas of interest include Consultation Liaison Psychiatry, catatonia, and mood disorders. She is primarily interested in studying the relationship between mental health and chronic diseases. Currently, she is in charge of medical data collection and participant screening for a study on a collaborative care intervention for cardiac inpatients with psychiatric comorbidities, as well as for four positive psychology interventions for individuals with acute coronary syndrome, diabetes, heart failure, and metabolic syndrome. She graduated from Universidad de los Andes School of Medicine in Bogot, Colombia and intends to apply for residency training in psychiatry this upcoming year.

Perla M. Romero, MD is a research fellow at the CPRP. She was born and raised in Bogot, Colombia, where she also attended Universidad de los Andes School of Medicine. During her studies, she was involved in several research projects, including an original investigation analyzing the association between armed conflict, violence and mental health. Her main interests include human behavior, neuroscience and mental health. Perla's main goal is to pursue a psychiatry training in the US, and intends to pursue an academic career dedicated to this specialty.

Juan Pablo Ospina, MD, is a research fellow at the CPRP. He graduated from Universidad de los Andes school of Medicine in Bogot, Colombia. He is interested in the intersection of Neurology and Psychiatry and in studying mind-brain-body interactions. At the CPRP, he oversees subject screening and medical data collection for several randomized clinical trials studying the impact of positive psychology and blended care interventions in patients with medical conditions including acute coronary syndrome, heart failure, diabetes and multiple sclerosis. Likewise, he contributes to the presentation of study findings in publications and poster sessions. In the future, he intends to apply to Neurology residency training.

Franklin King, MD, is an attending psychiatrist at Mass General and an Instructor in Psychiatry at Harvard Medical School. He joined the CPRP in 2018, after completing a fellowship in consult-liaison psychiatry at Mass General in 2018 and residency at MGH/McLean in 2017, where he also served as consult-liaison chief resident during his fourth year. He graduated from UMass Medical School in 2013. His clinical interests include disorders at the intersection of medicine and psychiatry, the mind-body interface, and neuropsychiatry.

Carol Mastromauro, MSW, LICSW, is one of the interventionists for the CPRP. She is a clinical research social worker who has been with the team for seven years. Carol specializes in anxiety and depression treatment and positive psychology interventions for cardiac populations. She has administered interventions to more than 200 subjects during her time at the CPRP, and recruited and evaluated over 350 cardiac inpatients for the SUCCEED and MOSAIC studies. Prior to joining the CPRP, Carol worked in geriatric research on memory disorders as well as working with Huntingtons disease patients and their families.

Rachel Millstein, PhD, MHS, is a clinical psychologist at Mass General and Assistant in Psychiatry at Harvard Medical School. She is the recipient of a National Institutes of Health K23 award to develop a multilevel intervention to promote health behaviors among patients with metabolic syndrome. Her research focuses on chronic disease prevention and the intersection of emotions and health. Rachel has authored many peer-reviewed articles and book chapters in these fields. Her clinical interests include evidence-based therapies, positive psychology, and mindfulness techniques for improving mood, anxiety, and well-being.

Emily Feig, PhD, is a research and clinical postdoctoral psychology fellow in her second year with the CPRP. She completed her doctoral training in clinical psychology at Drexel University and her doctoral internship in Health Psychology at Rush University Medical Center. Emily is an interventionist on the BEHOLD study. Her research interests focus on understanding risk factors for obesity and eating disorders, as well as improving adherence to health behaviors in individuals with obesity-related chronic disease. Clinically, Emily specializes in cognitive behavioral and acceptance-based therapies targeting anxiety, depression, and disordered eating.

Christina Massey, PhD, is a clinical psychologist at Mass General and Instructor at Harvard Medical School in her first year with the CPRP. She completed her doctoral training in clinical psychology with a specialization in forensic psychology at The Graduate Center, CUNY at John Jay College of Criminal Justice and her doctoral internship at Mass General. Christina is currently an interventionist on the BEHOLD study. Her clinical and research interests include evidence-based treatments, diagnostic and forensic assessment and evaluation, and investigating the long-term consequences (including resilience) of childhood adversity.

Wei-Jean Chung, PhD, is a clinical psychologist at Mass General and Instructor at Harvard Medical School. She received her doctoral training in clinical psychology at Adelphi University prior to completing her doctoral internship and postdoctoral fellowship at Mass General. She is currently an interventionist for the PEACE and BEHOLD Studies at the CPRP. In addition to her involvement with the CPRP, her clinical practice involves caring for people with serious mental illness and complex personality organization across multiple clinical services within Mass General Psychiatry, including Primary Care Psychiatry, the Dialectical Behavioral Therapy Team, the Psychological Evaluation and Research Laboratory, and the Mass General inpatient psychiatry service.

Lydia Brown, PhD, is a psychologist and postdoctoral researcher with an interest in links between positive emotional/cognitive qualities and health. She completed her PhD and clinical training at The University of Melbourne, Australia, where she continues to hold a joint academic position. She has a particular interest in self-compassion, as well as novel interventions that might simultaneously boost both mental and physical health in the second half of life.

Margaret C. Bell, RN, MPH, MS, works as a nurse care manager in the CPRPs Total Health Study, a blended care intervention trial for patients with comorbid heart disease and mood or anxiety disorders. She is a registered nurse with a masters degree in psychiatric nursing from Boston College in 1994. Her work at Boston College included publications on Russian immigrant adjustment, effect of post-partum depression on mother-child interaction and domestic violence in pregnant women. She has worked in health care in Jerusalem, Amsterdam, New York, New Hampshire and Boston as a public health nurse, student health nurse, and psychiatric nurse. For the last 20 years she has monitored and managed NIH multi-site research trials in hepatology and cardiac research.

Beth Pino-Mauch, RN, BSN, works as a nurse care manager in the CPRPs Total Health Study, a blended care intervention trial for patients with comorbid heart disease and mood or anxiety disorders. Beth graduated from Boston College in 1983. She has worked as a cardiac and critical care nurse for over 15 years. Beth has also worked for a Boston-based Academic Research Organization as both a Project Manager, and subsequently, a Clinical Nurse Reviewer of reported Serious Adverse Events in several FDA-monitored medical device trials for coronary intervention.

Melanie Freedman, BS, graduated cum laude from Northeastern University in 2015 with a degree in psychology. She is a senior member of the CPRP, serving as the primary research coordinator for the REACH for Health Study. In this role, she is responsible for recruitment, enrollment, and managing study materials. She is also serving as the sole interventionist for a pilot trial of a positive psychology intervention in patients with Multiple Sclerosis through the Partners MS Center (PI: Glanz). Previously, Melanie worked as a research assistant at the Lifespan Emotional Development Lab at Northeastern University, which investigated emotion regulation and attention throughout the lifespan. She then worked as a Resource Specialist on the inpatient psychiatric unit at MGH before joining the CPRP.

Diana Smith, BA, graduated magna cum laude from Harvard University in 2017, with a degree in cognitive neuroscience and evolutionary psychology. She is in her second year with the CPRP and primarily manages the Total Health study, a blended care intervention trial for patients with comorbid heart disease and mood or anxiety disorders. She is also the primary coordinator for an ongoing project (PI: Nock), which is a real-time assessment of suicidal thoughts among psychiatric inpatients. In addition to her role at the CPRP, she volunteers for Samaritans, a suicide prevention and crisis line in Boston. Diana is currently applying to MD/PhD programs to begin in Fall 2019.

Sonia Kim, BA, graduated from UCLA in 2015 summa cum laude with a degree in psychology. She is in her first year with the program and is serving as the primary research coordinator for the MAPP (a PP-MI behavioral intervention study for patients with metabolic syndrome) and NCCP (a pilot care management intervention project for patients with non-cardiac chest pain). Before joining the CPRP, she worked as a rehabilitation specialist at the Sound End Community Health Center, working with underserved population that suffers from severe psychiatric illnesses. Previously in college, she was involved in an fMRI research in Dr. Matthew Liebermans lab, investigating the neural and behavioral effects of neuropeptides on human social cognition.

Julia Golden, BA, graduated from Mount Holyoke College in 2015 summa cum laude with a degree in psychology. Currently in her first year with the program, she is serving as the primary research coordinator for the BEHOLD studies. In this role, she is responsible for recruiting and enrolling diabetes patients as well as for organizing and managing study-related data. Previously, Julia worked as a research assistant at the Institute of Living, Hartford Hospitals psychiatric division, and was involved in studies related to mood disorders and metabolic syndrome in young adult patients. This past year she completed a post-baccalaureate pre-medical program at the University of Virginia.

Carlyn Scheu, BS, graduated cum laude from the University of Denver in 2018 with a degree in biology and psychology. In her first year with the program, Carlyn works primarily on the Dexmedetomidine study, a trial for the use of a sedative drug in patients with probable Alzheimers disease. She is also the primary coordinator for the PATH study, which focuses on a positive psychology intervention for cancer patients who have had a hematopoietic stem cell transplant. Prior to her involvement with the CPRP, Carlyn worked as a research assistant for the Traumatic Stress Studies Group at the University of Denver, which seeks to understand complex consequences of trauma and how to improve outcomes for trauma survivors.

Brian Healy, PhD,is an Assistant Professor in the Department of Neurology at Harvard Medical School, a member of the Biostatistics Center at Mass General, and an Instructor in Biostatistics at the Harvard School of Public Health. Dr. Healy is also the lead biostatistician for the Partners Multiple Sclerosis Center, which is affiliated with Brigham and Women's Hospital. His primary research interest is statistical methods development and application for modeling of multiple sclerosis. He has been working with the CPRP for the past 5 years, and he has participated in the design and analysis of several studies.

Elizabeth Madva, MD, is a fourth year resident in the MGH/McLean psychiatry residency program and a member of the residency's Research Concentration Program and Clinician Educator Program. She is currently serving as the administrative chief resident and the Mass General Consultation-Liaison Psychiatry chief resident. She graduated from Weill Cornell Medical College in 2015 and from Yale University in 2008, magna cum laude, with a BA in Cognitive Science. She is a member of the Alpha Omega Alpha and Phi Beta Kappa honor societies. She began working with the CPRP in 2016 at the end of her first year of residency. Her clinical and research interests fall in the areas of consultation-liaison psychiatry and neuropsychiatry, with a special interest in somatic symptom and functional neurological disorders.

Hermioni Lokko, MD, MPP, is an Instructor in Psychiatry at Harvard Medical School (HMS) as well as, staff physician on the Medical Psychiatry Service at Brigham and Women's Hospital (BWH) and the Department of Psychosocial Oncology and Palliative Care at the Dana-Farber Cancer Institute (DFCI). She is also the Associate Training Director of the BWH/HMS psychiatry residency training Program. Her areas of interest include the impact of psychiatric illness, management strategies and palliative care in diverse cancer patients to develop innovative and practical psychological interventions for cancer patients and their care givers. She is currently the principal investigator for a Harvard Medical School funded project seeking to develop a positive psychology intervention to improve function and quality of life in hematopoietic stem cell transplant patients. She is an active co-investigator for the PEACE trial and assists with other projects at the CPRP. She is a graduate of the psychosomatic medicine/psycho-oncology fellowship at the BWH and DFCI, the adult psychiatry residency training program at the Mass General and McLean Hospital, Harvard Medical School and Harvard Kennedy School of Government.

Medical Students:

Residents:

Faculty/Fellows:

Research Coordinators:

Social Work and Nursing Interventionists:

Our work has also been generously supported by the esteemed Avery D. Weisman, MD, of the eponymous Mass General Psychiatry Consultation Service and a long-standing national leader in psychosomatic medicine. His support has allowed the CPRP to continue to investigate the associations between positive and negative emotional states and physical health and well-being, and we are forever indebted.

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Cardiac Psychiatry Research Program - Massachusetts ...

Theranostics 2017; 7(7):2067-2077. doi:10.7150/thno.19427

Review

Kiwon Ban1, Seongho Bae2, Young-sup Yoon2, 3

1. Department of Biomedical Sciences, City University of Hong Kong, Hong Kong;2. Department of Medicine, Division of Cardiology, Emory University, Atlanta, Georgia, USA;3. Severance Biomedical Science Institute, Yonsei University College of Medicine, Seoul, Korea.

This is an open access article distributed under the terms of the Creative Commons Attribution (CC BY-NC) license (https://creativecommons.org/licenses/by-nc/4.0/). See http://ivyspring.com/terms for full terms and conditions.

Cardiomyocytes (CMs) derived from human pluripotent stem cells (hPSCs) are considered a most promising option for cell-based cardiac repair. Hence, various protocols have been developed for differentiating hPSCs into CMs. Despite remarkable improvement in the generation of hPSC-CMs, without purification, these protocols can only generate mixed cell populations including undifferentiated hPSCs or non-CMs, which may elicit adverse outcomes. Therefore, one of the major challenges for clinical use of hPSC-CMs is the development of efficient isolation techniques that allow enrichment of hPSC-CMs. In this review, we will discuss diverse strategies that have been developed to enrich hPSC-CMs. We will describe major characteristics of individual hPSC-CM purification methods including their scientific principles, advantages, limitations, and needed improvements. Development of a comprehensive system which can enrich hPSC-CMs will be ultimately useful for cell therapy for diseased hearts, human cardiac disease modeling, cardiac toxicity screening, and cardiac tissue engineering.

Keywords: Cardiomyocytes, hPSCs

Heart failure is the leading cause of death worldwide [1]. Approximately 6 million people suffer from heart failure in the United States every year [1]. Despite this high incidence, existing surgical and pharmacological interventions for treating heart failure are limited because these approaches only delay the progression of the disease; they cannot directly repair the damaged hearts [2]. In the case of large myocardial infarction (MI), patients progress to heart failure and die within short time from the onset of symptoms [3].

The adult human heart has minimal regenerative capacity, because during mammalian development, the proliferative capacity of cardiomyocytes (CMs) progressively diminishes and becomes terminally differentiated shortly after birth [4].Therefore, once CMs are damaged, they are rarely restored [5]. When MI occurs, the infarcted area is easily converted to non-contractile scar tissue due to loss of CMs and replacement by fibrosis [6]. Development of a fibroblastic scar initiates a series of events that lead to adverse remodeling, hypertrophy, and eventual heart failure [2, 3, 7].

While heart transplantation is considered the most viable option for treating advanced heart failure, the number of available donor hearts is always less than needed [6]. Therefore, more realistic therapeutic options have been required [2]. Accordingly, over the past two decades, cell-based cardiac repair has been intensively pursued [2, 7]. Several different cell types have been tested and varied outcomes were obtained. Indeed, the key factor for successful cell-based cardiac repair is to find the optimal cell type that can restore normal heart function. Naturally, CMs have been considered the best cell type to repair a damaged heart [8]. In fact, many scientists hypothesized that implanted CMs would survive in damaged hearts and form junctions with host CMs and synchronously contract with the host myocardium [9]. In fact, animal studies with primary fetal or neonatal CMs demonstrated that transplanted CMs could survive in infarcted hearts [9-11]. These primary CMs reduced scar size, increased wall thickness, and improved cardiac contractile function with signs of electro-mechanical integration [9-11]. These studies strongly suggest that CMs can be a promising source to repair the heart. However, the short supply and ethical concerns disallow using primary human CMs. In a patient with ischemic cardiomyopathy, about 40-50% of the CMs are lost in 40 to 60 grams of heart tissue [7]. Even if we seek to regenerate a fairly small portion of the damaged myocardium, a large number of human primary CMs would be required, which is impossible.

Accordingly, CMs differentiated from human pluripotent stem cells (hPSCs) including both embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have emerged as a promising option for candidate CMs for cell therapy [12, 13]. hPSCs have many advantages as a source for CMs. First, hPSCs have obvious cardiomyogenic potential. hPSC derived-CMs (hPSC-CMs) possess a clear cardiac phenotype, displaying spontaneous contraction, cardiac excitation-contraction (EC) coupling, and expression of cardiac transcription factors, cardiac ion channels, and cardiac structural proteins [14, 15]. Second, undifferentiated hPSCs and their differentiated cardiac progeny display significant proliferation capacity, allowing generation of a large number of hPSC-CMs. Lastly, many pre-clinical studies demonstrated that implantation of hPSC-CMs can repair injured hearts and improve cardiac function [16-19]. Histologically, implanted hPSC-CMs are engrafted, aligned and coupled with the host CMs in a synchronized manner [16-19].

In the last two decades, various protocols for differentiating hPSCs into CMs have been developed to improve the efficiency, purity and clinical compatibility [20] [18]. The reported differentiation methods include, but are not limited to: differentiation via embryoid body (EB) formation [20], co-culture with END-2 cells [18], and monolayer culture [15, 21, 22]. The EB-mediated CM differentiation protocol is one of the most widely employed methods due to its simple procedure and low cost. However, it often becomes labor-intensive to produce scalable EBs for further differentiation, which makes it difficult for therapeutic applications. EB-mediated differentiation also produces inconsistent results, showing beating CMs from 5% to 70% of EBs. Recently, researchers developed monolayer methods to complement the problems of EB-based methods [15, 21, 22]. In one representative protocol, hPSCs are cultured at a high density (up to 80%) and treated with a high concentration of Activin A (100 ng/ml) for 1 day and BMP4 (10 ng/ml) for 4 days followed by continuous culture on regular RPMI media with B27 [15]. This protocol induces spontaneous beating at approximately 12 days and produces approximately 40% CMs after 3 weeks. These hPSC-CMs can be further cultured in RPMI-B27 medium for another 2-3 weeks without significant cell damage [15]. However, these protocols use media with proprietary formulations, which complicates clinical application. As shown, most monolayer-based methods employ B27, which is a complex mix of 21 components. Some of the components of B27, including bovine serum albumin (BSA), are animal-derived products, and the effects of B27 components on differentiation, maturation or subtype specification processes are poorly defined. In 2014, Burridge and his colleagues developed an advanced protocol that is defined, cost-effective and efficient [22]. By subtracting one component from B27 at a time and proceeding with cardiac differentiation, the researchers reported that BSA and L-ascorbic acid 2-phosphate are essential components in cardiac differentiation. Subsequently, by replacing BSA with rice-derived recombinant human albumin, the chemically defined medium with 3 components (CDM3) was produced. The application of a GSK-inhibitor, CHIR99021, for the first 2 days followed by 2 days of the Wnt-inhibitor Wnt-59 to cells is an optimal culture condition in CDM3 resulting in similar levels of live-cell yields and CM differentiation [22].

Despite remarkable improvement in the generation of hPSC-CMs, obtaining pure populations of hPSC-CMs still remains challenging. Currently available methods can only generate a mixture of cells which include not only CMs but other cell types. This is one of the most critical barriers for applications of hPSC-CMs in regenerative therapy, drug discovery, and disease investigation. For Instance, cardiac transplantation of non-pure hPSC-CMs mixed with undifferentiated hPSCs or other cell types may produce tumors or unwanted cell types in hearts [23-28]. Accordingly, a pure or enriched population of hPSC-CMs would be required, particularly for cardiac cell therapy. Enriched hPSC-CMs would also be more beneficial for myocardial repair due to improved electric and mechanical properties [29]. A pure, homogeneous population of hPSC-CMs would pose less arrhythmic risk and have enhanced contractile performance, and would be more useful in disease modeling as they better reflect native CM physiology. Finally, purified hPSC-CMs would better serve for testing drug efficacy and toxicity. Therefore, many researchers have tried to develop methods to purify CMs from cardiomyogenically differentiated hPSCs.

There are three important topics that are not addressed in this review. First is the beneficial role of other cell types such as endothelial cells and fibroblasts in the integration, survival, and function of CMs [30-32]. We did not discuss this issue because it would need a separate review due to the volume of material. While the roles of such cells are important, the value of having purified hPSC-CMs is not diminished. Although cell mixtures or tissue engineered products can be used, unless purified CMs are employed, they would form tumors or other cells/tissues when implanted in vivo. Our point here is that even if cardiomyocytes are mixed with non-CMs, all cells should be clearly defined and purified as well. If the mixture is made in a non-purified or non-defined manner (for example, an unsophisticated top-down approach), there would be undefined cells that are neither CMs, ECs, nor fibroblasts and these unidentified cells will make aberrant tissues or tumors. Second, we did not deal with maturation of hPSC-CMs because of its broad scope and depth [33, 34]. Third is direct reprogramming or conversion of somatic cells into CMs. There has been another advancement in the generation of CMs by directly reprogramming or converting somatic cells into CM-like cells by introducing a combination of cardiac transcription factors (TFs) or muscle-specific microRNAs (miRNAs) both in vitro and in vivo [35-41]. These cells are referred to as induced CMs (iCMs) or cardiac-like myocytes (iCLMs). While this is an important advancement, we did not cover this topic either due to its size. Accordingly, this review will focus on the various strategies for purifying or enriching hPSC-CMs reported to date (Figure 1).

Early on, researchers isolated hPSC-CMs manually under microscopy by mechanically separating out the beating areas from myogenically differentiating hPSC cultures [18, 20, 42]. This method usually generates 5-70% hPSC-CMs. Although generally crude, it can enrich even higher percentages of CMs with further culture. This manual isolation method has the advantage of being easy, but while it can be useful for small-scale research, it is very labor intensive and not scalable, precluding large scale research or clinical application.

Currently available strategies for enriching cardiomyocytes derived from human pluripotent stem cells.

Xu et el. reported that hPSC-CMs, due to their physical and structural properties, can be enriched by Percoll density gradient centrifugation [43]. Percoll was first formulated by Pertoft et al [44] and it was originally developed for the isolation of cells, organelles, or viruses by density centrifugation. The Percoll-based method has several advantages. The procedure for Percoll-based separation is very simple and easy, it is inexpensive, and its low viscosity allows more rapid sedimentation and lower centrifugal forces compared to a sucrose density gradient. Lastly, it can be prepared and kept for a long time in an isotonic solution to maintain osmolarity. Although Percoll separation has resulted in major improvements in hPSC-CM isolation procedures, it has clear limitations with regard to purity and scalability. Previous studies found that Percoll separation is only able to enrich 40 -70% of hPSC-CMs. It is also not compatible with large-scale enrichment of hPSC-CMs.

Another traditional method for purifying hPSC-CMs is based on the expression of a drug resistant gene or a fluorescent reporter gene such as eGFP or DsRed, which is driven by a cardiac specific promoter in genetically modified hPSC lines [45, 46]. Here, enrichment of hPSC-CMs can be achieved by either drug treatment to eliminate cells that do not express the drug resistant gene or with FACS to isolate fluorescent cells [47, 48].

Briefly, enrichment of PSC-CMs by genetically based selection was first reported by Klug et al [49]. The authors generated murine ES cell lines via permanent gene transfection of the aminoglycoside phosphotransferase gene driven by the MHC (MYH7) promoter. With this approach, highly purified murine ESC-CMs up to 99% were achieved. Next, several studies reported the use of various CM-specific promoters to enrich ESC-CMs such as Mhc (Myh6), Myh7, Ncx (Sodium Calcium exchanger) and Mlc2v (Myl2) [46, 50, 51]. In the case of hESCs, MHC/EGFP hESCs were generated by permanent transfection of the EGFP-tagged MHC promoter [52]. Similarly, an NKX2.5/eGFP hESC line was generated to enrich GFP positive CMs [53]. However, since MHC and NKX2.5 are expressed in general CMs, the resulting CMs contain a mixture of the three subtypes of CMs, nodal-, atrial-, and ventricular-like CMs. To enrich only ventricular-like CMs, Huber et al. generated MLC2v/GFP ESCs to be able to isolate MLC2v/GFP positive ventricular-like cells by FACS [52] [54-57]. In addition, the cGATA6 gene was used to purify nodal-like hESC-CMs [58]. Future studies should focus on testing new types of cardiac specific promoters and devising advanced selection procedures to improve this strategy.

While fluorescence-based cell sorting is more widely used, the drug selection method may be a better approach to enrich high purity of hPSC-CMs during differentiation/culture as it does not require FACS. The advantage is its capability for high-purity cell enrichment due to specific gene-based cell sorting. These highly pure cells can allow more precise mechanistic studies and disease modeling. Despite its many advantages, the primary weakness of genetic selection is genetic manipulation, which disallows its use for therapeutic application. Insertion of reporter genes into the host genome requires viral or nonviral transfection/transduction methods, which can induce mutagenesis and tumor formation [50, 59-61].

Practically, antibody-based cell enrichment is the best method for cell purification to date. When cell type-specific surface proteins or marker proteins are known, one can tag cells with antibodies against the proteins and sort the target cells by FACS or magnetic-activated cell sorting (MACS). The main advantage is its specificity and sensitivity, and its utility is well demonstrated in research and even in clinical therapy with hematopoietic cells [62]. Another advantage is that multiple surface markers can be used at the same time to isolate target cells when one marker is not sufficient. However, no studies have reported surface markers that are specific for CMs, even after many years. Recently, though, several researchers demonstrated that certain proteins can be useful for isolating hPSC-CMs.

In earlier studies, KDR (FLK1 or VEGFR2) and PDGFR- were used to isolate cardiac progenitor cells [63]. However, since these markers are also expressed on hematopoietic cells, endothelial cells, and smooth muscle cells, they could not enrich only hPSC-CMs. Next, two independent studies reported two surface proteins, SIRPA [64] and VCAM-1 [65], which it was claimed could specifically identify hPSC-CMs. Dubois et al. screened a panel of 370 known antibodies against CMs differentiated from hESCs and identified SIRPA as a specific surface protein expressed on hPSC-CMs [64]. FACS with anti-SIRPA antibody enabled the purification of CMs and cardiac precursors from cardiomyogenically differentiating hPSC cultures, producing cardiac troponin T (TNNT2, also known as cTNT)-positive cells, which are generally considered hPSC-CMs, with up to 98% purity. In addition, a study performed by Elliot and colleagues identified another cell surface marker, VCAM1 [53]. In this study, the authors used NKX2.5/eGFP hESCs to generate hPSC-CMs, allowing the cells to be sorted by their NKX2.5 expression. NKX2.5 is a well-known cardiac transcription factor and a specific marker for cardiac progenitor cells [66, 67]. To identify CM-specific surface proteins, the authors performed expression profiling analyses and found that expression levels of both VCAM1 and SIRPA were significantly upregulated in NKX2.5/eGFP+ cells. Flow cytometry results showed that both proteins were expressed on the cell surface of NKX2.5/eGFP+ cells. Differentiation day 14 NKX2.5/eGFP+ cells expressed VCAM1 (71 %) or SIRPA (85%) or both VCAM1 and SIRPA (37%). When the FACS-sorted SIRPA-VCAM1-, SIRPA+ or SIRPA+VCAM1+ cells were further cultured, only SIRPA+ or SIRPA+VCAM1+ cells showed NKX2.5/eGFP+ contracting portion. Of note, NKX2.5/eGFP and SIRPA positive cells showed higher expression of smooth muscle cell and endothelial cell markers indicating that cells sorted solely based on SIRPA expression may not be of pure cardiac lineage. Hence, the authors concluded that a more purified population of hPSC-CMs could be isolated by sorting with both cell surface markers. Despite significant improvements, it appears that these surface markers are not exclusively specific for CMs as these antibodies also mark other cell types including smooth muscle cells and endothelial cells. Furthermore, they are also known to be expressed in the brain and the lung, which raises concerns whether these surface proteins can be used as sole markers for the purification of hPSC-CMs compatible for clinical applications.

More recently, Protze et al. reported successful differentiation and enrichment of sinoatrial node-like pacemaker cells (SANLPCs) from differentiating hPSCs by using cell surface markers and an NKX2-5-reporter hPSC line [68]. They found that BMP signaling specified cardiac mesoderm toward the SANLPC fate and retinoic acid signaling enhanced the pacemaker phenotype. Furthermore, they showed that later inhibition of the FGF pathway, the TFG pathway, and the WNT pathway shifted cell fate into SANLPCs, and final cell sorting for SIRPA-positive and CD90-negative cells resulted in enrichment of SANLPCs up to ~83%. These SIRPA+CD90- cells showed the molecular, cellular and electrophysiological characteristics of SANLPCs [68]. While this study makes important progress in enriching SANLPCs by modulating signaling pathways, no specific surface markers for SANLPCs were identified and the yield was still short of what is usually expected for cells purified via FACS.

Hattori et al. developed a highly efficient non-genetic method for purifying hPSC-derived CMs, in which they employed a red fluorescent dye, tetramethylrhodamine methyl ester perchlorate (TMRM), that can label active mitochondria. Since CMs contain a large number of mitochondria, CMs from mice and marmosets (monkey) could be strongly stained with TMRM [69]. They further found that primary CMs from several different types of animals and CMs derived from both mESCs and hESCs were successfully purified by FACS up to 99% based on the TMRM signals. In addition to its efficiency for CM enrichment, TMRM did not affect cell viability and disappeared completely from the cells within 24 hrs. Importantly, injected hPSC-CMs purified in this way did not form teratoma in the heart tissues. However, since TMRM only functions in CMs with high mitochondrial density, this method cannot purify entire populations of hPSC-CMs [64]. While originally TMRM was claimed to be able to isolate mature hPSC-CMs, mounting evidence indicates that hPSC-CMs are similar to immature human CMs at embryonic or fetal stages. Therefore, both the exact phenotype of the cells isolated by TMRM and its utility are rather questionable [33, 34]. Two subsequent studies demonstrated that TMRM failed to accurately distinguish hPSC-CMs due to the insufficient amounts of mitochondria [64].

Employing the unique metabolic properties of CMs, Tohyama et al. developed an elegant purification method to enrich PSC-CMs [70]. This approach is based on the remarkable biochemical differences in lactate and glucose metabolism between CMs and non-CMs, including undifferentiated cells. Mammalian cells use glucose as their main energy source [71]. However, CMs are capable of energy production from different sources such as lactate or fatty acids [71]. A comparative transcriptome analysis was performed to detect metabolism-related genes which have different expression patterns between newborn mouse CMs and undifferentiated mouse ESCs. These results showed that CMs expressed genes encoding tricarboxylic acid (TCA) cycle enzymes more than genes related to lipid and amino acid synthesis and the pentose phosphate cycle compared to undifferentiated ESCs. To further prove this observation, they compared the metabolites of these pathways using fluxome analysis between CMs and other cell types such as ESCs, hepatocytes and skeletal muscle cells, and found that CMs have lower levels of metabolites related to lipid and amino acid synthesis and pentose phosphate. Subsequently, authors cultured newborn rat CMs and mouse ESCs in media with lactate, forcing the cells to use the TCA cycle instead of glucose, and they observed that CMs were the only cells to survive this condition for even 96 hrs. They further found that when PSC derivatives were cultured in lactate-supplemented and glucose-depleted culture medium, only CMs survived. Their yield of CM population was up to 99% and no tumors were formed when these CMs were transplanted into hearts. This lactate-based method has many advantages: its simple procedures, ease of application, no use of FACS for cell sorting, and relatively low cost. More recently, this method was applied to large-scale CM aggregates to ensure scalability. As a follow-up study, the same group recently reported a more refined lactate-based enrichment method which further depletes glutamine in addition to glucose [72]. The authors found that glutamine is essential for the survival of hPSCs since hPSCs are highly dependent on glycolysis for energy production rather than oxidative phosphorylation. The use of glutamine- and glucose-depleted lactate-containing media resulted in more highly purified hPSC-CMs with less than 0.001% of residual PSCs [72]. One concern of this lactate-based enrichment method is the health of the purified hPSC-CMs, because physiological and functional characteristics of hPSC-CMs cultured in glucose- and glutamine-depleted media for a long time may have functional impairment since CMs with mature mitochondria were not able to survive without glucose and glutamine, although they were able to use lactate to synthesize pyruvate and glutamate [72]. In addition, this lactate-based strategy can only be applied to hPSC- CMs, but not other hPSC derived cells such as neuron or -cells.

Our group also recently reported a new method to isolate hPSC-CMs by directly labelling cardiac specific mRNAs using nano-sized probes called molecular beacons (MBs) [29, 73, 74]. Designed to detect intracellular mRNA targets, MBs are dual-labeled antisense oligonucleotide (ODN) nano-scale probes with a DNA or RNA backbone, a Cy3 fluorophore at the 5' end, and a Black Hole quencher 2 (BHQ2) at the 3' end [75, 76]. They form a stem-loop (hairpin) structure in the absence of a complementary target, quenching the fluorescence of the reporter. Hybridization with the target mRNA opens the hairpin and physically separates the reporter from the quencher, allowing a fluorescence signal to be emitted upon excitation. The MB-based method can be applied to the purification of any cell type that has known specific gene(s) [77].

In one study [29], we designed five MBs targeting unique sites in TNNT2 or MYH6/7 mRNA in both mouse and human. To determine the most efficient transfection method to deliver MBs into living cells, various methods were tested and nucleofection was found to have the highest efficiency. Next, we tested the sensitivity and specificity of MBs using an immortalized mouse CM cell line, HL-1, and other cell types. Finally, we narrowed it down to one MB, MHC-MB, which showed >98% sensitivity and > 95% specificity. This MHC-MB was applied to cardiomyogenically differentiated mouse and human PSCs and FACS sorting was performed. The resultant MHC-MB-positive cells expressed cardiac proteins at ~97% when measured by flow cytometry. These sorted cells also demonstrated spontaneous contraction and all the molecular and electrophysiological signatures of human CMs. Importantly, when these purified CMs were injected into the mouse infarcted myocardium, they were well integrated into the myocardium without forming any tumors, and they improved cardiac function.

In a subsequent study [74], we refined a method to enrich ventricular CMs from differentiating PSCs (vCMs) by targeting a transcription factor which is not robustly expressed in cells. Since vCMs are the main source for generating cardiac contractile forces and the most frequently damaged in the heart, there has been great demand to develop a method that can obtain a pure population of vCMs for cardiac repair. Despite this critical unmet need, no studies have demonstrated the feasibility of isolating ventricular CMs without permanently altering their genome. Accordingly, we first designed MBs targeting the Iroquois homeobox protein 4 (Irx4) mRNA, a vCM specific transcription factor [78, 79]. After testing sensitivity and specificity, one IRX4-MB was selected and applied to myogenically differentiated mPSCs. The FACS-sorted IRX4-MB-positive cells exhibited vCM-like action potentials in more than 98% of cells when measured by several electrophysiological analyses including patch clamp and Ca2+ transient analyses. Furthermore, these cells maintained spontaneous contraction and expression of vCM-specific proteins.

The MB-based cell purification method is theoretically the most broadly applicable technology among the purification methods because it can isolate any target cells expressing any specific gene. Thus, the MB-based sorting technique can be applied to the isolation of other cell types such as neural-lineage cells or islet cells, which are critical elements in regenerative medicine but do not have specific surface proteins identified to date. In addition, theoretically, this technology may have the highest efficiency when MBs are designed to have the maximum sensitivity and specificity for the cells of interest, but not others. These characteristics are particularly important for cell therapy. Despite these advantages, the delivery method of MB into the cells needs to be improved. So far, nucleofection is the best delivery method, but caused some cell damage with < 70% cell viability. Thus, development of a safer delivery method will enable wider application of MB-based cell enrichment.

Recently, Miki and colleagues reported a novel method for purifying cells of interest based on endogenous miRNA activity [80]. Miki et al. employed several synthetic mRNA switches (= miRNA switch), which consist of synthetic mRNA sequences that include a recognition sequence for miRNA and an open reading frame that codes a desired gene, such as a regulatory protein that emits fluorescence or promotes cell death. If the miRNA recognition sequence binds to miRNA expressed in the desired cells, the expression of the regulatory protein is suppressed, thus distinguishing the cell type from others that do not contain the miRNA and express the protein.

Briefly, the authors first identified 109 miRNA candidates differentially expressed in distinct stages of hPSC-CMs (differentiation day 8 and 20). Next, they found that 14 miRNAs were co-expressed in hPSC-CMs at day 8 and day 20 and generated synthetic mRNAs that recognize these 14 miRNA, called miRNA switches. Among those miRNA switches, miR-1-, miR-208a-, and miR-499a-5p-switches successfully enriched hPSC-CMs with purity of sorted cells up to 96% determined by TNNT2 intracellular flow cytometry. Particularly, hPSC-CMs enriched by the miR-1-switch showed substantially higher expression of several cardiac specific genes/proteins and lower expression of non-CM genes/proteins compared with control cells. Patch clamp confirmed that these purified hPSC-CMs possessed both ventricular-like and atrial-like action potentials.

One of the major advantages of this technology is its wider applicability to other cell types. miRNA switches have the flexibility to design the open reading frame in the mRNA sequence such that any desired transgene can be incorporated into the miRNA switches to regulate the cell phenotype based on miRNA activity. The authors tested this possibility by incorporating BIM sequence, an apoptosis inducer, into the cardiac specific miR-1- and miR-208a switches and tested whether they could selectively induce apoptosis in non-CMs. They found that miR-1- and miR-208a-Bim-switches successfully enriched cTNT-positive hPSC-CMs without cell sorting. Enriched hPSC-CMs by 208a-Bim-switch were injected into the hearts of mice with acute MI and they engrafted, survived, expressed both cTNT and CX43, and formed gap junctions with the host myocardium. No teratoma was detected. In addition, other miRNA switches such as miR-126-, miR-122-5p-, and miR-375-switches targeting endothelial cells, hepatocytes, and -cells, respectively, successfully enriched these cell types differentiated from hPSCs. However, identification of specific miRNAs expressed only in the specific cell type of interest and verification of their specificity in target cells will be key issues for continuing to use this miRNA-based cell enrichment method.

Recent advances in biomedical engineering have contributed to developing systems that can isolate target cells using physicochemical properties of the cells. Microfluidic systems have been intensively applied for cell separation due to recent improvements in miniaturizing a cell culture system [81-83]. These advances made possible the design of automated microfluidic devices with cellular microenvironments and controlled fluid flows that save time and cost in experiments. Thus, there have been an increasing number of studies seeking to apply the microfluidic system for cell separation. Among the first, Singh et al. tested the possibility of using a microfluidic system for the separation of hPSC [84] by preparative detachment of hPSCs from differentiating cultures based on differences in the adhesion properties of different cell types. Distinct streams of buffer that generated varying levels of shear stress further allowed selective enrichment of hPSC colonies from mixed populations of adherent non-hPSCs, achieving up to 95% purity. Of note, this strategy produced hPSC survival rates almost two times higher than FACS, reaching 80%.

Subsequently, for hPSC-CMs purification, Xin et al. developed a microfluidic system with integrated ridge-like flow derivations and fishnet-like microcolumns for the enrichment of hiPSC-CMs [85]. This device is composed of a 250 mm-long microfluidic channel, which has two integrated parallel microcolumns with surfaces functionalized with anti-human TRA-1 antibody for undifferentiated hiPSC trapping. Aided by the ridge-like surface patterns on the upper wall of the channel, micro-streams are generated so that the cell suspension of mixed undifferentiated hiPSCs and hiPSC-CMs are forced to cross the functionalized fishnet-like microcolumns, resulting in trapping of undifferentiated hiPSCs due to the interaction between the hiPSCs and the columns, and the untrapped hiPSC-CMs are eventually separated. By modulating flow and coating with anti-human TRA-1 antibody, they were able to enrich CMs to more than 80% purity with 70% viability. While this study demonstrated that a microfluidic device could be used for purifying hPSC-CMs, it was not realistic because the authors used a mixture of only undifferentiated hiPSCs and hiPSC-CMs. In real cardiomyogenically differentiated hiPSCs, undifferentiated hiPSCs are rare and many intermediate stage cells or other cell types are present, so the idea that this simple device can select only hiPSC-CMs from a complex mixture is uncertain.

Overall, the advantages of microfluidic system based cell isolation include fast speed, improved cell viability and low cost owing to the automated microfluidic devices that can control cellular microenvironments and fluid flows [86-88]. However, microfluidic-based cell purification methods have limitations in terms of low purity and scalability [89-92]. In fact, there have been only a few studies demonstrating the feasibility that microfluidic device-based cell separation could achieve higher than 80% purity of target cells. Furthermore, currently available microfluidic devices allow only separation of a small number of cells (< 1011). To employ microfluidic devices for large-scale cell production, we need to develop a next generation of microfluidic devices that can achieve a throughput greater than 1011 sorted cells per hour with > 95% purity.

Having available a large quantity of a homogeneous population of cells of interest is an important factor in advancing biomedical research and clinical medicine, and is especially true for hPSC-CMs. While remarkable progress has been made in the methods for differentiating hPSCs into CMs, technologies to enrich hPSC-CMs, particularly those which are clinically applicable, have been emerging only over the last few years. Contamination with other cell types and even the heterogeneous nature of hPSC-CMs significantly hinder their use for several future applications such as cardiac drug toxicology screening, human cardiac disease modeling, and cell-based cardiac repair. For instance, cardiac drug-screening assays require pure populations of hPSC-CMs, so that the observed signals can be attributed to effects on human CMs. Studies of human cardiac diseases can also be more adequately interpreted with purified populations of patient derived hiPSC-CMs. Clinical applications with hPSC-CMs will need to be free of other PSC derivatives to minimize the risk of teratoma formation and other adverse outcomes.

Summary of representative methods for hPSC-CM purification

Schematic pictures of microfluidic device for enriching hiPSC-CMs. (A) The part of the device designed for trapping undifferentiated hiPSCs. (B) (Left) Illustration of the overall microfluidic device assembled with peristaltic pump, cell suspension reservoirs, and a serpentine channel. (Right) Magnified image showing a channel combining microcolumns and ridge-like flow derivation structures. Modified from Li et al. On chip purification of hiPSC-derived cardiomyocytes using a fishnet-like microstructure. Biofabrication. 2016 Sep 8;8(3): 035017

Therefore, development of reproducible, effective, non-mutagenic, scalable, and economical technologies for purifying hPSC-CMs, independent of hPSC lines or differentiation protocols, is a fundamental requirement for the success of hPSC-CM applications. Fortunately, new technologies based on the biological specificity of CMs such as MITO-tracker, molecular beacons, lactate-enriched-glucose depleted-media, and microRNA switches have been developed. In addition, technologies based on engineering principles have recently yielded a promising platform using microfluidic technology. While due to the short history of this field, more studies are needed to verify the utility of these technologies, the growing attention toward this research is a welcome move.

Another important question raised recently is how to non-genetically purify chamber-specific subtypes of CMs such as ventricular-like, atrial-like and nodal-like hPSC-CMs. So far, only a few studies have addressed this potential with human PSCs. We also showed that a molecular beacon-based strategy could enrich ventricular CMs differentiated from PSCs [74]. Another study demonstrated generation of SA-node like pacemaker cells by using a stepwise treatment of various morphogens and small molecules followed by cell sorting with several sub-specific surface markers. However, the yield of both studies was relatively low (<85%). Given the growing clinical importance of chamber-specific CMs, the strategies for purifying specific subtypes of CM that are independent of hPSC lines or differentiation protocols should be continuously developed. A recently reported cell surface capture-technology [93, 94] may facilitate identification of chamber specific CM proteins that will be useful for target CM isolation.

In summary, technological advances in the purification of hPSC-CMs have opened an avenue for realistic application of hPSC-CMs. Although initial success was achieved for purification of CMs from differentiating hPSC cultures, questions such as scalability, clinical compatibility, and cellular damage remain to be answered and isolation of human subtype CMs has yet to be demonstrated. While there are other challenges such as maturity, in vivo integration, and arrhythmogenecity, this development of purification technology represents major progress in the field and will provide unprecedented opportunities for cell-based therapy, disease modeling, drug discovery, and precision medicine. Furthermore, the availability of chamber-specific CMs with single cell analyses will facilitate more sophisticated investigation of human cardiac development and cardiac pathophysiology.

This work was supported by the Bio & Medical Technology Development Program of the National Research Foundation (NRF) funded by the Korean government (MSIP) (No 2015M3A9C6031514), the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (HI15C2782, HI16C2211) and grants from NHLBI (R01HL127759, R01HL129511), NIDDK (DP3-DK108245). This work was also supported by a CityU Start-up Grant (No 7200492), a CityU Research Project (No 9610355), and a Georgia Immuno Engineering Consortium through funding from Georgia Institute of Technology, Emory University, and the Georgia Research Alliance.

The authors have declared that no competing interest exists.

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Current Strategies and Challenges for Purification of ...

VetStem Technology: Summary

VetStem Regenerative Cell Therapy is based on a clinical technology licensed from Artecel Inc. Original patents are from the University of Pittsburgh and Duke University.

Adipose-derived regenerative cells are:

VetStem Regenerative Cell (VSRC) therapy delivers a functionally diverse cell population able to communicate with other cells in their local environment. Until recently, differentiation was thought to be the primary function of regenerative cells. However, the functions of regenerative cells are now known to be much more diverse and are implicated in a highly integrated and complex network. VSRC therapy should be viewed as a complex, yet balanced, approach to a therapeutic goal. Unlike traditional medicine, in which one drug targets one receptor, Regenerative Medicine, including VSRC therapy, can be applied in a wide variety of traumatic and developmental diseases. Regenerative cell functions include:

In general, in vitro studies demonstrate that MSCs limit inflammatory responses and promote anti-inflammatory pathways.

Multiple studies demonstrate that MSCs secrete bioactive levels of cytokines and growth factors that support angiogenesis, tissue remodeling, differentiation, and antiapoptotic events.25,28 MSCs secrete a number of angiogenesis-related cytokines such as:28

Adipose-derived MSC studies demonstrate a diverse plasticity, including differentiation into adipo-, osteo-, chondro-, myo-, cardiomyo-, endothelial, hepato-, neuro-, epithelial, and hematopoietic lineages, similar to that described for bone marrow derived MSCs.22 These data are supported by in vivo experiments and functional studies that demonstrated the regenerative capacity of adipose-derived MSCs to repair damaged or diseased tissue via transplant engraftment and differentiation.6,9,30

Homing (chemotaxis) is an event by which a cell migrates from one area of the body to a distant site where it may be needed for a given physiological event. Homing is an important function of MSCs and other progenitor cells and one mechanism by which intravenous or parenteral administration of MSCs permits an auto-transplanted therapeutic cell to effectively target a specific area of pathology.

Adipose-derived regenerative cells contain endothelial progenitor cells and MSCs that assist in angiogenesis and neovascularization by the secretion of cytokines, such as hepatic growth factor (HGF), vascular endothelial growth factor (VEGF), placental growth factor (PGF), transforming growth factor (TGF), fibroblast growth factor (FGF-2), and angiopoietin.25

Apoptosis is defined as a programmed cell death or cell suicide, an event that is genetically controlled.35 Under normal conditions, apoptosis determines the lifespan and coordinated removal of cells. Unlike during necrosis, apoptotic cells are typically intact during their removal (phagocytosis).

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What is VetStem Regenerative Medicine? | Why Use Adipose ...

A cell-by-cell search for cardiac stem cells has come up empty, suggesting that previous studies hinting at the existence of cardiac stem cells were mistaken. More significantly, the absence of cardiac stem cells indicates that heart muscle that is lost due to a heart attack cannot be replaced.

The sobering finding was reported by scientists based at the Hubrecht Institute, which is located in the Netherlands. The scientists, led by Hans Clevers, group leader at the Hubrecht Institute and professor of molecular genetics at the University Medical Center Utrecht, published their work this week in the Proceedings of the National Academy of Sciences.

Along with colleagues from cole Normale Suprieure de Lyon and the Francis Crick Institute London, the Hubrecht Institute scientists described how they applied the broadest and most direct definition of stem cell function in the mouse heart: the ability of a cell to replace lost tissue by cell division. In the heart, this means that any cell that can produce new heart muscle cells after a heart attack would be termed a cardiac stem cell.

In an attempt to find cardiac stem cells, the scientists generated a cell-by-cell map of all dividing cardiac cells before and after a myocardial infarction using advanced molecular and genetic technologies. Details of this work appeared in the PNAS article, which is titled, Profiling proliferative cells and their progeny in damaged murine hearts.

Cycling cardiomyocytes were only robustly observed in the early postnatal growth phase, while cycling cells in homoeostatic and damaged adult myocardium represented various noncardiomyocyte cell types, the articles authors indicated in a prepublication version of their paper. Proliferative postdamage fibroblasts expressing follistatin-like protein 1 (FSTL1) closely resemble neonatal cardiac fibroblasts and form the fibrotic scar. Genetic deletion of FSTL1 in cardiac fibroblasts results in postdamage cardiac rupture.

Ultimately, the researchers found no evidence for the existence of a quiescent circulating stem cell population, for transdifferentiation of other cell types toward cardiomyocytes, or for proliferation of significant numbers of cardiomyocytes in response to cardiac injury.

Most tissues of animals and humans contain stem cells that come to the rescue upon tissue damage: they rapidly produce large numbers of daughter cells to replace lost tissue cells. Cardiac tissues, however, appear to behave differently. According to the new study, the damaged heart incorporates many types of dividing cells, but none that are capable of generating new heart muscle. In fact, many of the false leads of past studies can now be explained: cells that were previously named cardiac stem cells now turn out to produce blood vessels or immune cells, but never heart muscle. Thus, the sobering conclusion is drawn that heart stem cells do not exist.

The authors make a second important observation. Connective tissue cells (also known as fibroblasts) that are intermingled with heart muscle cells respond vigorously to a myocardial infarction by undergoing multiple cell divisions. In doing so, they produce scar tissue that replaces the lost cardiac muscle.

While this scar tissue contains no muscle and thus does not contribute to the pump function of the heart, the fibrotic scar holds together the infarcted area. Indeed, when the formation of the scar tissue is blocked, the mice succumb to acute cardiac rupture. Thus, while scar formation is generally seen as a negative outcome of myocardial infarction, the authors stress the importance of the formation of scar tissue for maintaining the integrity of the heart.

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Adult Hearts Lack Cardiac Stem Cells - genengnews.com

Human life is dependent upon the hearts ability to pump forcefully and frequently enough, but heart failure signs can disturb its normal function. Most humans cannot live more than four minutes without a heartbeat or continuous blood-flow. At that time, brain cells begin to die because they lack adequately oxygenated blood-flow.

The human adult body requires, on average, 5.0 liters of re-circulated blood per minute. In the cardiology field, this metric is called the Cardiac Output, which is calculated as Stroke Volume (SV) x Heart Rate (HR). Another key metric is a patients Ejection-Fraction (EF %). A patients EF tells a cardiologist and other physicians if his or her heart is functioning normally or low normally. It is a measurement of ones heart contraction, with a normal EF range being 55-70%.

This number can also be combined with a patients heart rate to provide physicians with a baseline of a patients cardiac status. A normal range for an adult is 60-100 beats per minute, and this can be significantly higher during a normal pregnancy.

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For a cardiologist, cardiac metrics indicate if their services are required and allowthem to sign-off on pre-operative cardiac clearances. For other physicians, it tells them if the organ which they specialize in is being perfused adequately (for example, a nephrologist would be interested to know kidney perfusion). It can also indicate the degree to which decreased heart function may affect the severity or spread of disease.

When the heart fails to contract forcefully enough and its performance decreases to the point where its ability to circulate blood adequately is compromised (the EF% falls below 40%), this is considered heart failure. The clinical parameters of heart failure are clearly defined by the New York Heart Association (NYHA), which places patients in NYHA Class III & IV into the heart failure category.

An echocardiogram (often called an Echo), as opposed to an Electrocardiogram (EKG or ECG), allows technicians and physicians to visualize the beating heart. Video clips of the heart contracting are digitally recorded, and a patients EF and Cardiac Output (CO) can be measured with several diagnostic tools (Fractional Shortening via 2D or M-Mode measurements and Simpsons Method via 2D and 3D Quantification) on a cardiovascular ultrasound system.

When an experienced echo tech or cardiologist views a failing heart, it is immediately apparent. Based on my experience reading echocardiograms, I can see that the heart walls or heart muscles (myocardium) are not contracting as vigorously as they should.

For patients with a 5% EF range, any physical movement is extremely strenuous, and they can go into cardiac arrest at any moment, which is why they are usually on cardiac telemetry in a hospital setting. Most likely, a patient with 5% EF range would be awaiting a heart transplant, unless there is a medical condition preventing them from being eligible.

Once a patient falls into the heart failure range, they will be lethargic and have severe limits on activities. Other clinical manifestations of heart failure can include peripheral edema (i.e. swelling in the feet, legs, ankles, or stomach), pulmonary edema, and shortness of breath. In many cases, this can lead to depression.

In evaluating the frequency of heart failure in the U.S, statistics from the U.S. Centers for Disease Control (CDC) find that approximately 5.7 million adults are afflicted with this condition. Additionally, care for congestive heart failure costs an estimated $30.7B per year. Furthermore, the mortality rates of patients suffering from heart failure indicate its clinical severity, with 1 in 5 patients with this condition dying within a year of receiving the diagnosis.

A patient experiencing severe heart failure has limited treatment options, which are expensive, complicated, and have major lifestyle implications.

These limited options include:

Consequently, physicians need more effective weapons for treating heart failure in order to improve patients lives and reduce healthcare-related costs. CHF patients have disproportionate hospital readmission rates when compared to other major diseases.

Enter in the growing field of cardiac stem cell treatments, which introduce fundamentally new treatment options for heart failure patients. In cardiac stem cell treatments, stem cells are taken from a patients bone marrow or fat tissue in a sterile surgical procedure and injected via a catheter-wire into infarcted or poorly contracting muscular segments of the hearts main pumping chamber, the left ventricle (LV).

Over the course of a few months, the stem cells impact myocardial cells and begin to improve the contractility of the affected segments, most likely through paracrine signaling mechanisms and impacting the local microenvironment. This can bring a patients EF to low-normal or even normal levels. As a result, a patient can live a more normal life and return to many activities.

A very early clinical trial aimed at evaluating the potential and effectiveness of cardiac stem cell therapy in humans was conducted in 2006 utilizing a commercial product, VesCellTM. The parameters and results of this trial were documented in the American Heart Associations Circulation, Abstract 3682: Treatment of Patients with Severe Angina Pectoris Using Intracoronarily Injected Autologous Blood-Borne Angiogenic Cell Precursors.The subjects of this trial received an intracoronary injection of VesCellTM, an Autologous Angiogenic Cell Precursor (ACP)-based product.

The authors drew their conclusion regarding this study. VesCell therapy for chronic stable angina seems to be safe and improves anginal symptoms at 3 and 6 months. Larger studies are being initiated to evaluate the benefit of VesCell for the treatment of this and additional severe heart diseases. (Source: Tresukosol et al. Abstract 3682: Treatment of Patients with Severe Angina Pectoris Using Intracoronarily Injected Autologous Blood-Borne Angiogenic Cell Precursors. Circulation. October 31, 2006. Vol. 114, Issue Suppl 18. Link: http://circ.ahajournals.org/content/114/Suppl_18/II_786.4 )

Another early cardiac stem cell clinical trial was performed in 2009 by a Cedars-Sinai team based on technologies and discoveries made by Eduardo Marban, MD, PhD, and led by Raj Makkar, MD. In this study, they explored the safety of harvesting, expanding, and administering a patients cardiac stem cells to repair heart tissue injured by myocardial infarction.

Recently, the American College of Cardiology (ACC) also announced results of a ground-breaking clinical study to evaluate the efficacy and effectiveness of cardiac stem cell treatment for heart failure patients. As stated by Timothy Henry, M.D., Director of Cardiology at Cedars-Sinai Heart Institute and one of the studys lead authors, This is the largest double-blind, placebo-controlled stem cell trial for treatment of heart failure to be presentedBased on these positive results, we are encouraged that this is an attractive potential therapy for patients with class III and class IV heart failure.

Additionally, Dr. Charles Goldthwaite, Jr, published a whitepaper titled, Mending a Broken Heart: Stem Cells and Cardiac Repair, in which he draws the conclusion, Given the worldwide prevalence of cardiac dysfunction and the limited availability of tissue for cardiac transplantation, stem cells could ultimately fulfill a large-scale unmet clinical need and improve the quality of life for millions of people with CVD. However, the use of these cells in this setting is currently in its infancymuch remains to be learned about the mechanisms by which stem cells repair and regenerate myocardium, the optimal cell types, and modes of their delivery, and the safety issues that will accompany their use.

Clearly, there is a trend toward acceptance of cardiac stem cell therapies as an emerging treatment option. Several world-renowned institutes are now conducting clinical studies involving cardiac stem cell treatment, as well as applying for intellectual property protection (patents) pertaining to the techniques required in administrating the therapies.

The key questions at this point in time appear to be:

An important whitepaper pertaining to cardiac stem cells is Ischemic Cardiomyopathy Patients Treated with Autologous Angiogenic and Cardio-Regenerative Progenitor Cells, written by Dr. Athina Kyritsis, et al. In it, the physicians describe their objective as investigating the feasibility, safety, and clinical outcome of patients with Ischemic Cardiomyopathy treated with Autologous Angiogenic and Cardio-Regenerative Progenitor cells (ACPs).

The researchers state: In numerous human trials there is evidence of improvement in the ejection fractions of Cardiomyopathy patients treated with ACPs. Animal experiments not only show improvement in cardiac function, but also engraftment and differentiation of ACPs into cardiomyocytes, as well as neo-vascularization in infarcted myocardium. In our clinical experience, the process has shown to be safe as well as effective.

The authors also found that patients treated with this approach gained increases in cardiac ejection fraction from their starting measurements, with improvements in their cardiac ejection fraction of 21 points (75% increase) at rest and 28.5 points (80% increase) at stress. As a result of these finding, the authors conclude, ACPs can improve the ejection fraction in patients with severely reduced cardiac function with benefits sustained to six months.

In the practice of medicine, the focus should be on delivering excellent care to patients. If there are cardiac stem cell treatments available, then regulatory obstacles should be removed when sufficient clinical trial evidence has been provided to indicate safety and efficacy.

Cardiologist Zannos Grekos, MD, a pioneer in cardiac stem cell therapy since 2006, points to the vastly untapped promise of related therapies, commenting Those of us that have been involved with cardiac stem cell treatment for the last 10-plus years can see the incredible potential this approach has.

As of 2017, the U.S. healthcare system is under enormous pressure to deliver affordable healthcareto a growing population of patients, especially those who are fully or partially covered under Medicare or Medicaid (many have secondary coverage). Although we are in the infancy of its development, cardiac stem cell treatments represent a potentially powerful treatment alternative to patients with heart failure symptoms.

To learn more, view the resources below.

1) Regenocyte http://www.regenocyte.com

2) Cleveland Clinic Stem Cell Therapy for Heart Disease my.clevelandclinic.org/health/articles/stem-cell-therapy-heart-disease

3) Harvard Stem Cell Institute (HSCI) hsci.harvard.edu/heart-disease-0

4) Cedars Sinai Cardiac Stem Cell Treatment http://www.cedars-sinai.edu/Patients/Programs-and-Services/Heart-Institute/Clinical-Trials/Cardiac-Stem-Cell-Research.aspx

5) Johns Hopkins Medicine Cardiac Stem Cell Treatments http://www.hopkinsmedicine.org/stem_cell_research/cell_therapy/a_new_path_for_cardiac_stem_cells.html

What do you think about heart failure signs and cardiac stem cell therapies? Share your thoughts in the comments section below.

Up Next:European Society of Cardiology (ESC) Congress Presentation Reveals Results From Pre-Clinical Study Using CardioCells Stem Cells for Acute Myocardial Infarction

Guest Post: This is a guest article by Clifford M. Thornton, a Certified Cardiovascular Technologist, experienced Echocardiographer Technician, and journalist in the cardiac and medical device fields. His articles have been published in Inventors Digest, Global Innovation Magazine, and Modern Health Talk. He is enthusiastic about progress with cardiac stem cell therapies and their role in heart failure treatment.He can be reached byphone at 267-524-7144 or by email at[emailprotected].

Editors Note This post was originally published on March 14, 2017, and has been updated for quality and relevancy.

Heart Failure Signs | Cardiac Stem Cell Therapies for Heart Failure Treatment

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Heart Failure Signs | Cardiac Stem Cell Therapies: Heart ...

The old rats appeared newly invigorated after receiving their injections. As hoped, the cardiac stem cells improved heart function yet also provided additional benefits. The rats' fur fur, shaved for surgery, grew back more quickly than expected, and their chromosomal telomeres, which commonly shrink with age, lengthened.

The old rats receiving the cardiac stem cells also had increased stamina overall, exercising more than before the infusion.

"It's extremely exciting," said Dr. Eduardo Marbn, primary investigator on the research and director of the Cedars-Sinai Heart Institute. Witnessing "the systemic rejuvenating effects," he said, "it's kind of like an unexpected fountain of youth."

"We've been studying new forms of cell therapy for the heart for some 12 years now," Marbn said.

Some of this research has focused on cardiosphere-derived cells.

"They're progenitor cells from the heart itself," Marbn said. Progenitor cells are generated from stem cells and share some, but not all, of the same properties. For instance, they can differentiate into more than one kind of cell like stem cells, but unlike stem cells, progenitor cells cannot divide and reproduce indefinitely.

Since heart failure with preserved ejection fraction is similar to aging, Marbn decided to experiment on old rats, ones that suffered from a type of heart problem "that's very typical of what we find in older human beings: The heart's stiff, and it doesn't relax right, and it causes fluid to back up some," Marbn explained.

He and his team injected cardiosphere-derived cells from newborn rats into the hearts of 22-month-old rats -- that's elderly for a rat. Similar old rats received a placebo injection of saline solution. Then, Marbn and his team compared both groups to young rats that were 4 months old. After a month, they compared the rats again.

Even though the cells were injected into the heart, their effects were noticeable throughout the body, Marbn said

"The animals could exercise further than they could before by about 20%, and one of the most striking things, especially for me (because I'm kind of losing my hair) the animals ... regrew their fur a lot better after they'd gotten cells" compared with the placebo rats, Marbn said.

The rats that received cardiosphere-derived cells also experienced improved heart function and showed longer heart cell telomeres.

Why did it work?

The working hypothesis is that the cells secrete exosomes, tiny vesicles that "contain a lot of nucleic acids, things like RNA, that can change patterns of the way the tissue responds to injury and the way genes are expressed in the tissue," Marbn said.

It is the exosomes that act on the heart and make it better as well as mediating long-distance effects on exercise capacity and hair regrowth, he explained.

Looking to the future, Marbn said he's begun to explore delivering the cardiac stem cells intravenously in a simple infusion -- instead of injecting them directly into the heart, which would be a complex procedure for a human patient -- and seeing whether the same beneficial effects occur.

Dr. Gary Gerstenblith, a professor of medicine in the cardiology division of Johns Hopkins Medicine, said the new study is "very comprehensive."

"Striking benefits are demonstrated not only from a cardiac perspective but across multiple organ systems," said Gerstenblith, who did not contribute to the new research. "The results suggest that stem cell therapies should be studied as an additional therapeutic option in the treatment of cardiac and other diseases common in the elderly."

Todd Herron, director of the University of Michigan Frankel Cardiovascular Center's Cardiovascular Regeneration Core Laboratory, said Marbn, with his previous work with cardiac stem cells, has "led the field in this area."

"The novelty of this bit of work is, they started to look at more precise molecular mechanisms to explain the phenomenon they've seen in the past," said Herron, who played no role in the new research.

One strength of the approach here is that the researchers have taken cells "from the organ that they want to rejuvenate, so that makes it likely that the cells stay there in that tissue," Herron said.

He believes that more extensive study, beginning with larger animals and including long-term followup, is needed before this technique could be used in humans.

"We need to make sure there's no harm being done," Herron said, adding that extending the lifetime and improving quality of life amounts to "a tradeoff between the potential risk and the potential good that can be done."

Capicor hasn't announced any plans to do studies in aging, but the possibility exists.

After all, the cells have been proven "completely safe" in "over 100 human patients," so it would be possible to fast-track them into the clinic, Marbn explained: "I can't tell you that there are any plans to do that, but it could easily be done from a safety viewpoint."

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Cardiac stem cells rejuvenate rats' aging hearts ... - CNN

Stem cell, an undifferentiated cell that can divide to produce some offspring cells that continue as stem cells and some cells that are destined to differentiate (become specialized). Stem cells are an ongoing source of the differentiated cells that make up the tissues and organs of animals and plants. There is great interest in stem cells because they have potential in the development of therapies for replacing defective or damaged cells resulting from a variety of disorders and injuries, such as Parkinson disease, heart disease, and diabetes. There are two major types of stem cells: embryonic stem cells and adult stem cells, which are also called tissue stem cells.

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cardiovascular disease: Cardiac stem cells

Cardiac stem cells, which have the ability to differentiate (specialize) into mature heart cells and therefore could be used to repair damaged or diseased heart tissue, have garnered significant interest in the development of treatments for heart disease and cardiac defects. Cardiac stem

Embryonic stem cells (often referred to as ES cells) are stem cells that are derived from the inner cell mass of a mammalian embryo at a very early stage of development, when it is composed of a hollow sphere of dividing cells (a blastocyst). Embryonic stem cells from human embryos and from embryos of certain other mammalian species can be grown in tissue culture.

The most-studied embryonic stem cells are mouse embryonic stem cells, which were first reported in 1981. This type of stem cell can be cultured indefinitely in the presence of leukemia inhibitory factor (LIF), a glycoprotein cytokine. If cultured mouse embryonic stem cells are injected into an early mouse embryo at the blastocyst stage, they will become integrated into the embryo and produce cells that differentiate into most or all of the tissue types that subsequently develop. This ability to repopulate mouse embryos is the key defining feature of embryonic stem cells, and because of it they are considered to be pluripotentthat is, able to give rise to any cell type of the adult organism. If the embryonic stem cells are kept in culture in the absence of LIF, they will differentiate into embryoid bodies, which somewhat resemble early mouse embryos at the egg-cylinder stage, with embryonic stem cells inside an outer layer of endoderm. If embryonic stem cells are grafted into an adult mouse, they will develop into a type of tumour called a teratoma, which contains a variety of differentiated tissue types.

Mouse embryonic stem cells are widely used to create genetically modified mice. This is done by introducing new genes into embryonic stem cells in tissue culture, selecting the particular genetic variant that is desired, and then inserting the genetically modified cells into mouse embryos. The resulting chimeric mice are composed partly of host cells and partly of the donor embryonic stem cells. As long as some of the chimeric mice have germ cells (sperm or eggs) that have been derived from the embryonic stem cells, it is possible to breed a line of mice that have the same genetic constitution as the embryonic stem cells and therefore incorporate the genetic modification that was made in vitro. This method has been used to produce thousands of new genetic lines of mice. In many such genetic lines, individual genes have been ablated in order to study their biological function; in others, genes have been introduced that have the same mutations that are found in various human genetic diseases. These mouse models for human disease are used in research to investigate both the pathology of the disease and new methods for therapy.

Extensive experience with mouse embryonic stem cells made it possible for scientists to grow human embryonic stem cells from early human embryos, and the first human stem cell line was created in 1998. Human embryonic stem cells are in many respects similar to mouse embryonic stem cells, but they do not require LIF for their maintenance. The human embryonic stem cells form a wide variety of differentiated tissues in vitro, and they form teratomas when grafted into immunosuppressed mice. It is not known whether the cells can colonize all the tissues of a human embryo, but it is presumed from their other properties that they are indeed pluripotent cells, and they therefore are regarded as a possible source of differentiated cells for cell therapythe replacement of a patients defective cell type with healthy cells. Large quantities of cells, such as dopamine-secreting neurons for the treatment of Parkinson disease and insulin-secreting pancreatic beta cells for the treatment of diabetes, could be produced from embryonic stem cells for cell transplantation. Cells for this purpose have previously been obtainable only from sources in very limited supply, such as the pancreatic beta cells obtained from the cadavers of human organ donors.

The use of human embryonic stem cells evokes ethical concerns, because the blastocyst-stage embryos are destroyed in the process of obtaining the stem cells. The embryos from which stem cells have been obtained are produced through in vitro fertilization, and people who consider preimplantation human embryos to be human beings generally believe that such work is morally wrong. Others accept it because they regard the blastocysts to be simply balls of cells, and human cells used in laboratories have not previously been accorded any special moral or legal status. Moreover, it is known that none of the cells of the inner cell mass are exclusively destined to become part of the embryo itselfall of the cells contribute some or all of their cell offspring to the placenta, which also has not been accorded any special legal status. The divergence of views on this issue is illustrated by the fact that the use of human embryonic stem cells is allowed in some countries and prohibited in others.

In 2009 the U.S. Food and Drug Administration approved the first clinical trial designed to test a human embryonic stem cell-based therapy, but the trial was halted in late 2011 because of a lack of funding and a change in lead American biotech company Gerons business directives. The therapy to be tested was known as GRNOPC1, which consisted of progenitor cells (partially differentiated cells) that, once inside the body, matured into neural cells known as oligodendrocytes. The oligodendrocyte progenitors of GRNOPC1 were derived from human embryonic stem cells. The therapy was designed for the restoration of nerve function in persons suffering from acute spinal cord injury.

Embryonic germ (EG) cells, derived from primordial germ cells found in the gonadal ridge of a late embryo, have many of the properties of embryonic stem cells. The primordial germ cells in an embryo develop into stem cells that in an adult generate the reproductive gametes (sperm or eggs). In mice and humans it is possible to grow embryonic germ cells in tissue culture with the appropriate growth factorsnamely, LIF and another cytokine called fibroblast growth factor.

Some tissues in the adult body, such as the epidermis of the skin, the lining of the small intestine, and bone marrow, undergo continuous cellular turnover. They contain stem cells, which persist indefinitely, and a much larger number of transit amplifying cells, which arise from the stem cells and divide a finite number of times until they become differentiated. The stem cells exist in niches formed by other cells, which secrete substances that keep the stem cells alive and active. Some types of tissue, such as liver tissue, show minimal cell division or undergo cell division only when injured. In such tissues there is probably no special stem-cell population, and any cell can participate in tissue regeneration when required.

The epidermis of the skin contains layers of cells called keratinocytes. Only the basal layer, next to the dermis, contains cells that divide. A number of these cells are stem cells, but the majority are transit amplifying cells. The keratinocytes slowly move outward through the epidermis as they mature, and they eventually die and are sloughed off at the surface of the skin. The epithelium of the small intestine forms projections called villi, which are interspersed with small pits called crypts. The dividing cells are located in the crypts, with the stem cells lying near the base of each crypt. Cells are continuously produced in the crypts, migrate onto the villi, and are eventually shed into the lumen of the intestine. As they migrate, they differentiate into the cell types characteristic of the intestinal epithelium.

Bone marrow contains cells called hematopoietic stem cells, which generate all the cell types of the blood and the immune system. Hematopoietic stem cells are also found in small numbers in peripheral blood and in larger numbers in umbilical cord blood. In bone marrow, hematopoietic stem cells are anchored to osteoblasts of the trabecular bone and to blood vessels. They generate progeny that can become lymphocytes, granulocytes, red blood cells, and certain other cell types, depending on the balance of growth factors in their immediate environment.

Work with experimental animals has shown that transplants of hematopoietic stem cells can occasionally colonize other tissues, with the transplanted cells becoming neurons, muscle cells, or epithelia. The degree to which transplanted hematopoietic stem cells are able to colonize other tissues is exceedingly small. Despite this, the use of hematopoietic stem cell transplants is being explored for conditions such as heart disease or autoimmune disorders. It is an especially attractive option for those opposed to the use of embryonic stem cells.

Bone marrow transplants (also known as bone marrow grafts) represent a type of stem cell therapy that is in common use. They are used to allow cancer patients to survive otherwise lethal doses of radiation therapy or chemotherapy that destroy the stem cells in bone marrow. For this procedure, the patients own marrow is harvested before the cancer treatment and is then reinfused into the body after treatment. The hematopoietic stem cells of the transplant colonize the damaged marrow and eventually repopulate the blood and the immune system with functional cells. Bone marrow transplants are also often carried out between individuals (allograft). In this case the grafted marrow has some beneficial antitumour effect. Risks associated with bone marrow allografts include rejection of the graft by the patients immune system and reaction of immune cells of the graft against the patients tissues (graft-versus-host disease).

Bone marrow is a source for mesenchymal stem cells (sometimes called marrow stromal cells, or MSCs), which are precursors to non-hematopoietic stem cells that have the potential to differentiate into several different types of cells, including cells that form bone, muscle, and connective tissue. In cell cultures, bone-marrow-derived mesenchymal stem cells demonstrate pluripotency when exposed to substances that influence cell differentiation. Harnessing these pluripotent properties has become highly valuable in the generation of transplantable tissues and organs. In 2008 scientists used mesenchymal stem cells to bioengineer a section of trachea that was transplanted into a woman whose upper airway had been severely damaged by tuberculosis. The stem cells were derived from the womans bone marrow, cultured in a laboratory, and used for tissue engineering. In the engineering process, a donor trachea was stripped of its interior and exterior cell linings, leaving behind a trachea scaffold of connective tissue. The stem cells derived from the recipient were then used to recolonize the interior of the scaffold, and normal epithelial cells, also isolated from the recipient, were used to recolonize the exterior of the trachea. The use of the recipients own cells to populate the trachea scaffold prevented immune rejection and eliminated the need for immunosuppression therapy. The transplant, which was successful, was the first of its kind.

Research has shown that there are also stem cells in the brain. In mammals very few new neurons are formed after birth, but some neurons in the olfactory bulbs and in the hippocampus are continually being formed. These neurons arise from neural stem cells, which can be cultured in vitro in the form of neurospheressmall cell clusters that contain stem cells and some of their progeny. This type of stem cell is being studied for use in cell therapy to treat Parkinson disease and other forms of neurodegeneration or traumatic damage to the central nervous system.

Following experiments in animals, including those used to create Dolly the sheep, there has been much discussion about the use of somatic cell nuclear transfer (SCNT) to create pluripotent human cells. In SCNT the nucleus of a somatic cell (a fully differentiated cell, excluding germ cells), which contains the majority of the cells DNA (deoxyribonucleic acid), is removed and transferred into an unfertilized egg cell that has had its own nuclear DNA removed. The egg cell is grown in culture until it reaches the blastocyst stage. The inner cell mass is then removed from the egg, and the cells are grown in culture to form an embryonic stem cell line (generations of cells originating from the same group of parent cells). These cells can then be stimulated to differentiate into various types of cells needed for transplantation. Since these cells would be genetically identical to the original donor, they could be used to treat the donor with no problems of immune rejection. Scientists generated human embryonic stem cells successfully from SCNT human embryos for the first time in 2013.

While promising, the generation and use of SCNT-derived embryonic stem cells is controversial for several reasons. One is that SCNT can require more than a dozen eggs before one egg successfully produces embryonic stem cells. Human eggs are in short supply, and there are many legal and ethical problems associated with egg donation. There are also unknown risks involved with transplanting SCNT-derived stem cells into humans, because the mechanism by which the unfertilized egg is able to reprogram the nuclear DNA of a differentiated cell is not entirely understood. In addition, SCNT is commonly used to produce clones of animals (such as Dolly). Although the cloning of humans is currently illegal throughout the world, the egg cell that contains nuclear DNA from an adult cell could in theory be implanted into a womans uterus and come to term as an actual cloned human. Thus, there exists strong opposition among some groups to the use of SCNT to generate human embryonic stem cells.

Due to the ethical and moral issues surrounding the use of embryonic stem cells, scientists have searched for ways to reprogram adult somatic cells. Studies of cell fusion, in which differentiated adult somatic cells grown in culture with embryonic stem cells fuse with the stem cells and acquire embryonic stem-cell-like properties, led to the idea that specific genes could reprogram differentiated adult cells. An advantage of cell fusion is that it relies on existing embryonic stem cells instead of eggs. However, fused cells stimulate an immune response when transplanted into humans, which leads to transplant rejection. As a result, research has become increasingly focused on the genes and proteins capable of reprogramming adult cells to a pluripotent state. In order to make adult cells pluripotent without fusing them to embryonic stem cells, regulatory genes that induce pluripotency must be introduced into the nuclei of adult cells. To do this, adult cells are grown in cell culture, and specific combinations of regulatory genes are inserted into retroviruses (viruses that convert RNA [ribonucleic acid] into DNA), which are then introduced to the culture medium. The retroviruses transport the RNA of the regulatory genes into the nuclei of the adult cells, where the genes are then incorporated into the DNA of the cells. About 1 out of every 10,000 cells acquires embryonic stem cell properties. Although the mechanism is still uncertain, it is clear that some of the genes confer embryonic stem cell properties by means of the regulation of numerous other genes. Adult cells that become reprogrammed in this way are known as induced pluripotent stem cells (iPS).

Similar to embryonic stem cells, induced pluripotent stem cells can be stimulated to differentiate into select types of cells that could in principle be used for disease-specific treatments. In addition, the generation of induced pluripotent stem cells from the adult cells of patients affected by genetic diseases can be used to model the diseases in the laboratory. For example, in 2008 researchers isolated skin cells from a child with an inherited neurological disease called spinal muscular atrophy and then reprogrammed these cells into induced pluripotent stem cells. The reprogrammed cells retained the disease genotype of the adult cells and were stimulated to differentiate into motor neurons that displayed functional insufficiencies associated with spinal muscular atrophy. By recapitulating the disease in the laboratory, scientists were able to study closely the cellular changes that occurred as the disease progressed. Such models promise not only to improve scientists understanding of genetic diseases but also to facilitate the development of new therapeutic strategies tailored to each type of genetic disease.

In 2009 scientists successfully generated retinal cells of the human eye by reprogramming adult skin cells. This advance enabled detailed investigation of the embryonic development of retinal cells and opened avenues for the generation of novel therapies for eye diseases. The production of retinal cells from reprogrammed skin cells may be particularly useful in the treatment of retinitis pigmentosa, which is characterized by the progressive degeneration of the retina, eventually leading to night blindness and other complications of vision. Although retinal cells also have been produced from human embryonic stem cells, induced pluripotency represents a less controversial approach. Scientists have also explored the possibility of combining induced pluripotent stem cell technology with gene therapy, which would be of value particularly for patients with genetic disease who would benefit from autologous transplantation.

Researchers have also been able to generate cardiac stem cells for the treatment of certain forms of heart disease through the process of dedifferentiation, in which mature heart cells are stimulated to revert to stem cells. The first attempt at the transplantation of autologous cardiac stem cells was performed in 2009, when doctors isolated heart tissue from a patient, cultured the tissue in a laboratory, stimulated cell dedifferentiation, and then reinfused the cardiac stem cells directly into the patients heart. A similar study involving 14 patients who underwent cardiac bypass surgery followed by cardiac stem cell transplantation was reported in 2011. More than three months after stem cell transplantation, the patients experienced a slight but detectable improvement in heart function.

Patient-specific induced pluripotent stem cells and dedifferentiated cells are highly valuable in terms of their therapeutic applications because they are unlikely to be rejected by the immune system. However, before induced pluripotent stem cells can be used to treat human diseases, researchers must find a way to introduce the active reprogramming genes without using retroviruses, which can cause diseases such as leukemia in humans. A possible alternative to the use of retroviruses to transport regulatory genes into the nuclei of adult cells is the use of plasmids, which are less tumourigenic than viruses.

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stem cell | Definition, Types, Uses, Research, & Facts ...