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Clinical translation of stem cell therapies for heart disease requires electrical integration of transplanted cardiomyocytes. Generation of electrically matured human induced pluripotent stem cell–derived cardiomyocytes (hiPSC-CMs) is critical for electrical integration. Here, we found that hiPSC-derived endothelial cells (hiPSC-ECs) promoted the expression of selected maturation markers in hiPSC-CMs. Using tissue-embedded stretchable mesh nanoelectronics, we achieved a long-term stable map of human three-dimensional (3D) cardiac microtissue electrical activity. The results revealed that hiPSC-ECs accelerated the electrical maturation of hiPSC-CMs in 3D cardiac microtissues. Machine learning–based pseudotime trajectory inference of cardiomyocyte electrical signals further revealed the electrical phenotypic transition path during development. Guided by the electrical recording data, single-cell RNA sequencing identified that hiPSC-ECs promoted cardiomyocyte subpopulations with a more mature phenotype, and multiple ligand-receptor interactions were up-regulated between hiPSC-ECs and hiPSC-CMs, revealing a coordinated multifactorial mechanism of hiPSC-CM electrical maturation. Collectively, these findings show that hiPSC-ECs drive hiPSC-CM electrical maturation via multiple intercellular pathways.

Summary Recent studies suggest that metabolic regulation may improve differentiation of cardiomyocytes derived from induced pluripotent stem cells (iPSCs). AMP-activated protein kinase (AMPK) is a master regulator of metabolic activities. We investigated whether AMPK participates in iPSC-derived cardiomyocyte differentiation. We observed that AMPK phosphorylation at Thr172 increased at day 9 but then decreased after day 11 of differentiation to cardiomyocytes. Inhibition of AMPK with compound C significantly reduced mRNA and protein expression of cardiac troponins TNNT2 and TNNI3. Moreover, sustained AMPK activation using AICAR from days 9 to 14 of differentiation increased mRNA and protein expression of both TNNT2 and TNNI3. AICAR decreased acetylation of histone 3 at Lys9 and 56 and histone 4 at Lys16 (known target sites for nuclear-localized sirtuins [SIRT1, SIRT6]), suggesting that AMPK activation enhances sirtuin activity. Sustained AMPK activation during days 9–14 of differentiation induces sirtuin-mediated histone deacetylation and may enhance cardiomyocyte differentiation from iPSCs., Highlights • iPSC-derived cardiomyocytes transiently increased AMPK phosphorylation at Thr172 • Chemical inhibition of AMPK with compound C decreased TNNI3 and TNNT2 expression • Sustained activation of AMPK using AICAR increased expression of TNNT2 and TNNI3 • AICAR decreased acetylation of histones H3 (at Lys9 and Lys56) and H4 (at Lys16)., Lee and colleagues demonstrate that sustained activation of AMPK enhances differentiation of iPSC-derived cardiomyocytes. Sustained AMPK activation decreased histone acetylation at known target sites for nuclear-localized sirtuins, suggesting that AMPK activation enhances sirtuin activity. AMPK-induced sirtuin-mediated deacetylation of histone proteins may regulate chromatin accessibility and enhance cardiomyocyte differentiation.

Background: Current differentiation protocols to produce cardiomyocytes from human induced pluripotent stem cells (iPSCs) are capable of generating highly pure cardiomyocyte populations as determined by expression of cardiac troponin T. However, these cardiomyocytes remain immature, more closely resembling the fetal state, with a lower maximum contractile force, slower upstroke velocity, and immature mitochondrial function compared with adult cardiomyocytes. Immaturity of iPSC-derived cardiomyocytes may be a significant barrier to clinical translation of cardiomyocyte cell therapies for heart disease. During development, cardiomyocytes undergo a shift from a proliferative state in the fetus to a more mature but quiescent state after birth. The mechanistic target of rapamycin (mTOR)–signaling pathway plays a key role in nutrient sensing and growth. We hypothesized that transient inhibition of the mTOR-signaling pathway could lead cardiomyocytes to a quiescent state and enhance cardiomyocyte maturation. Methods: Cardiomyocytes were differentiated from 3 human iPSC lines using small molecules to modulate the Wnt pathway. Torin1 (0 to 200 nmol/L) was used to inhibit the mTOR pathway at various time points. We quantified contractile, metabolic, and electrophysiological properties of matured iPSC-derived cardiomyocytes. We utilized the small molecule inhibitor, pifithrin-α, to inhibit p53 signaling, and nutlin-3a, a small molecule inhibitor of MDM2 (mouse double minute 2 homolog) to upregulate and increase activation of p53. Results: Torin1 (200 nmol/L) increased the percentage of quiescent cells (G 0 phase) from 24% to 48% compared with vehicle control ( P Conclusions: Transient treatment of human iPSC-derived cardiomyocytes with Torin1 shifts cells to a quiescent state and enhances cardiomyocyte maturity.

Cardiovascular disease is a leading cause of death worldwide, and thus there remains great interest in regenerative approaches to treat heart failure. In the past 20 years, the field of heart regeneration has entered a renaissance period with remarkable progress in the understanding of endogenous heart regeneration, stem cell differentiation for exogenous cell therapy, and cell-delivery methods. In this review, we highlight how this new understanding can lead to viable strategies for human therapy. For the near term, drugs, electrical and mechanical devices, and heart transplantation will remain mainstays of cardiac therapies, but eventually regenerative therapies based on fundamental regenerative biology may offer more permanent solutions for patients with heart failure.

Clinical translation of stem cell therapies for heart disease is limited by a risk of potentially life-threatening ventricular arrhythmias seen following cardiomyocyte delivery in large animal models. Enhancing cardiomyocyte maturation may reduce this arrhythmogenic risk by reducing automaticity of delivered cardiomyocytes. We tested whether human induced pluripotent stem cell (hiPSC)-derived endothelial cells can enhance maturation and suppress automaticity of iPSC-derived cardiomyocytes in vitro. We found that co-culture of hiPSC-derived endothelial cells with hiPSC-derived cardiomyocytes significantly increased protein expression of cardiac troponin T, cardiac troponin I, Kir2.1, connexin 43, and CD36. In addition, using a stretchable mesh nanoelectronics device, we found that hiPSC-derived endothelial cells accelerated electrical maturation of hiPSC-derived cardiomyocytes and progressively suppressed cardiomyocyte automaticity in vitro (Figure). Using single cell RNA-seq, we identified a subpopulation of hiPSC-derived cardiomyocytes that is eliminated upon co-culture with hiPSC-derived endothelial cells. Further work will investigate whether this subpopulation of cardiomyocytes is responsible for automaticity of cardiomyocyte cultures. Figure. Induced pluripotent stem cell (iPSC)-derived cardiomyocytes alone (CM only) or iPSC-derived cardiomyocytes co-cultured with iPSC-derived endothelial cells (CM+EC) were seeded onto a stretchable mesh nanoelectronics device. Unstimulated voltage tracings at day 30 of cardiomyocyte differentiation show cardiomyocytes with a slower beating rate and more narrow action potential when co-cultured with iPSC-derived endothelial cells compared to iPSC-derived cardiomyocytes alone.

Directed differentiation of human pluripotent stem cells (PSC) into cardiomyocytes via manipulation of Wnt signaling leads to generation of immature cardiomyocytes, more closely resembling a fetal state. It has become increasingly apparent that metabolic parameters regulate of cardiomyocyte maturation. The forkhead box (FOX) family of transcription factors has previously been shown to regulate metabolic phenotype in neonatal cardiomyocytes through a balance between FOXO and FOXM proteins. Therefore, we hypothesized that dysregulation of FOXO-FOXM1 signaling inhibits maturation of iPSC-derived cardiomyocytes (iPSC-CMs). We cultured iPSC-CMs in 3D suspension culture using an orbital shaker. iPSC-CMs were treated with RCM-1 (FOXM1 inhibitor), LOM612 (FOXO nuclear translocator), or AS1842856 (FOXO inhibitor) starting 2 days after onset of beating. We found that inhibition of FOXO with AS1842856 resulted in loss of expression of cardiac-specific markers such as cardiac troponin T as well as loss of spontaneous beating. In contrast, inhibition of FOXM1 with RCM-1 or activation of FOXO with LOM612 resulted in retention of a cardiomyocyte phenotype with continued expression of cardiac troponin T but with significantly increased expression membrane protein expression of Kir2.1 (Figure), the protein largely responsible for maintaining the resting membrane potential in cardiomyocytes. These results suggest that inhibition of FOXM1 and/or activation of FOXO signaling may facilitate maturation of iPSC-derived cardiomyocytes. Figure. (A) % of TNNT2+ iPSC-derived cardiomyocytes that express Kir2.1 and (B) Mean fluorescence intensity of Kir2.1 in iPSC-derived cardiomyocytes after treatment with DMSO (vehicle control), RCM-1, LOM612, or AS1842856. *p

Administration of active growth differentiation factor 11 (GDF11) to aged mice can reduce cardiac hypertrophy, and low serum levels of GDF11 measured together with the related protein, myostatin (also known as GDF8), predict future morbidity and mortality in coronary heart patients. Using mice with a loxP-flanked (“floxed”) allele of Gdf11 and Myh6-driven expression of Cre recombinase to delete Gdf11 in cardiomyocytes, we tested the hypothesis that cardiac-specific Gdf11 deficiency might lead to cardiac hypertrophy in young adulthood. We observed that targeted deletion of Gdf11 in cardiomyocytes does not cause cardiac hypertrophy but rather leads to left ventricular dilation when compared with control mice carrying only the Myh6-cre or Gdf11-floxed alleles, suggesting a possible etiology for dilated cardiomyopathy. However, the mechanism underlying this finding remains unclear because of multiple confounding effects associated with the selected model. First, whole heart Gdf11 expression did not decrease in Myh6-cre; Gdf11-floxed mice, possibly because of upregulation of Gdf11 in noncardiomyocytes in the heart. Second, we observed Cre-associated toxicity, with lower body weights and increased global fibrosis, in Cre-only control male mice compared with flox-only controls, making it challenging to infer which changes in Myh6-cre;Gdf11-floxed mice were the result of Cre toxicity versus deletion of Gdf11. Third, we observed differential expression of cre mRNA in Cre-only controls compared with the cardiomyocyte-specific knockout mice, also making comparison between these two groups difficult. Thus, targeted Gdf11 deletion in cardiomyocytes may lead to left ventricular dilation without hypertrophy, but alternative animal models are necessary to understand the mechanism for these findings. NEW & NOTEWORTHY We observed that targeted deletion of growth differentiation factor 11 in cardiomyocytes does not cause cardiac hypertrophy but rather leads to left ventricular dilation compared with control mice carrying only the Myh6-cre or growth differentiation factor 11-floxed alleles. However, the mechanism underlying this finding remains unclear because of multiple confounding effects associated with the selected mouse model.

Current methods to differentiate cardiomyocytes from human pluripotent stem cells (PSCs) inadequately recapitulate complete development and result in PSC-derived cardiomyocytes (PSC-CMs) with an immature or fetal-like phenotype. Embryonic and fetal development are highly dynamic periods during which the developing embryo or fetus is exposed to changing nutrient, oxygen, and hormone levels until birth. It is becoming increasingly apparent that these metabolic changes initiate developmental processes to mature cardiomyocytes. Mitochondria are central to these changes, responding to these metabolic changes and transitioning from small, fragmented mitochondria to large organelles capable of producing enough ATP to support the contractile function of the heart. These changes in mitochondria may not simply be a response to cardiomyocyte maturation; the metabolic signals that occur throughout development may actually be central to the maturation process in cardiomyocytes. Here, we review methods to enhance maturation of PSC-CMs and highlight evidence from development indicating the key roles that mitochondria play during cardiomyocyte maturation. We evaluate metabolic transitions that occur during development and how these affect molecular nutrient sensors, discuss how regulation of nutrient sensing pathways affect mitochondrial dynamics and function, and explore how changes in mitochondrial function can affect metabolite production, the cell cycle, and epigenetics to influence maturation of cardiomyocytes.

BACKGROUND Two or more early rejections ( 1 y) have been associated with coronary artery vasculopathy (CAV) in pediatric heart transplant (HT) recipients. We hypothesized that clinical rejection defined by concurrent new-onset heart failure or left ventricular systolic dysfunction is more strongly associated with future CAV than rejection diagnosed on protocol biopsy. METHODS We identified all subjects

Today, cell replacement therapy using pluripotent stem cell-derived cardiomyocytes (PSC-CMs) remains a research endeavor, with several hurdles that must be overcome before delivery of PSC-CMs can become a therapeutic reality. In this review, we highlight major findings to date from pre-clinical studies involving delivery of PSC-CMs and consider remaining challenges that must be addressed for successful clinical translation. Our goal is to provide an overview of the current status of cardiomyocyte replacement therapy and what challenges must be addressed before successful clinical translation of such therapies will be possible.

The adult myocardium has a limited regenerative capacity following heart injury and the lost cells are primarily replaced by fibrotic scar tissue. Suboptimal efficiency of current clinical therapies to resurrect the infarcted heart results in injured heart enlargement and remodeling to maintain its physiological functions. These remodeling processes ultimately leads to ischemic cardiomyopathy and heart failure (HF). Recent therapeutic approaches (e.g., regenerative and nanomedicine) have shown promise to prevent HF post-myocardial infarction in animal models. However, these preclinical, clinical, and technological advancements have yet to yield substantial enhancements in the survival rate and quality of life of patients with severe ischemic injuries. This could be attributed largely to the considerable gap in knowledge between clinicians and nano-bioengineers. Development of highly effective cardiac regenerative therapies requires connecting and coordinating multiple fields, including cardiology, cellular and molecular biology, biochemistry and chemistry, and mechanical and materials sciences, among others. This review is particularly intended to bridge the knowledge gap between cardiologists and regenerative nanomedicine experts. Establishing this multidisciplinary knowledge base may help pave the way for developing novel, safer, and more effective approaches that will enable the medical community to reduce morbidity and mortality in HF patients.

Tightly regulated production of mature blood cells is essential for health and survival in vertebrates and dependent on discrete populations of blood-forming (hematopoietic) stem and progenitor cells. Prior studies suggested that inhibition of growth differentiation factor 11 (GDF11) through soluble activin receptor type II (ActRII) ligand traps or neutralizing antibodies promotes erythroid precursor cell maturation and red blood cell formation in contexts of homeostasis and anemia. As Gdf11 is expressed by mature hematopoietic cells, and erythroid precursor cell expression of Gdf11 has been implicated in regulating erythropoiesis, we hypothesized that genetic disruption of Gdf11 in blood cells might perturb normal hematopoiesis or recovery from hematopoietic insult. Contrary to these predictions, we found that deletion of Gdf11 in the hematopoietic lineage in mice does not alter erythropoiesis or erythroid precursor cell frequency under normal conditions or during hematopoietic recovery after irradiation and transplantation. In addition, although hematopoietic cell-derived Gdf11 may contribute to the pool of circulating GDF11 protein during adult homeostasis, loss of Gdf11 specifically in the blood system does not impair hematopoietic stem cell function or induce overt pathological consequences. Taken together, these results reveal that hematopoietic cell-derived Gdf11 is largely dispensable for native and transplant-induced blood formation.

Microvascular dysfunction in the heart and its association with periarteriolar fibrosis may contribute to the diastolic dysfunction seen in heart failure with preserved ejection fraction. Interleukin-33 (IL-33) prevents global myocardial fibrosis in a pressure overloaded left ventricle by acting via its receptor, ST2 (encoded by the gene, Il1rl1); however, whether this cytokine can also modulate periarteriolar fibrosis remains unclear. We utilized two approaches to explore the role of IL-33/ST2 in periarteriolar fibrosis. First, we studied young and old wild type mice to test the hypothesis that IL-33 and ST2 expression change with age. Second, we produced pressure overload in mice deficient in IL-33 or ST2 by transverse aortic constriction (TAC). With age, IL- 33 expression increased and ST2 expression decreased. These alterations accompanied increased periarteriolar fibrosis in aged mice. Mice deficient in ST2 but not IL-33 had a significant increase in periarteriolar fibrosis following TAC compared to wild type mice. Thus, loss of ST2 signaling rather than changes in IL-33 expression may contribute to periarteriolar fibrosis during aging or pressure overload, but manipulating this pathway alone may not prevent or reverse fibrosis.

Background— Previous studies suggest that children with myocarditis who receive heart transplantation (HT) may be at higher risk of post-transplant mortality compared with children who are transplanted for idiopathic dilated cardiomyopathy. We hypothesized that these differences are because of more severe heart failure at HT in children with myocarditis. Methods and Results— We identified 221 children with myocarditis and 1583 with idiopathic dilated cardiomyopathy who were Conclusions— Among children listed for HT, those with myocarditis have more severe heart failure than children with idiopathic dilated cardiomyopathy. After adjustment for severity of illness, myocarditis does not confer additional risk for wait-list or post-transplant mortality.

A pH- and temperature-responsive, injectable hydrogel has been designed to take advantage of the acidic microenvironment of ischemic myocardium. This system can improve therapeutic angiogenesis methods by providing spatio-temporal control of angiogenic growth factor delivery. The pH- and temperature-responsive random copolymer, poly(N-isopropylacrylamide-co-propylacrylic acid-co-butyl acrylate) (p[NIPAAm-co-PAA-co-BA]), was synthesized by reversible addition fragmentation chain transfer polymerization. This polymer was a liquid at pH 7.4 and 37 °C but formed a physical gel at pH 6.8 and 37 °C. Retention of biotinylated basic fibroblast growth factor (bFGF) between 0 and 7 days after injection into infarcted rat myocardium was 10-fold higher with hydrogel delivery versus saline. Following 28 days of treatment in vivo, capillary and arteriolar densities were increased 30-40% by polymer + bFGF treatment versus saline + bFGF or polymer-only controls. Treatment with polymer + bFGF for 28 days resulted in a 2-fold improvement in relative blood flow to the infarct region versus day 0, whereas saline + bFGF or polymer-only had no effect. Fractional shortening determined by echocardiography was significantly higher following treatment with polymer + bFGF (30 ± 1.4%) versus saline (25 ± 1.2%) and polymer alone (25 ± 1.8%). By responding to local changes in pH- and temperature in an animal model of ischemia, this hydrogel system provided sustained, local delivery of bFGF, improved angiogenesis, and achieved therapeutic effects in regional blood flow and cardiac function.

This study examines whether longitudinal antioxidant treatment initiated in prehypertensive spontaneously hypertensive rats (SHR) can attenuate vascular oxidant stress and prevent blood pressure elevation during development. Male SHR and age-matched Wistar-Kyoto rats (WKY) were treated from 6 to 11 weeks of age with Tempol (4-hydroxy-2,2,6,6-tetramethylpiperidinoxyl) (1 mmol/l in drinking water), a membrane-permeable superoxide dismutase mimetic. Mean systolic blood pressures (SBPs) were measured by tail-cuff Agonist-induced and basal O2- production was measured in thoracic aortas of 6- and 11-week-old SHR and WKY by lucigenin-derived chemiluminescence and oxidative fluorescent microscopy, respectively. SBP of 6-week-old SHR (131 +/- 5 mmHg) and WKY (130 +/- 4 mmHg) were not different; however, 11-week-old SHR SBP (171 +/- 4 mmHg) was significantly greater (p = .0001) than 11-week-old WKY SBP (143 +/- 5 mmHg). Tempol treatment completely, but reversibly, prevented this age-related rise in SHR SBP (SHR + Tempol: 137 +/- 4 mmHg; p < .0001 versus untreated SHR). Agonist-induced vascular O2- was increased in 6- (p = .03) and 11-week-old SHR (p < .0001) and 11-week-old WKY (p = .03) but not in 6-week-old WKY. Long-term Tempol treatment significantly lowered O2- production in both strains. Basal O2- measurements in both 6- and 11-week-old SHR were qualitatively increased compared with age-matched WKY; this increase in SHR was inhibited with in vitro Tempol treatment. These data show that antioxidant treatment to reduce oxidative stress prevents the age-related development of high blood pressure in an animal model of genetic hypertension.

There is an urgent clinical need to develop new therapeutic approaches to treat heart failure, but the biology of cardiovascular regeneration is complex. Model systems are required to advance our understanding of biological mechanisms of cardiac regeneration as well as to test therapeutic approaches to regenerate tissue and restore cardiac function following injury. An ideal model system should be inexpensive, easily manipulated, easily reproducible, physiologically representative of human disease, and ethically sound. In this review, we discuss computational, cell-based, tissue, and animal models that have been used to elucidate mechanisms of cardiovascular regenerative biology or to test proposed therapeutic methods to restore cardiac function following disease or injury.

A new sharply pH- and temperature-responsive hydrogel system was designed for delivering drugs to regions of local acidosis, as found in wound healing, tumor sites, or sites of ischemia. The reversible addition-fragmentation chain transfer (RAFT) polymerization technique was used to synthesize copolymers of N-isopropylacrylamide (NIPAAM) and propylacrylic acid (PAA) with feed ratios of PAA between 0 and 20 mol %. The pH-responsive viscoelastic properties of these materials as a function of pH and temperature were quantified by rheometry. At physiologic pH (7.4) and 5 wt %, the polymer did not form gels but rather remained soluble at temperatures as high as 50 degrees C. At lower pH values (pH ca. 5.5 and below), the polymer was liquid at 20 degrees C, but exhibited a sol-gel phase transformation with increasing temperature and existed as a physical gel at 37 degrees C. Incorporation of the hydrophobic monomer, butyl acrylate, into the random copolymer raised the pH of gel formation to greater than 6.0 at 37 degrees C. Drug loading studies demonstrated that p(NIPAAm-co-PAA) hydrogels are able to maintain the bioactivity of basic fibroblast growth factor following storage in hydrogel for 40 h and can provide sustained pH-dependent release of vascular endothelial growth factor over a period of at least three weeks. This hydrogel system will thus gel at controllable acidic pH values upon injection, and is designed to undergo gradual dissolution as it performs its drug delivery function and the ischemic site returns to physiological pH.

A developing therapy for complete or partial loss of function in various tissues and organs involves transplanting an appropriate cell population, capable of compensating for the existing deficiencies. Clinical application of this type of strategy is currently limited by the death or dedifferentiation of the transplanted cells after delivery to the recipient. A delay in thorough vascularization of the implant area creates an environment low in oxygen and other nutrients, and likely contributes to the initial death of transplanted cells. We have addressed this problem by sustained delivery of vascular endothelial growth factor (VEGF), an initiator of angiogenesis, from a porous polymer matrix utilized simultaneously for cell delivery. As expected from previous studies, VEGF delivered from these constructs elicited an enhanced angiogenic response over a 2-week period when implanted subcutaneously in SCID mice. Hepatocytes implanted using VEGF-containing matrices demonstrated significantly greater survival after 1 week in vivo as compared with cells implanted on matrices without growth factor. The results of this study therefore indicate that enhancing vascularization in the location of transplanted cells promotes their survival. In addition, this delivery system may be used in future studies to directly promote cell survival and function by also providing growth factors specific to the transplanted cells.

Regions of local acidosis, such as that seen in myocardial ischemia, offer a target for stimuli-sensitive drug delivery systems. Hydrogels are polymer networks that are capable of entrapping significant amounts of water and can be designed to respond to stimuli such as temperature, pH, light, and electric fields. These properties make them highly attractive for drug delivery systems because of their biocompatibility and potential for drug targeting. In particular, reversible hydrogels are attractive because they can be designed to be soluble under normal physiologic conditions but form a gel under other conditions, such as decreased pH. In this study, we investigated the sol-gel properties of a pH- and temperature-sensitive polymer and assessed the ability of this polymer to be developed into an injectable hydrogel drug delivery system for ischemic cardiac tissue. Poly(N-isopropylacrylamide-co-propyl acrylic acid) (p(NIPAAm-co-PAA)) was synthesized by reversible addition fragmentation chain transfer (RAFT) polymerization. The feed composition of the pH-sensitive component, PAA, was varied between 0 and 20 mol%. Polymer was characterized by gel permeation chromatography and proton nuclear magnetic resonance to determine the molecular weight and polymer composition, respectively. Polymer samples were dissolved in phosphate buffer, and then pH was adjusted between 4.5 and 7.0. One-milliliter samples were loaded onto a rheometer to quantify viscoelastic properties. P(NIPAAm-co-PAA) polymers of varying PAA content were successfully synthesized by RAFT. At physiologic pH (7.4), the polymer did not form a gel but rather remained in the soluble state at all temperatures. However, at low pH (below approximately pH 5.5), with increasing temperature (in a physiologically relevant range), the polymers underwent a phase change from a clear liquid, to a cloudy liquid, and finally to a gel. As the pH was decreased further, the gelation temperature for a given polymer composition also decreased. In conclusion, by varying the relative ratio of NIPAAm and PAA in p(NIPAAm-co-PAA), the temperature and pH at which a gel forms can be controlled. Future studies will investigate the drug-release properties of this system.

Stem cell therapy for cardiac disease is an exciting but highly controversial research area. Strategies such as cell transplantation and reprogramming have demonstrated both intriguing and sobering results. Yet as clinical trials proceed, our incomplete understanding of stem cell behavior is made evident by numerous unresolved matters, such as the mechanisms of cardiomyocyte turnover or the optimal therapeutic strategies to achieve clinical efficacy. In this Perspective, we consider how cardiac stem cell biology has led us into clinical trials, and we suggest that achieving true cardiac regeneration in patients may ultimately require resolution of critical controversies in experimental cardiac regeneration.