RESUMO
BACKGROUND: Current protocols generate highly pure human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) in vitro that recapitulate characteristics of mature in vivo cardiomyocytes. Yet, a risk of arrhythmias exists when hiPSC-CMs are injected into large animal models. Thus, understanding hiPSC-CM maturational mechanisms is crucial for clinical translation. Forkhead box (FOX) transcription factors regulate postnatal cardiomyocyte maturation through a balance between FOXO and FOXM1. We also previously demonstrated that p53 activation enhances hiPSC-CM maturation. Here, we investigate whether p53 activation modulates the FOXO/FOXM1 balance to promote hiPSC-CM maturation in 3-dimensional suspension culture. METHODS AND RESULTS: Three-dimensional cultures of hiPSC-CMs were treated with Nutlin-3a (p53 activator, 10 µM), LOM612 (FOXO relocator, 5 µM), AS1842856 (FOXO inhibitor, 1 µM), or RCM-1 (FOXM1 inhibitor, 1 µM), starting 2 days after onset of beating, with dimethyl sulfoxide (0.2% vehicle) as control. P53 activation promoted hiPSC-CM metabolic and electrophysiological maturation alongside FOXO upregulation and FOXM1 downregulation, in n=3 to 6 per group for all assays. FOXO inhibition significantly decreased expression of cardiac-specific markers such as TNNT2. In contrast, FOXO activation or FOXM1 inhibition promoted maturational characteristics such as increased contractility, oxygen consumption, and voltage peak maximum upstroke velocity, in n=3 to 6 per group for all assays. Further, by single-cell RNA sequencing of n=2 LOM612-treated cells compared with dimethyl sulfoxide, LOM612-mediated FOXO activation promoted expression of cardiac maturational pathways. CONCLUSIONS: We show that p53 activation promotes FOXO and suppresses FOXM1 during 3-dimensional hiPSC-CM maturation. These results expand our understanding of hiPSC-CM maturational mechanisms in a clinically-relevant 3-dimensional culture system.
Assuntos
Diferenciação Celular , Proteína Forkhead Box M1 , Células-Tronco Pluripotentes Induzidas , Miócitos Cardíacos , Proteína Supressora de Tumor p53 , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Proteína Forkhead Box M1/metabolismo , Proteína Forkhead Box M1/genética , Proteína Supressora de Tumor p53/metabolismo , Proteína Supressora de Tumor p53/genética , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Técnicas de Cultura de Células em Três Dimensões/métodos , Células Cultivadas , Transdução de Sinais , Proteína Forkhead Box O1/metabolismo , Proteína Forkhead Box O1/genéticaRESUMO
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.
Assuntos
Coração , Miocárdio/citologia , Miócitos Cardíacos/citologia , Regeneração/fisiologia , Medicina Regenerativa , Animais , Coração/fisiologia , Humanos , Medicina Regenerativa/métodos , Transplante de Células-Tronco/métodosRESUMO
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.
Assuntos
Cardiomiopatias/cirurgia , Miocárdio/patologia , Miócitos Cardíacos/transplante , Células-Tronco Pluripotentes/transplante , Regeneração , Medicina Regenerativa/tendências , Transplante de Células-Tronco/tendências , Animais , Cardiomiopatias/patologia , Cardiomiopatias/fisiopatologia , Diferenciação Celular , Difusão de Inovações , Previsões , Humanos , Recuperação de Função Fisiológica , Transplante de Células-Tronco/efeitos adversosRESUMO
BACKGROUND: Two or more early rejections (<1 y) or any late acute rejection (>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 <21 years old who received first HT at Boston Children's Hospital during 1986-2015 with at least 1 post-HT coronary angiogram. CAV was diagnosed using 2010 International Society for Heart and Lung Transplantation guidelines. Time to CAV diagnosis was assessed using a Cox model with occurrence of clinical rejection analyzed as a time-varying covariate. RESULTS: Of 228 study subjects, 106 remained rejection-free, 77 had rejection diagnosed only on protocol biopsy (≥2R cellular or antibody-mediated), and 45 had a clinical rejection. Subjects with rejection diagnosed only on protocol biopsy were not at higher risk of CAV (hazard ratio [HR] 1.09, 95% confidence interval [CI]: 0.54-2.09). In contrast, clinical rejection was significantly associated with risk of CAV (HR 4.84, 95% CI: 2.99-7.83). Late rejection was associated with a higher risk of CAV (HR 4.27, 95% CI: 2.42-7.51) if it was clinical rejection but not if it was diagnosed on protocol biopsy (HR 0.83, 95% CI: 0.51-1.37). CONCLUSIONS: Clinical rejection poses a far greater risk for future CAV than rejection on protocol biopsy in pediatric HT recipients. Preventing CAV should therefore become the focus of medical management after initial treatment and resolution of clinical rejection.
Assuntos
Doença da Artéria Coronariana/epidemiologia , Rejeição de Enxerto/diagnóstico , Insuficiência Cardíaca/cirurgia , Transplante de Coração/efeitos adversos , Complicações Pós-Operatórias/epidemiologia , Disfunção Ventricular Esquerda/epidemiologia , Adolescente , Aloenxertos/irrigação sanguínea , Aloenxertos/patologia , Biópsia , Criança , Pré-Escolar , Angiografia Coronária , Doença da Artéria Coronariana/etiologia , Doença da Artéria Coronariana/prevenção & controle , Vasos Coronários/diagnóstico por imagem , Vasos Coronários/patologia , Feminino , Seguimentos , Rejeição de Enxerto/complicações , Rejeição de Enxerto/patologia , Humanos , Lactente , Recém-Nascido , Masculino , Miocárdio/patologia , Complicações Pós-Operatórias/etiologia , Complicações Pós-Operatórias/prevenção & controle , Estudos Retrospectivos , Fatores de Risco , Transplante Homólogo , Disfunção Ventricular Esquerda/etiologia , Adulto JovemRESUMO
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 (G0 phase) from 24% to 48% compared with vehicle control (P<0.05). Torin1 significantly increased expression of selected sarcomere proteins (including TNNI3 [troponin I, cardiac muscle]) and ion channels (including Kir2.1) in a dose-dependent manner when Torin1 was initiated after onset of cardiomyocyte beating. Torin1-treated cells had an increased relative maximum force of contraction, increased maximum oxygen consumption rate, decreased peak rise time, and increased downstroke velocity. Torin1 treatment increased protein expression of p53, and these effects were inhibited by pifithrin-α. In contrast, nutlin-3a independently upregulated p53, led to an increase in TNNI3 expression and worked synergistically with Torin1 to further increase expression of both p53 and TNNI3. CONCLUSIONS: Transient treatment of human iPSC-derived cardiomyocytes with Torin1 shifts cells to a quiescent state and enhances cardiomyocyte maturity.
Assuntos
Células-Tronco Pluripotentes Induzidas/metabolismo , Miócitos Cardíacos/metabolismo , Naftiridinas/farmacologia , Serina-Treonina Quinases TOR/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Via de Sinalização Wnt/efeitos dos fármacos , Benzotiazóis/farmacologia , Linhagem Celular , Humanos , Imidazóis/farmacologia , Células-Tronco Pluripotentes Induzidas/citologia , Miócitos Cardíacos/citologia , Piperazinas/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Tolueno/análogos & derivados , Tolueno/farmacologia , Proteína Supressora de Tumor p53/antagonistas & inibidoresRESUMO
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.
Assuntos
Proteínas Morfogenéticas Ósseas/genética , Deleção de Genes , Fatores de Diferenciação de Crescimento/genética , Hematopoese , Animais , Células Cultivadas , Feminino , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Masculino , CamundongosAssuntos
Cardiomiopatias , Insuficiência Cardíaca , Medula Óssea , Criança , Humanos , Transplante de Células-TroncoRESUMO
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.
Assuntos
Cardiopatias/terapia , Mioblastos Cardíacos/citologia , Mioblastos Cardíacos/transplante , Miocárdio/citologia , Regeneração , Cardiopatias/patologia , Humanos , Miocárdio/patologiaRESUMO
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.
Assuntos
Fator 2 de Crescimento de Fibroblastos/administração & dosagem , Fator 2 de Crescimento de Fibroblastos/farmacologia , Hidrogel de Polietilenoglicol-Dimetacrilato/química , Infarto do Miocárdio/fisiopatologia , Neovascularização Fisiológica/efeitos dos fármacos , Acrilamidas/síntese química , Acrilamidas/química , Animais , Biotina/metabolismo , Circulação Coronária/efeitos dos fármacos , Modelos Animais de Doenças , Concentração de Íons de Hidrogênio/efeitos dos fármacos , Inflamação/complicações , Inflamação/patologia , Injeções , Masculino , Infarto do Miocárdio/complicações , Infarto do Miocárdio/diagnóstico por imagem , Infarto do Miocárdio/patologia , Ratos , Ratos Endogâmicos F344 , Sístole/efeitos dos fármacos , Sístole/fisiologia , UltrassonografiaRESUMO
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.
Assuntos
Acrilamidas/uso terapêutico , Proteínas Angiogênicas/administração & dosagem , Sistemas de Liberação de Medicamentos/métodos , Hidrogéis/síntese química , Hidrogéis/uso terapêutico , Concentração de Íons de Hidrogênio , Injeções , Temperatura , ViscosidadeRESUMO
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.