RESUMEN
BACKGROUND: Atrial fibrillation has an estimated prevalence of 1.5-2%, making it the most common cardiac arrhythmia. The processes that cause and sustain the disease are still not completely understood. An association between atrial fibrillation and systemic, as well as local, inflammatory processes has been reported. However, the exact mechanisms underlying this association have not been established. While it is understood that inflammatory macrophages can influence cardiac electrophysiology, a direct, causative relationship to atrial fibrillation has not been described. This study investigated the pro-arrhythmic effects of activated M1 macrophages on human induced pluripotent stem cell (hiPSC)-derived atrial cardiomyocytes, to propose a mechanistic link between inflammation and atrial fibrillation. METHODS: Two hiPSC lines from healthy individuals were differentiated to atrial cardiomyocytes and M1 macrophages and integrated in an isogenic, pacing-free, atrial fibrillation-like coculture model. Electrophysiology characteristics of cocultures were analysed for beat rate irregularity, electrogram amplitude and conduction velocity using multi electrode arrays. Cocultures were additionally treated using glucocorticoids to suppress M1 inflammation. Bulk RNA sequencing was performed on coculture-isolated atrial cardiomyocytes and compared to meta-analyses of atrial fibrillation patient transcriptomes. RESULTS: Multi electrode array recordings revealed M1 to cause irregular beating and reduced electrogram amplitude. Conduction analysis further showed significantly lowered conduction homogeneity in M1 cocultures. Transcriptome sequencing revealed reduced expression of key cardiac genes such as SCN5A, KCNA5, ATP1A1, and GJA5 in the atrial cardiomyocytes. Meta-analysis of atrial fibrillation patient transcriptomes showed high correlation to the in vitro model. Treatment of the coculture with glucocorticoids showed reversal of phenotypes, including reduced beat irregularity, improved conduction, and reversed RNA expression profiles. CONCLUSIONS: This study establishes a causal relationship between M1 activation and the development of subsequent atrial arrhythmia, documented as irregularity in spontaneous electrical activation in atrial cardiomyocytes cocultured with activated macrophages. Further, beat rate irregularity could be alleviated using glucocorticoids. Overall, these results point at macrophage-mediated inflammation as a potential AF induction mechanism and offer new targets for therapeutic development. The findings strongly support the relevance of the proposed hiPSC-derived coculture model and present it as a first of its kind disease model.
Asunto(s)
Fibrilación Atrial , Técnicas de Cocultivo , Células Madre Pluripotentes Inducidas , Macrófagos , Miocitos Cardíacos , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/citología , Miocitos Cardíacos/metabolismo , Fibrilación Atrial/metabolismo , Fibrilación Atrial/patología , Macrófagos/metabolismo , Fenotipo , Diferenciación Celular , Atrios Cardíacos/patología , Atrios Cardíacos/metabolismo , Atrios Cardíacos/citologíaRESUMEN
Safety pharmacology is an essential part of drug development aiming to identify, evaluate and investigate undesirable pharmacodynamic properties of a drug primarily prior to clinical trials. In particular, cardiovascular adverse drug reactions (ADR) have halted many drug development programs. Safety pharmacology has successfully implemented a screening strategy to detect cardiovascular liabilities, but there is room for further refinement. In this setting, we present the INSPIRE project, a European Training Network in safety pharmacology for Early Stage Researchers (ESRs), funded by the European Commission's H2020-MSCA-ITN programme. INSPIRE has recruited 15 ESR fellows that will conduct an individual PhD-research project for a period of 36 months. INSPIRE aims to be complementary to ongoing research initiatives. With this as a goal, an inventory of collaborative research initiatives in safety pharmacology was created and the ESR projects have been designed to be complementary to this roadmap. Overall, INSPIRE aims to improve cardiovascular safety evaluation, either by investigating technological innovations or by adding mechanistic insight in emerging safety concerns, as observed in the field of cardio-oncology. Finally, in addition to its hands-on research pillar, INSPIRE will organize a number of summer schools and workshops that will be open to the wider community as well. In summary, INSPIRE aims to foster both research and training in safety pharmacology and hopes to inspire the future generation of safety scientists.
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Sistema Cardiovascular/efectos de los fármacos , Desarrollo de Medicamentos/métodos , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/prevención & control , Farmacología/métodos , Humanos , SeguridadRESUMEN
Chagas disease (ChD) is one of the most neglected tropical diseases, with cardiomyopathy being the main cause of death in Trypanosoma cruzi-infected patients. As the parasite actively replicates in cardiomyocytes (CMs), the heart remains a key target organ in the pathogenesis of ChD. Here we modeled ChD using human induced pluripotent stem cell-derived CMs (iPSC-CMs) to understand the complex interplay between the parasite and host cells. We showed that iPSC-CMs can get infected with the T. cruzi Y strain and that all parasite cycle stages can be identified in our model system. Importantly, characterization of T. cruzi-infected iPSC-CMs showed significant changes in their gene expression profile, cell contractility, and distribution of key cardiac markers. Moreover, these infected iPSC-CMs exhibited a pro-inflammatory profile as indicated by significantly elevated cytokine levels and cell-trafficking regulators. We believe our iPSC-CM model is a valuable platform to explore new treatment strategies for ChD.
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Cardiomiopatía Chagásica/metabolismo , Células Madre Pluripotentes Inducidas , Modelos Biológicos , Miocitos Cardíacos , Trypanosoma cruzi/metabolismo , Cardiomiopatía Chagásica/patología , Cardiomiopatía Chagásica/terapia , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/parasitología , Células Madre Pluripotentes Inducidas/patología , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/parasitología , Miocitos Cardíacos/patologíaRESUMEN
The ability to differentiate human pluripotent stem cells (hPSCs) into cardiomyocytes (CMs) makes them an attractive source for repairing injured myocardium, disease modeling, and drug testing. Although current differentiation protocols yield hPSC-CMs to >90% efficiency, hPSC-CMs exhibit immature characteristics. With the goal of overcoming this limitation, we tested the effects of varying passive stretch on engineered heart muscle (EHM) structural and functional maturation, guided by computational modeling. Human embryonic stem cells (hESCs, H7 line) or human induced pluripotent stem cells (IMR-90 line) were differentiated to hPSC-derived cardiomyocytes (hPSC-CMs) in vitro using a small molecule based protocol. hPSC-CMs were characterized by troponin+ flow cytometry as well as electrophysiological measurements. Afterwards, 1.2 × 106 hPSC-CMs were mixed with 0.4 × 106 human fibroblasts (IMR-90 line) (3:1 ratio) and type-I collagen. The blend was cast into custom-made 12-mm long polydimethylsiloxane reservoirs to vary nominal passive stretch of EHMs to 5, 7, or 9 mm. EHM characteristics were monitored for up to 50 days, with EHMs having a passive stretch of 7 mm giving the most consistent formation. Based on our initial macroscopic observations of EHM formation, we created a computational model that predicts the stress distribution throughout EHMs, which is a function of cellular composition, cellular ratio, and geometry. Based on this predictive modeling, we show cell alignment by immunohistochemistry and coordinated calcium waves by calcium imaging. Furthermore, coordinated calcium waves and mechanical contractions were apparent throughout entire EHMs. The stiffness and active forces of hPSC-derived EHMs are comparable with rat neonatal cardiomyocyte-derived EHMs. Three-dimensional EHMs display increased expression of mature cardiomyocyte genes including sarcomeric protein troponin-T, calcium and potassium ion channels, ß-adrenergic receptors, and t-tubule protein caveolin-3. Passive stretch affects the structural and functional maturation of EHMs. Based on our predictive computational modeling, we show how to optimize cell alignment and calcium dynamics within EHMs. These findings provide a basis for the rational design of EHMs, which enables future scale-up productions for clinical use in cardiovascular tissue engineering. Stem Cells 2018;36:265-277.
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Biología Computacional/métodos , Miocardio/citología , Línea Celular , Citometría de Flujo , Humanos , Miocardio/metabolismo , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Ingeniería de Tejidos/métodosRESUMEN
There is growing interest in using embryonic stem cell (ESC) and induced pluripotent stem cell (iPSC) derivatives for tissue regeneration. However, an increased understanding of human immune responses to stem cell-derived allografts is necessary for maintaining long-term graft persistence. To model this alloimmunity, humanized mice engrafted with human hematopoietic and immune cells could prove to be useful. In this study, an in-depth analysis of graft-infiltrating human lymphocytes and splenocytes revealed that humanized mice incompletely model human immune responses toward allogeneic stem cells and their derivatives. Furthermore, using an "allogenized" mouse model, we show the feasibility of reconstituting immunodeficient mice with a functional mouse immune system and describe a key role of innate immune cells in the rejection of mouse stem cell allografts.
Asunto(s)
Trasplante de Células Madre Hematopoyéticas/métodos , Inmunidad Innata/inmunología , Células Madre Pluripotentes/metabolismo , Acondicionamiento Pretrasplante/métodos , Animales , Modelos Animales de Enfermedad , Rechazo de Injerto , Humanos , RatonesRESUMEN
RATIONALE: Targeted genetic engineering using programmable nucleases such as transcription activator-like effector nucleases (TALENs) is a valuable tool for precise, site-specific genetic modification in the human genome. OBJECTIVE: The emergence of novel technologies such as human induced pluripotent stem cells (iPSCs) and nuclease-mediated genome editing represent a unique opportunity for studying cardiovascular diseases in vitro. METHODS AND RESULTS: By incorporating extensive literature and database searches, we designed a collection of TALEN constructs to knockout 88 human genes that are associated with cardiomyopathies and congenital heart diseases. The TALEN pairs were designed to induce double-strand DNA break near the starting codon of each gene that either disrupted the start codon or introduced a frameshift mutation in the early coding region, ensuring faithful gene knockout. We observed that all the constructs were active and disrupted the target locus at high frequencies. To illustrate the utility of the TALEN-mediated knockout technique, 6 individual genes (TNNT2, LMNA/C, TBX5, MYH7, ANKRD1, and NKX2.5) were knocked out with high efficiency and specificity in human iPSCs. By selectively targeting a pathogenic mutation (TNNT2 p.R173W) in patient-specific iPSC-derived cardiac myocytes, we demonstrated that the knockout strategy ameliorates the dilated cardiomyopathy phenotype in vitro. In addition, we modeled the Holt-Oram syndrome in iPSC-cardiac myocytes in vitro and uncovered novel pathways regulated by TBX5 in human cardiac myocyte development. CONCLUSIONS: Collectively, our study illustrates the powerful combination of iPSCs and genome editing technologies for understanding the biological function of genes, and the pathological significance of genetic variants in human cardiovascular diseases. The methods, strategies, constructs, and iPSC lines developed in this study provide a validated, readily available resource for cardiovascular research.
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Enfermedades Cardiovasculares/genética , Técnicas de Inactivación de Genes/métodos , Biblioteca de Genes , Ingeniería Genética/métodos , Células Madre Pluripotentes Inducidas/fisiología , Secuencia de Bases , Enfermedades Cardiovasculares/terapia , Células Cultivadas , Marcación de Gen/métodos , Humanos , Células Madre Pluripotentes Inducidas/trasplanteRESUMEN
Tyrosine kinase inhibitors (TKIs), despite their efficacy as anticancer therapeutics, are associated with cardiovascular side effects ranging from induced arrhythmias to heart failure. We used human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), generated from 11 healthy individuals and 2 patients receiving cancer treatment, to screen U.S. Food and Drug Administration-approved TKIs for cardiotoxicities by measuring alterations in cardiomyocyte viability, contractility, electrophysiology, calcium handling, and signaling. With these data, we generated a "cardiac safety index" to reflect the cardiotoxicities of existing TKIs. TKIs with low cardiac safety indices exhibit cardiotoxicity in patients. We also derived endothelial cells (hiPSC-ECs) and cardiac fibroblasts (hiPSC-CFs) to examine cell type-specific cardiotoxicities. Using high-throughput screening, we determined that vascular endothelial growth factor receptor 2 (VEGFR2)/platelet-derived growth factor receptor (PDGFR)-inhibiting TKIs caused cardiotoxicity in hiPSC-CMs, hiPSC-ECs, and hiPSC-CFs. With phosphoprotein analysis, we determined that VEGFR2/PDGFR-inhibiting TKIs led to a compensatory increase in cardioprotective insulin and insulin-like growth factor (IGF) signaling in hiPSC-CMs. Up-regulating cardioprotective signaling with exogenous insulin or IGF1 improved hiPSC-CM viability during cotreatment with cardiotoxic VEGFR2/PDGFR-inhibiting TKIs. Thus, hiPSC-CMs can be used to screen for cardiovascular toxicities associated with anticancer TKIs, and the results correlate with clinical phenotypes. This approach provides unexpected insights, as illustrated by our finding that toxicity can be alleviated via cardioprotective insulin/IGF signaling.
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Cardiotoxicidad/patología , Ensayos Analíticos de Alto Rendimiento/métodos , Células Madre Pluripotentes Inducidas/metabolismo , Inhibidores de Proteínas Quinasas/toxicidad , Biomarcadores/metabolismo , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Insulina/farmacología , Factor I del Crecimiento Similar a la Insulina/farmacología , Modelos Biológicos , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Fosforilación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Sarcómeros/metabolismo , Transducción de Señal/efectos de los fármacos , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
The measurement of the electrophysiology of human pluripotent stem cell-derived cardiomyocytes is critical for their biomedical applications, from disease modeling to drug screening. Yet, a method that enables the high-throughput intracellular electrophysiology measurement of single cardiomyocytes in adherent culture is not available. To address this area, we have fabricated vertical nanopillar electrodes that can record intracellular action potentials from up to 60 single beating cardiomyocytes. Intracellular access is achieved by highly localized electroporation, which allows for low impedance electrical access to the intracellular voltage. Herein, we demonstrate that this method provides the accurate measurement of the shape and duration of intracellular action potentials, validated by patch clamp, and can facilitate cellular drug screening and disease modeling using human pluripotent stem cells. This study validates the use of nanopillar electrodes for myriad further applications of human pluripotent stem cell-derived cardiomyocytes such as cardiomyocyte maturation monitoring and electrophysiology-contractile force correlation.
RESUMEN
Understanding individual susceptibility to drug-induced cardiotoxicity is key to improving patient safety and preventing drug attrition. Human induced pluripotent stem cells (hiPSCs) enable the study of pharmacological and toxicological responses in patient-specific cardiomyocytes (CMs) and may serve as preclinical platforms for precision medicine. Transcriptome profiling in hiPSC-CMs from seven individuals lacking known cardiovascular disease-associated mutations and in three isogenic human heart tissue and hiPSC-CM pairs showed greater inter-patient variation than intra-patient variation, verifying that reprogramming and differentiation preserve patient-specific gene expression, particularly in metabolic and stress-response genes. Transcriptome-based toxicology analysis predicted and risk-stratified patient-specific susceptibility to cardiotoxicity, and functional assays in hiPSC-CMs using tacrolimus and rosiglitazone, drugs targeting pathways predicted to produce cardiotoxicity, validated inter-patient differential responses. CRISPR/Cas9-mediated pathway correction prevented drug-induced cardiotoxicity. Our data suggest that hiPSC-CMs can be used in vitro to predict and validate patient-specific drug safety and efficacy, potentially enabling future clinical approaches to precision medicine.
Asunto(s)
Perfilación de la Expresión Génica , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Miocitos Cardíacos/citología , Tacrolimus/efectos adversos , Tiazolidinedionas/efectos adversos , Sistemas CRISPR-Cas/genética , Muerte Celular/efectos de los fármacos , Edición Génica , Genoma Humano , Ventrículos Cardíacos/efectos de los fármacos , Ventrículos Cardíacos/metabolismo , Humanos , Secuencias Invertidas Repetidas/genética , Miocitos Cardíacos/metabolismo , Rosiglitazona , Resultado del TratamientoRESUMEN
Human induced pluripotent stem cells (hiPSCs) have revolutionized the field of human disease modeling, with an enormous potential to serve as paradigm shifting platforms for preclinical trials, personalized clinical diagnosis, and drug treatment. In this review, we describe how hiPSCs could transition cardiac healthcare away from simple disease diagnosis to prediction and prevention, bridging the gap between basic and clinical research to bring the best science to every patient.
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Enfermedades Cardiovasculares/terapia , Células Madre Pluripotentes Inducidas , Medicina de Precisión , HumanosRESUMEN
Doxorubicin is an anthracycline chemotherapy agent effective in treating a wide range of malignancies, but it causes a dose-related cardiotoxicity that can lead to heart failure in a subset of patients. At present, it is not possible to predict which patients will be affected by doxorubicin-induced cardiotoxicity (DIC). Here we demonstrate that patient-specific human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) can recapitulate the predilection to DIC of individual patients at the cellular level. hiPSC-CMs derived from individuals with breast cancer who experienced DIC were consistently more sensitive to doxorubicin toxicity than hiPSC-CMs from patients who did not experience DIC, with decreased cell viability, impaired mitochondrial and metabolic function, impaired calcium handling, decreased antioxidant pathway activity, and increased reactive oxygen species production. Taken together, our data indicate that hiPSC-CMs are a suitable platform to identify and characterize the genetic basis and molecular mechanisms of DIC.
Asunto(s)
Antibióticos Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Neoplasias de la Mama/tratamiento farmacológico , Doxorrubicina/farmacología , Insuficiencia Cardíaca/inducido químicamente , Mitocondrias Cardíacas/efectos de los fármacos , Miocitos Cardíacos/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Adulto , Anciano , Antibióticos Antineoplásicos/efectos adversos , Calcio/metabolismo , Cardiotoxicidad/genética , Supervivencia Celular/efectos de los fármacos , Daño del ADN/efectos de los fármacos , Susceptibilidad a Enfermedades , Doxorrubicina/efectos adversos , Femenino , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Insuficiencia Cardíaca/genética , Humanos , Células Madre Pluripotentes Inducidas , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Persona de Mediana Edad , Mitocondrias Cardíacas/metabolismo , Miocitos Cardíacos/metabolismo , Polimorfismo de Nucleótido Simple , Especies Reactivas de Oxígeno/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , TranscriptomaRESUMEN
The advent of human induced pluripotent stem cell (hiPSC) technology has revitalized the efforts in the past decade to realize more fully the potential of human embryonic stem cells for scientific research. Adding to the possibility of generating an unlimited amount of any cell type of interest, hiPSC technology now enables the derivation of cells with patient-specific phenotypes. Given the introduction and implementation of the large-scale Precision Medicine Initiative, hiPSC technology will undoubtedly have a vital role in the advancement of cardiovascular research and medicine. In this Review, we summarize the progress that has been made in the field of hiPSC technology, with particular emphasis on cardiovascular disease modelling and drug development. The growing roles of hiPSC technology in the practice of precision medicine will also be discussed.
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Enfermedades Cardiovasculares/tratamiento farmacológico , Células Madre Pluripotentes Inducidas , Modelos Cardiovasculares , Medicina de Precisión , Enfermedades Cardiovasculares/genética , Enfermedades Cardiovasculares/prevención & control , Diferenciación Celular , Técnicas de Reprogramación Celular , Descubrimiento de Drogas , Células Endoteliales/citología , Humanos , Miocitos Cardíacos/citología , Miocitos del Músculo Liso/citologíaRESUMEN
The generation of cardiomyocytes from human induced pluripotent stem cells (hiPSCs) provides a source of cells that accurately recapitulate the human cardiac pathophysiology. The application of these cells allows for modeling of cardiovascular diseases, providing a novel understanding of human disease mechanisms and assessment of therapies. Here, we describe a stepwise protocol developed in our laboratory for the generation of hiPSCs from patients with a specific disease phenotype, long-term hiPSC culture and cryopreservation, differentiation of hiPSCs to cardiomyocytes, and assessment of disease phenotypes. Our protocol combines a number of innovative tools that include a codon-optimized mini intronic plasmid (CoMiP), chemically defined culture conditions to achieve high efficiencies of reprogramming and differentiation, and calcium imaging for assessment of cardiomyocyte phenotypes. Thus, this protocol provides a complete guide to use a patient cohort on a testable cardiomyocyte platform for pharmacological drug assessment.
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Cardiomiopatía Dilatada/patología , Reprogramación Celular , Fibroblastos/citología , Células Madre Pluripotentes Inducidas/citología , Modelos Biológicos , Miocitos Cardíacos/citología , Antígenos de Superficie/genética , Antígenos de Superficie/metabolismo , Biomarcadores/metabolismo , Calcio/metabolismo , Cardiomiopatía Dilatada/genética , Cardiomiopatía Dilatada/metabolismo , Diferenciación Celular , Criopreservación , Dermis/citología , Dermis/metabolismo , Fibroblastos/metabolismo , Expresión Génica , Humanos , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/metabolismo , Péptidos y Proteínas de Señalización Intercelular/farmacología , Imagen Molecular , Mutación , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Plásmidos/química , Plásmidos/metabolismo , Cultivo Primario de Células , Proteoglicanos/genética , Proteoglicanos/metabolismo , Factores de Transcripción SOXB1/genética , Factores de Transcripción SOXB1/metabolismo , Pase Seriado , Troponina T/genética , Troponina T/metabolismoRESUMEN
ß-adrenergic signaling pathways mediate key aspects of cardiac function. Its dysregulation is associated with a range of cardiac diseases, including dilated cardiomyopathy (DCM). Previously, we established an iPSC model of familial DCM from patients with a mutation in TNNT2, a sarcomeric protein. Here, we found that the ß-adrenergic agonist isoproterenol induced mature ß-adrenergic signaling in iPSC-derived cardiomyocytes (iPSC-CMs) but that this pathway was blunted in DCM iPSC-CMs. Although expression levels of several ß-adrenergic signaling components were unaltered between control and DCM iPSC-CMs, we found that phosphodiesterases (PDEs) 2A and PDE3A were upregulated in DCM iPSC-CMs and that PDE2A was also upregulated in DCM patient tissue. We further discovered increased nuclear localization of mutant TNNT2 and epigenetic modifications of PDE genes in both DCM iPSC-CMs and patient tissue. Notably, pharmacologic inhibition of PDE2A and PDE3A restored cAMP levels and ameliorated the impaired ß-adrenergic signaling of DCM iPSC-CMs, suggesting therapeutic potential.
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Cardiomiopatía Dilatada/genética , Cardiomiopatía Dilatada/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 2/genética , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 2/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3/genética , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3/metabolismo , Células Madre Pluripotentes Inducidas/fisiología , Receptores Adrenérgicos beta/metabolismo , Agonistas Adrenérgicos beta/farmacología , Cardiomiopatía Dilatada/patología , Diferenciación Celular , Células Cultivadas , Epigénesis Genética , Frecuencia Cardíaca/efectos de los fármacos , Humanos , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/patología , Isoproterenol/farmacología , Modelos Cardiovasculares , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutación , Contracción Miocárdica/efectos de los fármacos , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/patología , Miocitos Cardíacos/fisiología , Transducción de Señal , Troponina T/genética , Troponina T/metabolismo , Regulación hacia ArribaRESUMEN
Cardiomyocytes from human embryonic stem cells (hESC-CMs) and induced pluripotent stem cells (hiPSC-CMs) represent new models for drug discovery. Although hypertrophy is a high-priority target, we found that hiPSC-CMs were systematically unresponsive to hypertrophic signals such as the α-adrenoceptor (αAR) agonist phenylephrine (PE) compared to hESC-CMs. We investigated signaling at multiple levels to understand the underlying mechanism of this differential responsiveness. The expression of the normal α1AR gene, ADRA1A, was reversibly silenced during differentiation, accompanied by ADRA1B upregulation in either cell type. ADRA1B signaling was intact in hESC-CMs, but not in hiPSC-CMs. We observed an increased tonic activity of inhibitory kinase pathways in hiPSC-CMs, and inhibition of antihypertrophic kinases revealed hypertrophic increases. There is tonic suppression of cell growth in hiPSC-CMs, but not hESC-CMs, limiting their use in investigation of hypertrophic signaling. These data raise questions regarding the hiPSC-CM as a valid model for certain aspects of cardiac disease.
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Adrenérgicos/farmacología , Tamaño de la Célula/efectos de los fármacos , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Miocitos Cardíacos/efectos de los fármacos , Agonistas alfa-Adrenérgicos/farmacología , Agonistas Adrenérgicos beta/farmacología , Antagonistas Adrenérgicos beta/farmacología , Línea Celular , Células Madre Embrionarias/citología , Células Madre Embrionarias/efectos de los fármacos , Células Madre Embrionarias/metabolismo , Expresión Génica/efectos de los fármacos , Humanos , Hipertrofia , Imidazoles/farmacología , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Isoproterenol/farmacología , Microscopía Confocal , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Fenilefrina/farmacología , Receptores Adrenérgicos alfa 1/genética , Receptores Adrenérgicos alfa 1/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genéticaRESUMEN
RATIONALE: Viral myocarditis is a life-threatening illness that may lead to heart failure or cardiac arrhythmias. A major causative agent for viral myocarditis is the B3 strain of coxsackievirus, a positive-sense RNA enterovirus. However, human cardiac tissues are difficult to procure in sufficient enough quantities for studying the mechanisms of cardiac-specific viral infection. OBJECTIVE: This study examined whether human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) could be used to model the pathogenic processes of coxsackievirus-induced viral myocarditis and to screen antiviral therapeutics for efficacy. METHODS AND RESULTS: hiPSC-CMs were infected with a luciferase-expressing coxsackievirus B3 strain (CVB3-Luc). Brightfield microscopy, immunofluorescence, and calcium imaging were used to characterize virally infected hiPSC-CMs for alterations in cellular morphology and calcium handling. Viral proliferation in hiPSC-CMs was quantified using bioluminescence imaging. Antiviral compounds including interferonß1, ribavirin, pyrrolidine dithiocarbamate, and fluoxetine were tested for their capacity to abrogate CVB3-Luc proliferation in hiPSC-CMs in vitro. The ability of these compounds to reduce CVB3-Luc proliferation in hiPSC-CMs was consistent with reported drug effects in previous studies. Mechanistic analyses via gene expression profiling of hiPSC-CMs infected with CVB3-Luc revealed an activation of viral RNA and protein clearance pathways after interferonß1 treatment. CONCLUSIONS: This study demonstrates that hiPSC-CMs express the coxsackievirus and adenovirus receptor, are susceptible to coxsackievirus infection, and can be used to predict antiviral drug efficacy. Our results suggest that the hiPSC-CM/CVB3-Luc assay is a sensitive platform that can screen novel antiviral therapeutics for their effectiveness in a high-throughput fashion.
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Antivirales/uso terapéutico , Enterovirus Humano B/aislamiento & purificación , Infecciones por Enterovirus/tratamiento farmacológico , Modelos Cardiovasculares , Miocarditis/tratamiento farmacológico , Miocitos Cardíacos/patología , Células Madre Pluripotentes/patología , Antivirales/farmacología , Calcio/metabolismo , Proliferación Celular , Células Cultivadas , Evaluación Preclínica de Medicamentos , Infecciones por Enterovirus/metabolismo , Humanos , Técnicas In Vitro , Miocarditis/metabolismo , Miocarditis/virología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/virología , Células Madre Pluripotentes/efectos de los fármacos , Células Madre Pluripotentes/virología , ARN Viral/metabolismo , Resultado del TratamientoRESUMEN
A major research focus in the field of cardiovascular medicine is the prospect of using stem cells and progenitor cells for cardiac regeneration. With the advent of induced pluripotent stem cell (iPSC) technology, major efforts are also underway to use iPSCs to model heart disease, to screen for new drugs, and to test candidate drugs for cardiotoxicity. Here, we discuss recent advances in the exciting fields of stem cells and cardiovascular disease.
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Descubrimiento de Drogas , Cardiopatías/terapia , Modelos Cardiovasculares , Trasplante de Células Madre , Células Madre/citología , Humanos , Cicatrización de HeridasRESUMEN
Existing methods for human induced pluripotent stem cell (hiPSC) cardiac differentiation are efficient but require complex, undefined medium constituents that hinder further elucidation of the molecular mechanisms of cardiomyogenesis. Using hiPSCs derived under chemically defined conditions on synthetic matrices, we systematically developed an optimized cardiac differentiation strategy, using a chemically defined medium consisting of just three components: the basal medium RPMI 1640, L-ascorbic acid 2-phosphate and rice-derived recombinant human albumin. Along with small molecule-based induction of differentiation, this protocol produced contractile sheets of up to 95% TNNT2(+) cardiomyocytes at a yield of up to 100 cardiomyocytes for every input pluripotent cell and was effective in 11 hiPSC lines tested. This chemically defined platform for cardiac specification of hiPSCs will allow the elucidation of cardiomyocyte macromolecular and metabolic requirements and will provide a minimal system for the study of maturation and subtype specification.
Asunto(s)
Miocitos Cardíacos/citología , Diferenciación Celular , Medios de Cultivo , Humanos , Células Madre Pluripotentes Inducidas/citologíaRESUMEN
Cardiac regeneration strategies and de novo generation of cardiomyocytes have long been significant areas of research interest in cardiovascular medicine. In this review, we outline a variety of common cell sources and methods used to regenerate cardiomyocytes and highlight the important role that key Circulation Research articles have played in this flourishing field.