RESUMEN
AIMS: Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have proven valuable for studies in drug discovery and safety, although limitations regarding their structural and electrophysiological characteristics persist. In this study, we investigated the electrophysiological properties of Pluricyte® CMs, a commercially available hiPSC-CMs line with a ventricular phenotype, and assessed arrhythmia incidence by IKr block at the single-cell and 2D monolayer level. METHODS AND RESULTS: Action potentials were measured at different pacing frequencies, using dynamic clamp. Through voltage-clamp experiments, we determined the properties of INa, IKr, and ICaL. Intracellular Ca2+ measurements included Ca2+-transients at baseline and during caffeine perfusion. Effects of IKr block were assessed in single hiPSC-CMs and 2D monolayers (multi-electrode arrays). Action-potential duration (APD) and its rate dependence in Pluricyte® CMs were comparable to those reported for native human CMs. INa, IKr, and ICaL revealed amplitudes, kinetics, and voltage dependence of activation/inactivation similar to other hiPSC-CM lines and, to some extent, to native CMs. Near-physiological Ca2+-induced Ca2+ release, response to caffeine and excitation-contraction coupling gain characterized the cellular Ca2+-handling. Dofetilide prolonged the APD and field-potential duration, and induced early afterdepolarizations. Beat-to-beat variability of repolarization duration increased significantly before the first arrhythmic events in single Pluricyte® CMs and 2D monolayers, and predicted pending arrhythmias better than action-potential prolongation. CONCLUSION: Taking their ion-current characteristics and Ca2+ handling into account, Pluricyte® CMs are suitable for in vitro studies on action potentials and field potentials. Beat-to-beat variability of repolarization duration proved useful to evaluate the dynamics of repolarization instability and demonstrated its significance as proarrhythmic marker in hiPSC-CMs during IKr block.
Asunto(s)
Células Madre Pluripotentes Inducidas , Potenciales de Acción , Arritmias Cardíacas , Fenómenos Electrofisiológicos , Humanos , Miocitos CardíacosRESUMEN
Differentiated derivatives of human pluripotent stem cells (hPSCs) are often considered immature because they resemble foetal cells more than adult, with hPSC-derived cardiomyocytes (hPSC-CMs) being no exception. Many functional features of these cardiomyocytes, such as their cell morphology, electrophysiological characteristics, sarcomere organization and contraction force, are underdeveloped compared with adult cardiomyocytes. However, relatively little is known about how their gene expression profiles compare with the human foetal heart, in part because of the paucity of data on the human foetal heart at different stages of development. Here, we collected samples of matched ventricles and atria from human foetuses during the first and second trimester of development. This presented a rare opportunity to perform gene expression analysis on the individual chambers of the heart at various stages of development, allowing us to identify not only genes involved in the formation of the heart, but also specific genes upregulated in each of the four chambers and at different stages of development. The data showed that hPSC-CMs had a gene expression profile similar to first trimester foetal heart, but after culture in conditions shown previously to induce maturation, they cluster closer to the second trimester foetal heart samples. In summary, we demonstrate how the gene expression profiles of human foetal heart samples can be used for benchmarking hPSC-CMs and also contribute to determining their equivalent stage of development.
Asunto(s)
Diferenciación Celular/fisiología , Feto/fisiología , Regulación del Desarrollo de la Expresión Génica/fisiología , Miocardio/metabolismo , Miocitos Cardíacos/metabolismo , Células Madre Pluripotentes/citología , Transcriptoma , Feto/metabolismo , Perfilación de la Expresión Génica , HumanosRESUMEN
Recent methodological advances have improved the ease and efficiency of generating human induced pluripotent stem cells (hiPSCs), but this now typically results in a greater number of hiPSC clones being derived than can be wholly characterized. It is therefore imperative that methods are developed which facilitate rapid selection of hiPSC clones most suited for the downstream research aims. Here we describe a combination of procedures enabling the simultaneous screening of multiple clones to determine their genomic integrity as well as their cardiac differentiation potential within two weeks of the putative reprogrammed colonies initially appearing. By coupling splinkerette-PCR with Ion Torrent sequencing, we could ascertain the number and map the proviral integration sites in lentiviral-reprogrammed hiPSCs. In parallel, we developed an effective cardiac differentiation protocol that generated functional cardiomyocytes within 10 days without requiring line-specific optimization for any of the six independent human pluripotent stem cell lines tested. Finally, to demonstrate the scalable potential of these procedures, we picked 20 nascent iPSC clones and performed these independent assays concurrently. Before the clones required passaging, we were able to identify clones with a single integrated copy of the reprogramming vector and robust cardiac differentiation potential for further analysis.
Asunto(s)
Diferenciación Celular , Reprogramación Celular , Células Madre Pluripotentes Inducidas/citología , Miocitos Cardíacos/citología , Provirus/genética , Integración Viral/genética , Southern Blotting , Proliferación Celular , Células Cultivadas , Pulpa Dental/citología , Pulpa Dental/metabolismo , Dermis/citología , Dermis/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Ensayos Analíticos de Alto Rendimiento , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Miocitos Cardíacos/metabolismoRESUMEN
It has been known for over 20 years that foetal calf serum can induce hypertrophy in cultured cardiomyocytes but this is rarely considered when examining cardiomyocytes derived from pluripotent stem cells (PSC). Here, we determined how serum affected cardiomyocytes from human embryonic- (hESC) and induced pluripotent stem cells (hiPSC) and hiPSC from patients with hypertrophic cardiomyopathy linked to a mutation in the MYBPC3 gene. We first confirmed previously published hypertrophic effects of serum on cultured neonatal rat cardiomyocytes demonstrated as increased cell surface area and beating frequency. We then found that serum increased the cell surface area of hESC- and hiPSC-derived cardiomyocytes and their spontaneous contraction rate. Phenylephrine, which normally induces cardiac hypertrophy, had no additional effects under serum conditions. Likewise, hiPSC-derived cardiomyocytes from three MYBPC3 patients which had a greater surface area than controls in the absence of serum as predicted by their genotype, did not show this difference in the presence of serum. Serum can thus alter the phenotype of human PSC derived cardiomyocytes under otherwise defined conditions such that the effects of hypertrophic drugs and gene mutations are underestimated. It is therefore pertinent to examine cardiac phenotypes in culture media without or in low concentrations of serum.
Asunto(s)
Cardiomiopatía Hipertrófica/patología , Proteínas Portadoras/genética , Medios de Cultivo/química , Células Madre Embrionarias/fisiología , Células Madre Pluripotentes Inducidas/fisiología , Miocitos Cardíacos/fisiología , Suero/química , Potenciales de Acción , Animales , Animales Recién Nacidos , Calcio/metabolismo , Cardiomiopatía Hipertrófica/genética , Cardiomiopatía Hipertrófica/metabolismo , Proteínas Portadoras/metabolismo , Estudios de Casos y Controles , Diferenciación Celular , Células Cultivadas , Dermis/citología , Dermis/metabolismo , Células Madre Embrionarias/citología , Fibroblastos/citología , Fibroblastos/metabolismo , Humanos , Procesamiento de Imagen Asistido por Computador , Células Madre Pluripotentes Inducidas/citología , Ratones , Mutación/genética , Miocitos Cardíacos/citología , Fenotipo , RatasRESUMEN
NKX2-5 is expressed in the heart throughout life. We targeted eGFP sequences to the NKX2-5 locus of human embryonic stem cells (hESCs); NKX2-5(eGFP/w) hESCs facilitate quantification of cardiac differentiation, purification of hESC-derived committed cardiac progenitor cells (hESC-CPCs) and cardiomyocytes (hESC-CMs) and the standardization of differentiation protocols. We used NKX2-5 eGFP(+) cells to identify VCAM1 and SIRPA as cell-surface markers expressed in cardiac lineages.
Asunto(s)
Separación Celular/métodos , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Proteínas de Homeodominio/metabolismo , Mioblastos Cardíacos/citología , Miocitos Cardíacos/citología , Factores de Transcripción/metabolismo , Antígenos de Diferenciación/genética , Antígenos de Diferenciación/metabolismo , Biomarcadores/análisis , Diferenciación Celular , Perfilación de la Expresión Génica , Proteína Homeótica Nkx-2.5 , Proteínas de Homeodominio/genética , Humanos , Mioblastos Cardíacos/metabolismo , Miocitos Cardíacos/metabolismo , Reacción en Cadena de la Polimerasa , Receptores Inmunológicos/genética , Receptores Inmunológicos/metabolismo , Factores de Transcripción/genética , Molécula 1 de Adhesión Celular Vascular/genética , Molécula 1 de Adhesión Celular Vascular/metabolismoRESUMEN
Low efficiency of transfection limits the ability to genetically manipulate human embryonic stem cells (hESCs), and differences in cell derivation and culture methods require optimization of transfection protocols. We transiently transferred multiple independent hESC lines with different growth requirements to standardized feeder-free culture, and optimized conditions for clonal growth and efficient gene transfer without loss of pluripotency. Stably transfected lines retained differentiation potential, and most lines displayed normal karyotypes.
Asunto(s)
Células Madre Embrionarias/metabolismo , Técnicas de Transferencia de Gen , Técnicas de Cultivo de Célula , Diferenciación Celular , Línea Celular , Colágeno , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Combinación de Medicamentos , Fibroblastos/citología , Fibroblastos/metabolismo , Silenciador del Gen , Proteínas HMGB/genética , Proteínas HMGB/metabolismo , Humanos , Laminina , Proteoglicanos , ARN Interferente Pequeño/genética , Reproducibilidad de los Resultados , Factores de Transcripción SOXB1 , Factores de Tiempo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , TripsinaRESUMEN
The absence of identified cell surface proteins and corresponding antibodies to most differentiated derivatives of human embryonic stem cells (hESCs) has largely limited selection of specific cell types from mixed cell populations to genetic approaches. Here, we describe the use of mass spectrometry (MS)-based proteomics on cell membrane proteins isolated from hESCs that were differentiated into cardiomyocytes to identify candidate proteins for this particular lineage. Quantitative MS distinguished cardiomyocyte-specific plasma membrane proteins that were highly enriched or detected only in cardiomyocytes derived from hESCs and human fetal hearts compared with a heterogeneous pool of hESC-derived differentiated cells. For several candidates, cardiomyocyte-specific expression and cell surface localization were verified by conventional antibody-based methodologies. Using an antibody against elastin microfibril interfacer 2 (EMILIN2), we demonstrate that cardiomyocytes can be sorted from live cell populations. Besides showing that MS-based membrane proteomics is a powerful tool to identify candidate proteins that allow purification of specific cell lineages from heterogeneous populations, this approach generated a plasma membrane proteome profile suggesting signaling pathways that control cell behavior.
Asunto(s)
Biomarcadores/análisis , Células Madre Embrionarias/metabolismo , Proteínas de la Membrana/análisis , Miocitos Cardíacos/metabolismo , Proteómica/métodos , Anticuerpos/metabolismo , Biomarcadores/metabolismo , Western Blotting , Diferenciación Celular , Separación Celular , Células Madre Embrionarias/citología , Fibrilinas , Glicoproteínas/metabolismo , Humanos , Marcaje Isotópico , Espectrometría de Masas , Proteínas de la Membrana/metabolismo , Proteínas de Microfilamentos/metabolismo , Microscopía Fluorescente , Miocitos Cardíacos/citologíaRESUMEN
Human embryonic stem cells (hESCs) are often cocultured on mitotically inactive fibroblast feeder cells to maintain their undifferentiated state. Under these growth conditions, hESCs form multilayered colonies of morphologically heterogeneous cells surrounded by flattened mesenchymal cells. In contrast, hESCs grown in feeder cell-conditioned medium on Matrigel instead tend to grow as monolayers with uniform morphology. Using mass spectrometry and immunofluorescence microscopy, we showed that hESCs under these conditions primarily express proteins belonging to epithelium-related cell-cell adhesion complexes, including adherens junctions, tight junctions, desmosomes, and gap junctions. This indicates that monolayers of hESCs cultured under feeder-free conditions retain a homogeneous epithelial phenotype similar to that of the upper central cell layer of colonies maintained on feeder cells. Notably, feeder-free hESCs also coexpressed vimentin, which is usually associated with mesenchyme, suggesting that these cells may have undergone epithelium-to-mesenchyme transitions, indicating differentiation. However, if grown on a "soft" substrate (Hydrogel), intracellular vimentin levels were substantially reduced. Moreover, when hESCs were transferred back to feeder cells, expression of vimentin was again absent from the epithelial cell population. These results imply that on tissue culture substrates, vimentin expression is most likely a stress-induced response, unrelated to differentiation. Disclosure of potential conflicts of interest is found at the end of this article.
Asunto(s)
Membrana Celular/metabolismo , Células Madre Embrionarias/citología , Células Epiteliales/citología , Proteínas de la Membrana/metabolismo , Antígenos de Diferenciación/metabolismo , Adhesión Celular , Diferenciación Celular , Células Cultivadas , Técnicas de Cocultivo/métodos , Colágeno/metabolismo , Combinación de Medicamentos , Células Madre Embrionarias/metabolismo , Células Epiteliales/metabolismo , Humanos , Laminina/metabolismo , Espectrometría de Masas , Microscopía Fluorescente , Proteoglicanos/metabolismoRESUMEN
Defined growth conditions are essential for many applications of human embryonic stem cells (hESC). Most defined media are presently used in combination with Matrigel, a partially defined extracellular matrix (ECM) extract from mouse sarcoma. Here, we defined ECM requirements of hESC by analyzing integrin expression and ECM production and determined integrin function using blocking antibodies. hESC expressed all major ECM proteins and corresponding integrins. We then systematically replaced Matrigel with defined medium supplements and ECM proteins. Cells attached efficiently to natural human vitronectin, fibronectin, and Matrigel but poorly to laminin + entactin and collagen IV. Integrin-blocking antibodies demonstrated that alphaVbeta5 integrins mediated adhesion to vitronectin, alpha5beta1 mediated adhesion to fibronectin, and alpha6beta1 mediated adhesion to laminin + entactin. Fibronectin in feeder cell-conditioned medium partially supported growth on all natural matrices, but in defined, nonconditioned medium only Matrigel or (natural and recombinant) vitronectin was effective. Recombinant vitronectin was the only defined functional alternative to Matrigel, supporting sustained self-renewal and pluripotency in three independent hESC lines.
Asunto(s)
Células Madre Embrionarias/citología , Receptores de Vitronectina/fisiología , Vitronectina/farmacología , Animales , Adhesión Celular , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Colágeno , Medios de Cultivo , Combinación de Medicamentos , Células Madre Embrionarias/metabolismo , Proteínas de la Matriz Extracelular/biosíntesis , Humanos , Laminina , Ratones , Proteoglicanos , Proteínas Recombinantes/farmacologíaRESUMEN
Human embryonic stem cells (hESC) can differentiate to cardiomyocytes in vitro but with generally poor efficiency. Here, we describe a novel method for the efficient generation of cardiomyocytes from hESC in a scalable suspension culture process. Differentiation in serum-free medium conditioned by the cell line END2 (END2-CM) readily resulted in differentiated cell populations with more than 10% cardiomyocytes without further enrichment. By screening candidate molecules, we have identified SB203580, a specific p38 MAP kinase inhibitor, as a potent promoter of hESC-cardiogenesis. SB203580 at concentrations <10 microM, induced more than 20% of differentiated cells to become cardiomyocytes and increased total cell numbers, so that the overall cardiomyocyte yield was approximately 2.5-fold higher than controls. Gene expression indicated that early mesoderm formation was favored in the presence of SB203580. Accordingly, transient addition of the inhibitor at the onset of differentiation only was sufficient to determine the hESC fate. Patch clamp electrophysiology showed that the distribution of cardiomyocyte phenotypes in the population was unchanged by the compound. Interestingly, cardiomyogenesis was strongly inhibited at SB203580 concentrations > or =15 microM. Thus, modulation of the p38MAP kinase pathway, in combination with factors released by END2 cells, plays an essential role in early lineage determination in hESC and the efficiency of cardiomyogenesis. Our findings contribute to transforming human cardiomyocyte generation from hESC into a robust and scalable process.
Asunto(s)
Células Madre Embrionarias/efectos de los fármacos , Corazón/embriología , Imidazoles/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Piridinas/farmacología , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Diferenciación Celular/efectos de los fármacos , Línea Celular , Medio de Cultivo Libre de Suero , Células Madre Embrionarias/citología , Humanos , Técnicas de Placa-Clamp , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacosRESUMEN
Safety pharmacology studies that evaluate drug candidates for potential cardiovascular liabilities remain a critical component of drug development. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have recently emerged as a new and promising tool for preclinical hazard identification and risk assessment of drugs. Recently, Pluriomics organized its first User Meeting entitled 'Combining Pluricyte® Cardiomyocytes & MEA for Safety Pharmacology applications', consisting of scientific sessions and live demonstrations, which provided the opportunity to discuss the application of hiPSC-CMs (Pluricyte® Cardiomyocytes) in cardiac safety assessment to support early decision making in safety pharmacology. This report summarizes the outline and outcome of this Pluriomics User Meeting, which took place on November 24-25, 2016 in Leiden (The Netherlands). To reflect the content of the communications presented at this meeting we have cited key scientific articles and reviews.
Asunto(s)
Potenciales de Acción/efectos de los fármacos , Evaluación Preclínica de Medicamentos/métodos , Miocitos Cardíacos/efectos de los fármacos , Cardiotoxicidad/prevención & control , Línea Celular , Evaluación Preclínica de Medicamentos/instrumentación , Evaluación Preclínica de Medicamentos/normas , Electrodos , Guías como Asunto , Humanos , Células Madre Pluripotentes Inducidas/fisiología , Contracción Miocárdica/efectos de los fármacos , Miocitos Cardíacos/fisiología , Técnicas de Placa-Clamp/instrumentación , Técnicas de Placa-Clamp/métodosRESUMEN
Oncolytic adenoviruses are exploited as possible anticancer agents in clinical trails. To monitor adenoviral gene expression, a real-time RT-PCR method with a LightCycler was developed that allows the rapid and easy quantification of a number of early and late adenoviral genes in infected tumor cells. Primers were designed that can amplify the spliced forms of the genes encoding E1A13S, DNA polymerase (Pol), pre-terminal protein (pTP), adenoviral death protein (ADP), Hexon (Hex) and Penton (Pent) genes. Standard curves were generated using two-fold serial dilutions of cDNAs derived from non-small cell lung cancer (NSCLC) H460 cells infected for 24h with wild-type adenovirus serotype 5. For all genes correlation coefficients of the standard curves of 0.984 or higher were obtained. The dynamic range of the assay was sufficient to allow the quantitative determination of adenoviral gene expression during a lytic cycle. This RT-PCR assay could be used as a research tool to study the effect of host-cell factors or exogenous treatments on adenoviral gene expression. As example, it is shown that the procedure is suitable to detect changes in adenoviral gene expression in infected H460 cells treated with paclitaxel that is known to enhance the antitumor effect of oncolytic adenoviruses.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/genética , ADN Viral/análisis , Expresión Génica/fisiología , Neoplasias Pulmonares/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Adenoviridae/clasificación , Adenoviridae/genética , Antineoplásicos Fitogénicos/farmacología , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , ADN Viral/genética , Expresión Génica/efectos de los fármacos , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/patología , Técnicas de Amplificación de Ácido Nucleico , Paclitaxel/farmacología , Serotipificación , Factores de TiempoRESUMEN
Human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) can differentiate to cardiomyocytes in vitro, offering unique opportunities to investigate cardiac development and disease as well as providing a platform to perform drug and toxicity tests. Initial cardiac differentiation methods were based on either inductive co-culture or aggregation as embryoid bodies, often in the presence of fetal calf serum. More recently, monolayer differentiation protocols have evolved as feasible alternatives and are often performed in completely defined culture medium and substrates. Thus, our ability to efficiently and reproducibly generate cardiomyocytes from multiple different hESC and hiPSC lines has improved significantly.We have developed a directed differentiation monolayer protocol that can be used to generate cultures comprising ~50% cardiomyocytes, in which both the culture of the undifferentiated human pluripotent stem cells (hPSCs) and the differentiation procedure itself are defined and serum-free. The differentiation method is also effective for hPSCs maintained in other culture systems. In this chapter, we outline the differentiation protocol and describe methods to assess cardiac differentiation efficiency as well as to identify and quantify the yield of cardiomyocytes.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Reprogramación Celular , Células Madre Embrionarias/citología , Inhibidores Enzimáticos/farmacología , Miocitos Cardíacos/citología , Células Madre Pluripotentes/citología , Activinas/farmacología , Biomarcadores/metabolismo , Proteína Morfogenética Ósea 4/farmacología , Diferenciación Celular , Colágeno/química , Combinación de Medicamentos , Células Madre Embrionarias/efectos de los fármacos , Células Madre Embrionarias/metabolismo , Expresión Génica , Compuestos Heterocíclicos con 3 Anillos/farmacología , Humanos , Péptidos y Proteínas de Señalización Intercelular/farmacología , Laminina/química , Imagen Molecular , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Células Madre Pluripotentes/efectos de los fármacos , Células Madre Pluripotentes/metabolismo , Cultivo Primario de Células , Proteoglicanos/química , Piridinas/farmacología , Pirimidinas/farmacología , Troponina T/genética , Troponina T/metabolismoRESUMEN
Almost 7 years after their first derivation from human embryos, a pressing urgency to deliver the promises of therapies based on human embryonic stem cells (hESC) has arisen. Protocols have been developed to support long-term growth of undifferentiated cells and partially direct differentiation to specific cell lineages. The stage has almost been set for the next step: transplantation in animal models of human disease. Here, we review the state-of-the-art with respect to the transplantation of embryonic stem cell-derived heart cells in animals. One problem affecting progress in this area and functional analysis in vivo in general, is the availability of genetically marked hESC. There are only a few cell lines that express reporter genes ubiquitously, and none is associated with particular lineages; a major hurdle has been the resistance of hESC to established infection and chemical transfection methodologies to introduce ectopic genes. The methods that have been successful are reviewed. We also describe the processes for generating a new, genetically-modified hESC line that constitutively expresses GFP as well as some of its characteristics, including its ability to form cardiomyocytes with electrophysiological properties of ventricular-like cells.
Asunto(s)
Terapia Genética , Cardiopatías/cirugía , Miocitos Cardíacos/citología , Trasplante de Células Madre , Células Madre Totipotentes/citología , Animales , Línea Celular , Cardiopatías/genética , Humanos , Miocitos Cardíacos/fisiología , Miocitos Cardíacos/trasplante , Células Madre Totipotentes/fisiologíaRESUMEN
INTRODUCTION: The field of cardiac safety pharmacology has been experiencing exciting changes over the recent years. Drug induced arrhythmia of the torsade des pointes types has been the reason for the denial of approval of novel drug candidates. The aim of cardiac safety pharmacology is to detect undesirable pharmacodynamic drug effects within and above the therapeutic range. A special focus is on the identification of potential arrhythmogenic effects within the drug discovery chain. AREAS COVERED: Here, the authors discuss the relevance of induced pluripotent stem (iPS) cell derived cardiomyocytes for safety pharmacology. The technology of obtaining functional cardiomyocytes from somatic cells of healthy donors and patients with inherited diseases is the basis for diverse disease models in multi-level safety pharmacology screening. The reader will gain an overview of stem cell based technologies in cardiac safety pharmacology in cardiac and disease modeling by iPS cell derived cardiomyocytes from patients with an inherited cardiac syndrome. EXPERT OPINION: iPS cell derived cardiomyocytes - especially from patients with increased risk of cardiac arrhythmia - are on the verge of offering new options for drug testing. More reliable assays can be expected to predict the arrhythmogenic risk of drug candidates in humans. However, this technology is still new and extensive validation studies are due.
Asunto(s)
Arritmias Cardíacas/inducido químicamente , Bioensayo , Canales de Potasio Éter-A-Go-Go/antagonistas & inhibidores , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Miocitos Cardíacos/efectos de los fármacos , Pruebas de Toxicidad/métodos , Potenciales de Acción , Animales , Arritmias Cardíacas/genética , Arritmias Cardíacas/metabolismo , Arritmias Cardíacas/fisiopatología , Células Cultivadas , Canal de Potasio ERG1 , Canales de Potasio Éter-A-Go-Go/genética , Canales de Potasio Éter-A-Go-Go/metabolismo , Predisposición Genética a la Enfermedad , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Miocitos Cardíacos/metabolismo , Técnicas de Placa-Clamp , Reproducibilidad de los Resultados , Medición de Riesgo , Factores de RiesgoRESUMEN
Recent advances in pluripotent stem cell biology now make it possible to generate human cardiomyocytes in vitro from both healthy individuals and from patients with cardiac abnormalities. This offers unprecedented opportunities to study cardiac disease development 'in a dish' and establish novel platforms for drug discovery, either to prevent disease progression or to reverse it. In this review paper, we discuss some of the genetic diseases that affect the heart and illustrate how these new paradigms could assist our understanding of cardiac pathogenesis and aid in drug discovery. In particular, we highlight the limitations of other commonly used model systems in predicting the consequences of drug exposure on the human heart.
Asunto(s)
Cardiomiopatías/genética , Descubrimiento de Drogas , Cardiopatías Congénitas/genética , Cardiopatías Congénitas/metabolismo , Miocitos Cardíacos/citología , Células Madre Pluripotentes/citología , Potenciales de Acción , Animales , Cardiomiopatías/metabolismo , Cardiomiopatías/patología , Línea Celular , Modelos Animales de Enfermedad , Electrofisiología , Humanos , Ratones , Miocitos Cardíacos/metabolismo , Células Madre Pluripotentes/metabolismo , Transducción de SeñalRESUMEN
Generic methods for genetic manipulation of human embryonic stem cells (hESCs) are important for both present research and future commercial applications. To date, differences in cell derivation and culture have required independent optimization of transfection and transduction protocols and some lines have remained refractile to all methods. Here we describe a culture protocol that has been extensively tested in 12 different hESC lines (1, 2) and shown to support efficient gene transfer independent of the method of gene delivery or history of the cell line. The system is based on Matrigel monolayer culture and conditioned medium from mouse embryonic feeder cells (MEFs) and entails transient high-density culture followed by rapid adaptation to low density for gene transfer. Under these conditions, plasmid transfection, virus infection, and siRNA transfection are highly effective. Stable genetically modified hESC lines can be generated with plasmid transfection, viral infection, or electroporation without loss of pluripotency or differentiation potential. The majority of lines generated in this system display a normal karyotype.
Asunto(s)
Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Técnicas de Transferencia de Gen , Animales , Técnicas de Cultivo de Célula/métodos , Diferenciación Celular , Línea Celular , Separación Celular , Criopreservación , Medios de Cultivo , Medios de Cultivo Condicionados , Electroporación/métodos , Citometría de Flujo , Humanos , Ratones , Plásmidos/genética , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , ARN Interferente Pequeño/genética , Transducción Genética/métodos , Transfección/métodosRESUMEN
Recent withdrawals of prescription drugs from clinical use because of unexpected side effects on the heart have highlighted the need for more reliable cardiac safety pharmacology assays. Block of the human Ether-a-go go Related Gene (hERG) ion channel in particular is associated with life-threatening arrhythmias, such as Torsade de Pointes (TdP). Here we investigated human cardiomyocytes derived from pluripotent (embryonic) stem cells (hESC) as a renewable, scalable, and reproducible system on which to base cardiac safety pharmacology assays. Analyses of extracellular field potentials in hESC-derived cardiomyocytes (hESC-CM) and generation of derivative field potential duration (FPD) values showed dose-dependent responses for 12 cardiac and noncardiac drugs. Serum levels in patients of drugs with known effects on QT interval overlapped with prolonged FPD values derived from hESC-CM, as predicted. We thus propose hESC-CM FPD prolongation as a safety criterion for preclinical evaluation of new drugs in development. This is the first study in which dose responses of such a wide range of compounds on hESC-CM have been generated and shown to be predictive of clinical effects. We propose that assays based on hESC-CM could complement or potentially replace some of the preclinical cardiac toxicity screening tests currently used for lead optimization and further development of new drugs.
Asunto(s)
Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos , Células Madre Embrionarias/citología , Miocitos Cardíacos/citología , Miocitos Cardíacos/efectos de los fármacos , Animales , Línea Celular , Evaluación Preclínica de Medicamentos , Electrofisiología , Humanos , Lidocaína/toxicidad , Síndrome de QT Prolongado/inducido químicamente , Ratones , Técnicas de Placa-Clamp , Quinidina/toxicidad , Sotalol/toxicidadRESUMEN
In recent years the differentiation efficiency of human embryonic stem cells (hESCs) to cardiomyocytes has improved considerably. In general, hESC-derived cardiomyocytes are formed in aggregates, which require dissociation for follow-up experimental analyses and (clinical) applications. Here, we show that inhibition of the Rho-associated kinase (ROCK) by Y-27632 improved survival of dissociated hESC-derived differentiated cells. A maximum effect on cell survival was already observed within the first 24 hours. Hereafter, no further differences in the percentage of apoptotic and proliferating cells were observed with or without ROCK-inhibitor treatment. Improved survival was observed in both cardiomyocyte as well as non-cardiomyocyte cell populations. Viable cardiomyocytes were indicated by the appearance of beating, sarcomeric organization of cardiac-specific proteins, and fluorescence of a mitochondrion-selective dye. These results facilitate development of applications of hESC-derived cardiomyocytes in multiple research areas. Furthermore, these findings may be applied to other cell types differentiated from hESCs or other stem cells.
Asunto(s)
Amidas/farmacología , Células Madre Embrionarias/citología , Células Madre Embrionarias/efectos de los fármacos , Miocitos Cardíacos/citología , Miocitos Cardíacos/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Piridinas/farmacología , Quinasas Asociadas a rho/antagonistas & inhibidores , Diferenciación Celular , Línea Celular , Separación Celular , Supervivencia Celular , Células Madre Embrionarias/enzimología , Humanos , Miocitos Cardíacos/enzimología , Quinasas Asociadas a rho/metabolismoRESUMEN
Stem cells derived from pre-implantation human embryos or from somatic cells by reprogramming are pluripotent and self-renew indefinitely in culture. Pluripotent stem cells are unique in being able to differentiate to any cell type of the human body. Differentiation towards the cardiac lineage has attracted significant attention, initially with a strong focus on regenerative medicine. Although an important research area, the heart has proven challenging to repair by cardiomyocyte replacement. However, the ability to reprogramme adult cells to pluripotent stem cells and genetically manipulate stem cells presented opportunities to develop models of human disease. The availability of human cardiomyocytes from stem cell sources is expected to accelerate the discovery of cardiac drugs and safety pharmacology by offering more clinically relevant human culture models than presently available. Here we review the state-of-the-art using stem cell-derived human cardiomyocytes in drug discovery, drug safety pharmacology, and regenerative medicine.