RESUMEN
Human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) resemble fetal cardiomyocytes and electrical stimulation (ES) has been explored to mature the differentiated cells. Here, we hypothesize that ES applied at the beginning of the differentiation process, triggers both differentiation of the hiPSC-CMs into a specialized conduction system (CS) phenotype and cell maturation. We applied ES for 15 days starting on day 0 of the differentiation process and found an increased expression of transcription factors and proteins associated with the development and function of CS including Irx3, Nkx2.5 and contactin 2, Hcn4 and Scn5a, respectively. We also found activation of intercalated disc proteins (Nrap and ß-catenin). We detected ES-induced CM maturation as indicated by increased Tnni1 and Tnni3 expression. Confocal micrographs showed a shift towards expression of the gap junction protein connexin 40 in ES hiPSC-CM compared to the more dominant expression of connexin 43 in controls. Finally, analysis of functional parameters revealed that ES hiPSC-CMs exhibited faster action potential (AP) depolarization, longer intracellular Ca2+ transients, and slower AP duration at 90% of repolarization, resembling fast conducting fibers. Altogether, we provided evidence that ES during the differentiation of hiPSC to cardiomyocytes lead to development of cardiac conduction-like cells with more mature cytoarchitecture. Thus, hiPSC-CMs exposed to ES during differentiation can be instrumental to develop CS cells for cardiac disease modelling, screening individual drugs on a precison medicine type platform and support the development of novel therapeutics for arrhythmias.
Asunto(s)
Potenciales de Acción/fisiología , Calcio/metabolismo , Células Madre Pluripotentes Inducidas/fisiología , Miocitos Cardíacos/fisiología , Biomarcadores/metabolismo , Diferenciación Celular , Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Conexinas/genética , Conexinas/metabolismo , Contactina 2/genética , Contactina 2/metabolismo , Estimulación Eléctrica , Expresión Génica , Sistema de Conducción Cardíaco/citología , Sistema de Conducción Cardíaco/fisiología , Proteína Homeótica Nkx-2.5/genética , Proteína Homeótica Nkx-2.5/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/genética , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/metabolismo , Células Madre Pluripotentes Inducidas/citología , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Miocitos Cardíacos/citología , Canal de Sodio Activado por Voltaje NAV1.5/genética , Canal de Sodio Activado por Voltaje NAV1.5/metabolismo , Canales de Potasio/genética , Canales de Potasio/metabolismo , Cultivo Primario de Células , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Troponina I/genética , Troponina I/metabolismo , beta Catenina/genética , beta Catenina/metabolismo , Proteína alfa-5 de Unión ComunicanteRESUMEN
Cardiomyocytes that are derived from human-induced pluripotent stem cells (iPSC-CM) are an exciting tool to investigate cardiomyopathy disease mechanisms at the cellular level as well as to screen for potential side effects of novel drugs. However, currently their benefit is limited due to their fairly immature differentiation status under conventional culture conditions. This review is mainly aimed at researchers outside of the iPSC-CM field and will describe potential pitfalls and which features at the level of the myofibrils would be desired to make them a more representative model system. We will also discuss different strategies that may help to achieve these.