Non-Cardiomyocytes Influence the Electrophysiological Maturation of Human Embryonic Stem Cell-Derived Cardiomyocytes During Differentiation
Non-Cardiomyocytes Influence the Electrophysiological Maturation of Human Embryonic Stem Cell-Derived Cardiomyocytes During Differentiation
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DOI：
10.1089/scd.2009.0349
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发表时间：
2010-06-01
影响因子：
4
通讯作者：
Chen, Huei-sheng Vincent
中科院分区：
文献类型：
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作者：
Kim, Changsung;Majdi, Maryam;Chen, Huei-sheng Vincent
在胎儿心脏发育过程中，各种类型的心肌细胞都经历了自律性和电特性的变化。人胚胎干细胞衍生的心肌细胞（hESC-CM），像胎儿心肌细胞一样，在电生理上是不成熟的，并表现出自律性。我们使用hESC-CM来研究自动性机制的发育变化，并确定电生理成熟是否由内在发育时钟驱动和/或通过与胚状体（EB）中的非心肌细胞的相互作用来调节。我们通过慢病毒工程化的嘌呤霉素抗性在不同分化阶段从EB中分离出纯的hESC-CM群体。使用药理学试剂，钙（Ca 2+）成像，和细胞内记录技术，我们发现，细胞内Ca 2+循环机制开发早期，并有助于占主导地位的自主性整个hESC-CM分化。肌膜离子通道在EB内进一步分化后进化，并在控制hESC-CM的自律性和电生理特性方面发挥越来越大的作用。与细胞内Ca 2+处理蛋白的发展相反，当hESC-CM早期从EB中分离并保持在培养物中而不与非心肌细胞进一步相互作用时，hESC-CM的离子通道发展和电生理成熟没有发生。将非心肌细胞添加回早期分离的hESC-CM挽救了电生理成熟的停滞，表明EB中的非心肌细胞驱动早期hESC-CM的电生理成熟。EB中的非心肌细胞包含已知影响早期心脏发育的胚胎心脏中存在的大多数细胞类型。我们的研究首次证明了非心肌细胞影响培养物中早期hESC-CM的电生理成熟。定义这些外在信号的性质将有助于未成熟hESC-CM的定向成熟，以减轻基于细胞的治疗的致瘤风险。
Various types of cardiomyocytes undergo changes in automaticity and electrical properties during fetal heart development. Human embryonic stem cell-derived cardiomyocytes (hESC-CMs), like fetal cardiomyocytes, are electrophysiologically immature and exhibit automaticity. We used hESC-CMs to investigate developmental changes in mechanisms of automaticity and to determine whether electrophysiological maturation is driven by an intrinsic developmental clock and/or is regulated by interactions with non-cardiomyocytes in embryoid bodies (EBs). We isolated pure populations of hESC-CMs from EBs by lentivirus-engineered Puromycin resistance at various stages of differentiation. Using pharmacological agents, calcium (Ca2+) imaging, and intracellular recording techniques, we found that intracellular Ca2+-cycling mechanisms developed early and contributed to dominant automaticity throughout hESC-CM differentiation. Sarcolemmal ion channels evolved later upon further differentiation within EBs and played an increasing role in controlling automaticity and electrophysiological properties of hESC-CMs. In contrast to the development of intracellular Ca2+-handling proteins, ion channel development and electrophysiological maturation of hESC-CMs did not occur when hESC-CMs were isolated from EBs early and maintained in culture without further interaction with non-cardiomyocytes. Adding back non-cardiomyocytes to early-isolated hESC-CMs rescued the arrest of electrophysiological maturation, indicating that non-cardiomyocytes in EBs drive electrophysiological maturation of early hESC-CMs. Non-cardiomyocytes in EBs contain most cell types present in the embryonic heart that are known to influence early cardiac development. Our study is the first to demonstrate that non-cardiomyocytes influence electrophysiological maturation of early hESC-CMs in cultures. Defining the nature of these extrinsic signals will aid in the directed maturation of immature hESC-CMs to mitigate arrhythmogenic risks of cell-based therapies.