Research and Development Unit, National Heart Centre Singapore, 17, Third Hospital Avenue, Mistri Wing, Singapore 168752.
Cardiovasc Res. 2011 Sep 1;91(4):577-86. doi: 10.1093/cvr/cvr132. Epub 2011 May 12.
Generation of human induced pluripotent stem cell (hiPSC) lines by reprogramming of fibroblast cells with virus-free methods offers unique opportunities for translational cardiovascular medicine. The aim of the study was to reprogramme fibroblast cells to hiPSCs and to study cardiomyogenic properties and ion channel characteristics of the virus-free hiPSC-derived cardiomyocytes.
The hiPSCs generated by episomal vectors generated teratomas in severe combined immunodeficient mice, readily formed embryoid bodies, and differentiated into cardiomyocytes with comparable efficiency to human embryonic stem cells. Temporal gene expression of these hiPSCs indicated that differentiation of cardiomyocytes was initiated by increasing expression of cardio/mesodermal markers followed by cardiac-specific transcription factors, structural, and ion channel genes. Furthermore, the cardiomyocytes showed characteristic cross-striations of sarcomeric proteins and expressed calcium-handling and ion channel proteins, confirming their cardiac ontogeny. Microelectrode array recordings established the electrotonic development of a functional syncytium that responded predictably to pharmacologically active drugs. The cardiomyocytes showed a chronotropic dose-response (0.1-10 µM) to isoprenaline and Bay K 8644. Furthermore, carbamycholine (5 µM) suppressed the response to isoprenaline, while verapamil (2.5 µM) blocked Bay K 8644-induced inotropic activity. Moreover, verapamil (1 µM) reduced the corrected field potential duration by 45%, tetrodotoxin (10 µM) shortened the minimal field potential by 40%, and E-4031 (50 nM) prolonged field repolarization.
Virus-free hiPSCs differentiate efficiently into cardiomyocytes with cardiac-specific molecular, structural, and functional properties that recapitulate the developmental ontogeny of cardiogenesis. These results, coupled with the potential to generate patient-specific hiPSC lines, hold great promise for the development of an in vitro platform for drug pharmacogenomics, disease modelling, and regenerative medicine.
通过无病毒方法重编程成纤维细胞产生人类诱导多能干细胞（hiPSC）系为转化心血管医学提供了独特的机会。本研究的目的是将成纤维细胞重编程为 hiPSC，并研究无病毒 hiPSC 衍生的心肌细胞的心肌生成特性和离子通道特性。
用附加型载体产生的 hiPSC 在严重联合免疫缺陷小鼠中产生畸胎瘤，容易形成胚状体，并分化为心肌细胞的效率与人类胚胎干细胞相当。这些 hiPSC 的时间基因表达表明，心肌细胞的分化是通过增加心/中胚层标志物的表达开始的，随后是心脏特异性转录因子、结构和离子通道基因。此外，心肌细胞表现出肌节蛋白的特征性交叉条纹，并表达钙处理和离子通道蛋白，证实了它们的心脏发生。微电极阵列记录建立了功能性合胞体的电兴奋发育，该合胞体可对药理学活性药物进行可预测的反应。心肌细胞对异丙肾上腺素和 Bay K 8644 表现出变时剂量反应（0.1-10 μM）。此外，卡巴胆碱（5 μM）抑制异丙肾上腺素的反应，而维拉帕米（2.5 μM）阻断 Bay K 8644 诱导的变力活性。此外，维拉帕米（1 μM）使校正的场电位持续时间减少 45%，河豚毒素（10 μM）使最小场电位缩短 40%，E-4031（50 nM）延长场复极。
无病毒 hiPSC 高效分化为具有心脏特异性分子、结构和功能特性的心肌细胞，这些特性再现了心脏发生的发育发生。这些结果，加上生成患者特异性 hiPSC 系的潜力，为药物药物基因组学、疾病建模和再生医学的体外平台的发展带来了巨大的希望。
