Induction of diverse cardiac cell types by reprogramming fibroblasts with cardiac transcription factors
Induction of diverse cardiac cell types by reprogramming fibroblasts with cardiac transcription factors
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DOI：
10.1242/dev.114025
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发表时间：
2014-11-01
期刊：
影响因子：
4.6
通讯作者：
Munshi, Nikhil V.
中科院分区：
文献类型：
--
作者：
Nam, Young-Jae;Lubczyk, Christina;Munshi, Nikhil V.
Gata 4（G）、Hand 2（H）、Mef 2c（M）和Tbx 5（T）等多种心源性转录因子的组合可以在体外和体内将成纤维细胞重编程为诱导的心肌样细胞（iCLM）。考虑到最佳心脏功能依赖于不同但功能上相互关联的心房、心室和起搏器（PM）心肌细胞（CM），还有待观察哪些亚型是通过直接重编程产生的，以及这一过程是否可以用来产生特定的感兴趣CM。在这里，我们采用PM特异性Hcn 4-GFP报告小鼠和一系列CM亚型特异性标记来研究通过原代成纤维细胞重编程产生的细胞表型范围。出乎意料的是，我们发现，由于肌节蛋白表达和组织不足，针对Hcn 4-GFP表达优化的四种转录因子（4F）的组合不产生跳动的PM细胞。然而，应用严格的单细胞标准GHMT重编程细胞，我们观察到诱导不同的细胞表型，包括那些类似于所有三个主要的心脏亚型（即心房，心室和起搏器）的不成熟形式。此外，我们证明了GHMT诱导的细胞是直接重编程的，而不是从Nxk2.5（+）祖细胞中间体产生的。两者合计，我们的研究结果表明显着程度的固有的GHMT重编程的可塑性，并提供了一个起点CM亚型特异性重编程协议的优化。
Various combinations of cardiogenic transcription factors, including Gata4 (G), Hand2 (H), Mef2c (M) and Tbx5 (T), can reprogram fibroblasts into induced cardiac-like myocytes (iCLMs) in vitro and in vivo. Given that optimal cardiac function relies on distinct yet functionally interconnected atrial, ventricular and pacemaker (PM) cardiomyocytes (CMs), it remains to be seen which subtypes are generated by direct reprogramming and whether this process can be harnessed to produce a specific CM of interest. Here, we employ a PM-specific Hcn4-GFP reporter mouse and a spectrum of CM subtype-specific markers to investigate the range of cellular phenotypes generated by reprogramming of primary fibroblasts. Unexpectedly, we find that a combination of four transcription factors (4F) optimized for Hcn4-GFP expression does not generate beating PM cells due to inadequate sarcomeric protein expression and organization. However, applying strict single-cell criteria to GHMT-reprogrammed cells, we observe induction of diverse cellular phenotypes, including those resembling immature forms of all three major cardiac subtypes (i.e. atrial, ventricular and pacemaker). In addition, we demonstrate that cells induced by GHMT are directly reprogrammed and do not arise from an Nxk2.5(+) progenitor cell intermediate. Taken together, our results suggest a remarkable degree of plasticity inherent to GHMT reprogramming and provide a starting point for optimization of CM subtype-specific reprogramming protocols.