Induction of human cardiomyocyte-like cells from fibroblasts by defined factors
Induction of human cardiomyocyte-like cells from fibroblasts by defined factors
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DOI：
10.1073/pnas.1304053110
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发表时间：
2013-07-30
影响因子：
11.1
通讯作者：
Ieda, Masaki
中科院分区：
文献类型：
--
作者：
Wada, Rie;Muraoka, Naoto;Ieda, Masaki
心脏病仍然是全球主要的死亡原因。由于心脏组织的再生能力有限，心脏再生疗法已成为一种有吸引力的方法。将人类心脏成纤维细胞（HCFs）直接重编程为心肌细胞可能为此具有巨大潜力。我们之前报道过，通过转导三种转录因子：Gata4、Mef2c和Tbx5（统称为GMT），可以在体外和体内从小鼠心脏成纤维细胞直接产生诱导性心肌样细胞（iCMs）。在本研究中，我们试图确定人类成纤维细胞是否也能通过特定因子转化为iCMs。我们最初发现GMT不足以在HCFs中诱导心脏细胞，这促使我们通过定量RT - PCR分析多种心脏特异性基因的诱导来筛选其他促进心脏重编程的因子。与单独使用GMT相比，在GMT中添加Mesp1和Myocd能更有效地在HCFs中上调更广泛的心脏基因。用GMT、Mesp1和Myocd（GMTMM）转导的HCFs和人类皮肤成纤维细胞将细胞形态从纺锤形变为杆状或多边形，表达多种心脏特异性蛋白质，增加了广泛的心脏基因并同时抑制了成纤维细胞基因，且表现出自发性Ca2 +振荡。此外，这些细胞在与小鼠心肌细胞共培养时成熟到能够表现出动作电位并同步收缩。5 - 乙炔基 - 2'-脱氧尿苷检测显示，由此产生的iCMs不经过有丝分裂细胞状态。这些发现表明，人类成纤维细胞可以通过特定因子直接转化为iCMs，这可能有助于未来在再生医学中的应用。
Heart disease remains a leading cause of death worldwide. Owing to the limited regenerative capacity of heart tissue, cardiac regenerative therapy has emerged as an attractive approach. Direct reprogramming of human cardiac fibroblasts (HCFs) into cardiomyocytes may hold great potential for this purpose. We reported previously that induced cardiomyocyte-like cells (iCMs) can be directly generated from mouse cardiac fibroblasts in vitro and vivo by transduction of three transcription factors: Gata4, Mef2c, and Tbx5, collectively termed GMT. In the present study, we sought to determine whether human fibroblasts also could be converted to iCMs by defined factors. Our initial finding that GMT was not sufficient for cardiac induction in HCFs prompted us to screen for additional factors to promote cardiac reprogramming by analyzing multiple cardiac-specific gene induction with quantitative RT-PCR. The addition of Mesp1 and Myocd to GMT up-regulated a broader spectrum of cardiac genes in HCFs more efficiently compared with GMT alone. The HCFs and human dermal fibroblasts transduced with GMT, Mesp1, and Myocd (GMTMM) changed the cell morphology from a spindle shape to a rod-like or polygonal shape, expressed multiple cardiac-specific proteins, increased a broad range of cardiac genes and concomitantly suppressed fibroblast genes, and exhibited spontaneous Ca2+ oscillations. Moreover, the cells matured to exhibit action potentials and contract synchronously in coculture with murine cardiomyocytes. A 5-ethynyl-2'-deoxyuridine assay revealed that the iCMs thus generated do not pass through a mitotic cell state. These findings demonstrate that human fibroblasts can be directly converted to iCMs by defined factors, which may facilitate future applications in regenerative medicine.