Optimization of direct fibroblast reprogramming to cardiomyocytes using calcium activity as a functional measure of success.
Optimization of direct fibroblast reprogramming to cardiomyocytes using calcium activity as a functional measure of success.
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DOI：
10.1016/j.yjmcc.2013.04.004
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发表时间：
2013-07
影响因子：
5
通讯作者：
Gearhart JD
中科院分区：
文献类型：
--
作者：
Addis RC;Ifkovits JL;Pinto F;Kellam LD;Esteso P;Rentschler S;Christoforou N;Epstein JA;Gearhart JD
成纤维细胞直接转化为诱导心肌细胞(ICM)在再生医学中具有巨大的潜力。最近的出版物报道了重大的进展，但重编程的评估依赖于非功能性方法，如心肌细胞标志物的流式细胞术或由心肌细胞特异性启动子驱动的GFP表达。这是一个实用性的问题：在重新编程实验中筛选的大量细胞中，最严格的措施--检测细胞兴奋的电生理学和自发收缩心肌细胞的存在--并不容易量化。然而，兴奋和收缩由心肌细胞的第三个功能特征联系在一起：细胞内钙水平的节律性振荡。我们开始使用可量化的钙报告程序来优化成纤维细胞到ICM的直接转化，以快速评估功能转分化。我们构建了一个钙指示剂GCaMP由心肌细胞特异性肌钙蛋白T启动子驱动的报告系统。以钙活性为主要指标，我们在小鼠胚胎成纤维细胞中比较了几种已发表的转录因子组合和新的组合。最有效的组合是Hand2、NKX2.5、Gata4、MEF2C和Tbx5(HNGMT)。这种组合的效率是单独GMT的50倍，并产生具有心肌细胞标志物表达、强劲的钙振荡和在重编程因子失活后持续数周的自发搏动的ICM。在成年小鼠心脏成纤维细胞向ICM的转分化方面，HNGMT也明显比先前发表的因子组合更有效。钙功能的定量是鉴定和评价直接重编程所产生的心肌细胞的一种方便有效的手段。使用这一严格的结果衡量标准，我们得出结论，HNGMT比以前发表的方法更有效地生产ICM。
Direct conversion of fibroblasts to induced cardiomyocytes (iCMs) has great potential for regenerative medicine. Recent publications have reported significant progress, but the evaluation of reprogramming has relied upon non-functional measures such as flow cytometry for cardiomyocyte markers or GFP expression driven by a cardiomyocyte-specific promoter. The issue is one of practicality: the most stringent measures - electrophysiology to detect cell excitation and the presence of spontaneously contracting myocytes - are not readily quantifiable in the large numbers of cells screened in reprogramming experiments. However, excitation and contraction are linked by a third functional characteristic of cardiomyocytes: the rhythmic oscillation of intracellular calcium levels. We set out to optimize direct conversion of fibroblasts to iCMs with a quantifiable calcium reporter to rapidly assess functional transdifferentiation. We constructed a reporter system in which the calcium indicator GCaMP is driven by the cardiomyocyte-specific Troponin T promoter. Using calcium activity as our primary outcome measure, we compared several published combinations of transcription factors along with novel combinations in mouse embryonic fibroblasts. The most effective combination consisted of Hand2, Nkx2.5, Gata4, Mef2c, and Tbx5 (HNGMT). This combination is >50-fold more efficient than GMT alone and produces iCMs with cardiomyocyte marker expression, robust calcium oscillation, and spontaneous beating that persists for weeks following inactivation of reprogramming factors. HNGMT is also significantly more effective than previously published factor combinations for the transdifferentiation of adult mouse cardiac fibroblasts to iCMs. Quantification of calcium function is a convenient and effective means for the identification and evaluation of cardiomyocytes generated by direct reprogramming. Using this stringent outcome measure, we conclude that HNGMT produces iCMs more efficiently than previously published methods.
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通讯作者：
Cserjesi, Peter
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