A universal system for highly efficient cardiac differentiation of human induced pluripotent stem cells that eliminates interline variability.
A universal system for highly efficient cardiac differentiation of human induced pluripotent stem cells that eliminates interline variability.
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DOI：
10.1371/journal.pone.0018293
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发表时间：
2011-04-08
期刊：
影响因子：
3.7
通讯作者：
Zambidis ET
中科院分区：
文献类型：
--
作者：
Burridge PW;Thompson S;Millrod MA;Weinberg S;Yuan X;Peters A;Mahairaki V;Koliatsos VE;Tung L;Zambidis ET
从人诱导多能干细胞（hiPSC）产生心肌细胞对于患者特异性心脏毒性药物测试、疾病建模和心脏再生具有很大的希望。然而，用于将hiPSC分化为心脏谱系的现有方案是低效的并且高度可变的。我们描述了一种用于将人胚胎干细胞（hESC）和hiPSC分化为心脏谱系的高效系统。该系统消除了各种人多能干细胞（hPSC）的心脏分化能力的变异性，包括使用非病毒、非整合方法从CD 34+脐带血产生的hiPSC。我们系统地和严格地优化了>45个实验变量以开发通用心脏分化系统，该系统在加速的9天内从测试的4个hESC和7个hiPSC系产生收缩的人胚状体（hEB），效率提高至94.7±2.4%，包括使用非整合附加型质粒衍生自新生儿CD 34+脐带血和成人成纤维细胞的hiPSC。这种成本有效的分化方法采用在化学成分确定的培养基中强制聚集hEB形成，沿着阶段性暴露于生理（5%）氧，以及优化浓度的中胚层形态发生素BMP 4和FGF 2、聚乙烯醇、血清和胰岛素。使用这些方法获得的收缩hEB由高百分比（64-89%）的心肌肌钙蛋白I+细胞组成，其显示功能性心肌细胞的超微结构特性和对心脏活性药物响应的均匀电生理特征。这种用于hiPSC心脏分化的高效且具有成本效益的通用系统允许潜在地无限制地产生适用于基于hPSC的药物开发、心脏病建模和用于再生医学的未来一代临床安全的非病毒人类心脏细胞的功能性心肌细胞。
The production of cardiomyocytes from human induced pluripotent stem cells (hiPSC) holds great promise for patient-specific cardiotoxicity drug testing, disease modeling, and cardiac regeneration. However, existing protocols for the differentiation of hiPSC to the cardiac lineage are inefficient and highly variable. We describe a highly efficient system for differentiation of human embryonic stem cells (hESC) and hiPSC to the cardiac lineage. This system eliminated the variability in cardiac differentiation capacity of a variety of human pluripotent stem cells (hPSC), including hiPSC generated from CD34+ cord blood using non-viral, non-integrating methods. We systematically and rigorously optimized >45 experimental variables to develop a universal cardiac differentiation system that produced contracting human embryoid bodies (hEB) with an improved efficiency of 94.7±2.4% in an accelerated nine days from four hESC and seven hiPSC lines tested, including hiPSC derived from neonatal CD34+ cord blood and adult fibroblasts using non-integrating episomal plasmids. This cost-effective differentiation method employed forced aggregation hEB formation in a chemically defined medium, along with staged exposure to physiological (5%) oxygen, and optimized concentrations of mesodermal morphogens BMP4 and FGF2, polyvinyl alcohol, serum, and insulin. The contracting hEB derived using these methods were composed of high percentages (64–89%) of cardiac troponin I+ cells that displayed ultrastructural properties of functional cardiomyocytes and uniform electrophysiological profiles responsive to cardioactive drugs. This efficient and cost-effective universal system for cardiac differentiation of hiPSC allows a potentially unlimited production of functional cardiomyocytes suitable for application to hPSC-based drug development, cardiac disease modeling, and the future generation of clinically-safe nonviral human cardiac cells for regenerative medicine.
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