Chemically Defined Culture and Cardiomyocyte Differentiation of Human Pluripotent Stem Cells.
Chemically Defined Culture and Cardiomyocyte Differentiation of Human Pluripotent Stem Cells.
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DOI：
10.1002/0471142905.hg2103s87
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发表时间：
2015-10-06
影响因子：
--
通讯作者：
Wu JC
中科院分区：
文献类型：
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作者：
Burridge PW;Holmström A;Wu JC
自从首次发现人类多能干细胞（hPSCs）可以分化为心肌细胞以来，人们一直在努力优化这一过程发生的条件。优化这一过程的最有效方法之一是简化培养基配方，消除复杂的动物来源成分，以帮助揭示精确的潜在机制。在这里，我们描述了我们最新的成本效益和高效的方法，用于培养多能态的人造血干细胞，使用化学定义的E8培养基的改良变体。我们为这些条件下的细胞处理提供了精确的指导方针，包括非酶EDTA传代，这已经为随后的心肌细胞分化进行了优化。我们深入描述了我们的单层化学定义的小分子分化方案的最新版本，包括基于代谢选择的心肌细胞纯化和添加三碘甲状腺原氨酸以增强心肌细胞成熟。最后，我们描述了一种分离hpsc来源的心肌细胞，冷冻保存和解冻的方法。
Since the first discovery that human pluripotent stem cells (hPSCs) can differentiate to cardiomyocytes, efforts have been made to optimize the conditions under which this process occurs. One of the most effective methodologies to optimize this process is reductionist simplification of the medium formula, which eliminates complex animal-derived components to help reveal the precise underlying mechanisms. Here we describe our latest cost-effective and efficient methodology for the culture of hPSCs in the pluripotent state using a modified variant of chemically defined E8 medium. We provide exact guidelines for cell handling under these conditions, including non-enzymatic EDTA passaging, which have been optimized for subsequent cardiomyocyte differentiation. We describe in depth the latest version of our monolayer chemically defined small molecule differentiation protocol, including metabolic selection-based cardiomyocyte purification and the addition of triiodothyronine to enhance cardiomyocyte maturation. Finally, we describe a method for the dissociation of hPSC-derived cardiomyocytes, cryopreservation, and thawing.