Lei Ieng Lam, Bu Lei, Wang Zhong
Cardiac Surgery, University of Michigan.
Leon H Charney Division of Cardiology, New York University School of Medicine.
J Vis Exp. 2015 Jan 12(95):52047. doi: 10.3791/52047.
Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes, smooth muscle cells (SMC), and endothelial cells and hold great promise in cell therapy against heart disease. Among various methods to isolate CPCs, differentiation of embryonic stem cell (ESC) into CPCs attracts great attention in the field since ESCs can provide unlimited cell source. As a result, numerous strategies have been developed to derive CPCs from ESCs. In this protocol, differentiation and purification of embryonic CPCs from both mouse and human ESCs is described. Due to the difficulty of using cell surface markers to isolate embryonic CPCs, ESCs are engineered with fluorescent reporters activated by CPC-specific cre recombinase expression. Thus, CPCs can be enriched by fluorescence-activated cell sorting (FACS). This protocol illustrates procedures to form embryoid bodies (EBs) from ESCs for CPC specification and enrichment. The isolated CPCs can be subsequently cultured for cardiac lineage differentiation and other biological assays. This protocol is optimized for robust and efficient derivation of CPCs from both mouse and human ESCs.
心脏祖细胞（CPCs）具有分化为心肌细胞、平滑肌细胞（SMC）和内皮细胞的能力，在针对心脏病的细胞治疗中具有巨大潜力。在分离CPCs的各种方法中，胚胎干细胞（ESC）向CPCs的分化在该领域引起了极大关注，因为ESC可以提供无限的细胞来源。因此，已经开发了许多从ESC中获得CPCs的策略。在本方案中，描述了从小鼠和人类ESC中分化和纯化胚胎CPCs的方法。由于使用细胞表面标志物分离胚胎CPCs存在困难，因此对ESC进行工程改造，使其带有由CPC特异性cre重组酶表达激活的荧光报告基因。这样，CPCs就可以通过荧光激活细胞分选（FACS）进行富集。本方案阐述了从ESC形成胚状体（EBs）以实现CPCs的特化和富集的程序。随后可以对分离出的CPCs进行培养，用于心脏谱系分化和其他生物学分析。本方案针对从小鼠和人类ESC中稳健且高效地获得CPCs进行了优化。
