Cryopreservation of Human Embryonic Stem Cells Derived-Cardiomyocytes Induced by BMP2 in Serum-Free Condition
Cryopreservation of Human Embryonic Stem Cells Derived-Cardiomyocytes Induced by BMP2 in Serum-Free Condition
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DOI：
10.1177/1933719110385130
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发表时间：
2011-03-01
影响因子：
2.9
通讯作者：
Choi, Young Min
中科院分区：
文献类型：
--
作者：
Kim, Yoon Young;Ku, Seung-Yup;Choi, Young Min
虽然先前的研究表明心肌细胞可以从人胚胎干细胞(HESCs)中产生，但目前还没有hESC来源的心肌细胞的冷冻保存方案。在这里，我们报道了在无血清条件下使用BMP2直接分化产生hESC来源的CMS，以及使用Rho相关激酶(ROCK)抑制剂成功地冷冻保存来源的CMS。用激活素A和骨形态发生蛋白2诱导人胚胎干细胞分化5d。中胚层基因brachyury从第3天开始表达，心脏特异性标志物NKX2.5和cTn1在第14天被检测到。此外，这些心脏祖细胞从第10天开始表达离子通道相关的转录产物如HCN1和HCN2。从第14天开始观察到搏动簇，最长持续35天。采用大体积冷冻保存法，在用岩石抑制剂Y-27632处理后，在第12天和第16天(打浆前和打浆后)对人胚胎干细胞来源的细胞质进行两个阶段的冷冻。I2组CMS解冻后存活率高于I6d组，12d组部分细胞团恢复收缩。透射电子显微镜分析，第I、2天组超微结构改变较轻。我们的结果为在无血清条件下使用BMP2进行心脏谱系分化以及长期保存hESC来源的CMS提供了一个深入的见解。
Although previous studies showed that cardiomyocytes (CMs) can be generated from human embryonic stem cells (hESCs), the protocols for cryopreservation of hESC-derived CMs is not available to date. Here, we report on the efficient generation of hESC-derived CMs by direct differentiation using BMP2 in a serum-free condition, along with successful cryopreservation of derived CMs using Rho-associated kinase (ROCK) inhibitor. To induce differentiation, hESCs were treated with activin A and BMP2 for 5 days. A mesodermal gene, Brachyury, was expressed from day 3, and cardiac-specific markers such as Nkx2.5 and cTn1 were detected at day 14. Furthermore, these cardiac progenitors expressed ion channel-related transcripts such as HCN1 and HCN2 from day 10. Beating clusters were observed from 14 days of differentiation for up to 35 days. Using mass cryopreservation, we froze hESC-derived CMs at 2 stages, at day 12 and 16 (prebeating and postbeating), after treating with ROCK inhibitor, Y-27632. Postthaw survival of CMs was higher in day I 2 group compared to day I 6, and some cell clusters from day 12 group recovered their contraction. From transmission electron microscope (TEM) analysis, less ultrastructural alterations were observed in day I 2 group. Our results provide an insight into the use of BMP2 for cardiac lineage differentiation in a serum-free condition and a possibility of long-term storage of hESC-derived CMs.