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首页> 外文期刊>The international journal of developmental biology >Common culture conditions for maintenance and cardiomyocyte differentiation of the human embryonic stem cell lines, BG01 and HUES-7
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Common culture conditions for maintenance and cardiomyocyte differentiation of the human embryonic stem cell lines, BG01 and HUES-7

机译：人类胚胎干细胞系BG01和HUES-7维持和心肌细胞分化的常见培养条件

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摘要

Development of generic differentiation protocols that function in a range of independently-derived human embryonic stem cell (hESC) lines remains challenging due to considerable diversity in culture methods practiced between lines. Maintenance of BG01 and HUES-7 has routinely been on mouse embryonic fibroblast (MEF) feeder layers using manual- and trypsin-passaging, respectively. We adapted both lines to trypsin-passaging on feeders or on Matrigel in feeder-free conditions and assessed proliferation and cardiac differentiation. On feeders, undifferentiated proliferation of BG01 and HUES-7 was supported by all three media tested (BG-SK, HUES-C and HUES-nL), although incidence of karyotypic instability increased in both lines in BG-SK. On Matrigel, KSR-containing conditioned medium (CM) promoted undifferentiated cell proliferation, while differentiation occurred in CM containing Plasmanate or ES-screened Fetal Bovine Serum (FBS) and in unconditioned medium containing 100 ng/ml bFGF. Matrigel cultures were advantageous for transfection but detrimental to embryoid body (EB) formation. However, transfer of hESCs from Matrigel back to feeders and culturing to confluence was found to rescue EB formation. EBs formed efficiently when hESCs on feeders were treated with collagenase, harvested by scraping and then cultured in suspension in CM. Subsequent culture in FBS-containing medium produced spontaneously contracting EBs, for which the mean beat rate was 37.2 +/- 2.3 and 41.1 +/- 3.1 beats / min for BG01-EBs and HUES-7-EBs, respectively. Derived cardiomyocytes expressed cardiac genes and responded to pharmacological stimulation. Therefore the same culture and differentiation conditions functioned in two independently-derived hESC lines. Similar studies in other lines may facilitate development of universal protocols.
机译：由于在品系之间实践的培养方法存在很大差异，因此在一系列独立来源的人类胚胎干细胞（hESC）品系中起作用的通用分化方案的开发仍然具有挑战性。 BG01和HUES-7的常规维护工作分别是通过手动和胰蛋白酶传代在小鼠胚胎成纤维细胞（MEF）饲养层上进行。我们将这两种品系都调整为在无饲养者的条件下在饲养者或Matrigel上进行胰蛋白酶传代，并评估了增殖和心脏分化。在饲养层上，所有三种测试的培养基（BG-SK，HUES-C和HUES-nL）均支持BG01和HUES-7的未分化增殖，尽管在BG-SK的两个品系中核型不稳定性的发生率均增加。在Matrigel上，含KSR的条件培养基（CM）促进未分化的细胞增殖，而分化发生在含血浆或经ES筛选的胎牛血清（FBS）的CM和含100 ng / ml bFGF的无条件培养基中。基质胶培养物对于转染是有利的，但是不利于胚状体（EB）的形成。然而，已发现将hESC从Matrigel转移回饲养器并培养至汇合可拯救EB的形成。当用胶原酶处理饲养细胞上的hESC，通过刮取收获，然后在CM中悬浮培养时，EB有效形成。随后在含FBS的培养基中培养产生自发收缩的EB，对于BG01-EB和HUES-7-EB，其平均搏动速率分别为37.2 +/- 2.3和41.1 +/- 3.1搏动/分钟。衍生的心肌细胞表达心脏基因并响应药理刺激。因此，相同的培养和分化条件在两个独立来源的hESC系中起作用。其他领域的类似研究可能会促进通用协议的开发。

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