Thomson Alison, Wojtacha Davina, Hewitt Zoë, Priddle Helen, Sottile Virginie, Di Domenico Alex, Fletcher Judy, Waterfall Martin, Corrales Néstor López, Ansell Ray, McWhir Jim
Division of Gene Function and Development, Roslin Institute, Roslin, Midlothian, Scotland.
Cloning Stem Cells. 2008 Mar;10(1):89-106. doi: 10.1089/clo.2007.0072.
Human embryonic stem cells (hESCs) are thought to be susceptible to chromosomal rearrangements as a consequence of single cell dissociation. Compared in this study are two methods of dissociation that do not generate single cell suspensions (collagenase and EDTA) with an enzymatic procedure using trypsin combined with the calcium-specific chelator EGTA (TEG), that does generate a single cell suspension, over 10 passages. Cells passaged by single cell dissociation using TEG retained a normal karyotype. However, cells passaged using EDTA, without trypsin, acquired an isochromosome p7 in three replicates of one experiment. In all of the TEG, collagenase and EDTA-treated cultures, cells retained consistent telomere length and potentiality, demonstrating that single cell dissociation can be used to maintain karyotypically and phenotypically normal hESCs. However, competitive genomic hybridization revealed that subkaryotypic deletions and amplifications could accumulate over time, reinforcing that present culture regimes remain suboptimal. In all cultures the cell surface marker CD30, reportedly expressed on embryonal carcinoma but not karyoptically normal ESCs, was expressed on hESCs with both normal and abnormal karyotype, but was upregulated on the latter.
人类胚胎干细胞（hESCs）被认为因单细胞解离而易于发生染色体重排。在本研究中，将两种不会产生单细胞悬液的解离方法（胶原酶和乙二胺四乙酸）与一种使用胰蛋白酶结合钙特异性螯合剂乙二醇双四乙酸（TEG）的酶促程序进行比较，后者的确会产生单细胞悬液，且持续超过10代。使用TEG通过单细胞解离传代的细胞保持了正常的核型。然而，在一个实验的三次重复中，使用不含胰蛋白酶的乙二胺四乙酸传代的细胞获得了一条7号等臂染色体。在所有经TEG、胶原酶和乙二胺四乙酸处理的培养物中，细胞保持了一致的端粒长度和潜能，表明单细胞解离可用于维持核型和表型正常的hESCs。然而，竞争性基因组杂交显示，亚核型缺失和扩增可能会随着时间积累，这进一步证明目前的培养方案仍不够理想。在所有培养物中，细胞表面标志物CD30在核型正常和异常的hESCs上均有表达，据报道该标志物在胚胎癌上表达，但在核型正常的ESCs上不表达，不过在核型异常的hESCs上表达上调。
