Increased risk of genetic and epigenetic instability in human embryonic stem cells associated with specific culture conditions.
Increased risk of genetic and epigenetic instability in human embryonic stem cells associated with specific culture conditions.
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DOI：
10.1371/journal.pone.0118307
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Laurent LC
中科院分区：
文献类型：
--
作者：
Garitaonandia I;Amir H;Boscolo FS;Wambua GK;Schultheisz HL;Sabatini K;Morey R;Waltz S;Wang YC;Tran H;Leonardo TR;Nazor K;Slavin I;Lynch C;Li Y;Coleman R;Gallego Romero I;Altun G;Reynolds D;Dalton S;Parast M;Loring JF;Laurent LC
人多能干细胞（human pluripotent stem cells，hPSC）的自我更新和分化能力使其成为细胞移植治疗、药物开发以及细胞分化和发育研究的有希望的材料来源。然而，许多这些应用所需的大量细胞需要hPSC培养物的广泛扩增，这是一个与遗传和表观遗传改变相关的过程。我们对hESC和hiPSC进行了组合研究，以比较酶促传代与机械传代以及无饲养层与小鼠胚胎成纤维细胞饲养层基质对hPSC的遗传和表观遗传稳定性以及表型特征的影响。在涉及超过100次连续传代的广泛实验中，我们观察到酶促传代和无饲养层培养均与畸胎瘤中的遗传不稳定性、更高的细胞增殖率和OCT 4/POU 5 F1阳性细胞的持久性相关，其中酶促传代具有更强的效果。除了饲养层上的机械传代外，在所有培养条件的组合中，我们注意到含有肿瘤抑制基因TP 53的基因组区域中的复发性缺失，这与TP 53的mRNA表达降低相关，以及与TP 53途径活性降低一致的几个下游基因表达的改变。在人胚胎干细胞培养中，我们还观察到了与培养相关的基因表达和DNA甲基化的变化。还在hiPSC培养物中观察到酶促传代和无饲养层条件的影响。我们的研究结果强调了需要仔细评估培养条件对临床治疗细胞的影响。
The self-renewal and differentiation capacities of human pluripotent stem cells (hPSCs) make them a promising source of material for cell transplantation therapy, drug development, and studies of cellular differentiation and development. However, the large numbers of cells necessary for many of these applications require extensive expansion of hPSC cultures, a process that has been associated with genetic and epigenetic alterations. We have performed a combinatorial study on both hESCs and hiPSCs to compare the effects of enzymatic vs. mechanical passaging, and feeder-free vs. mouse embryonic fibroblast feeder substrate, on the genetic and epigenetic stability and the phenotypic characteristics of hPSCs. In extensive experiments involving over 100 continuous passages, we observed that both enzymatic passaging and feeder-free culture were associated with genetic instability, higher rates of cell proliferation, and persistence of OCT4/POU5F1-positive cells in teratomas, with enzymatic passaging having the stronger effect. In all combinations of culture conditions except for mechanical passaging on feeder layers, we noted recurrent deletions in the genomic region containing the tumor suppressor gene TP53, which was associated with decreased mRNA expression of TP53, as well as alterations in the expression of several downstream genes consistent with a decrease in the activity of the TP53 pathway. Among the hESC cultures, we also observed culture-associated variations in global gene expression and DNA methylation. The effects of enzymatic passaging and feeder-free conditions were also observed in hiPSC cultures. Our results highlight the need for careful assessment of the effects of culture conditions on cells intended for clinical therapies.
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影响因子：
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作者：
Ben-Yosef, Dalit;Boscolo, Francesca S.;Laurent, Louise C.
通讯作者：
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Lahesmaa, Riitta