Establishment of a feeder and serum-free culture system for human embryonic stem cells
Establishment of a feeder and serum-free culture system for human embryonic stem cells
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DOI：
10.1017/s0967199419000625
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发表时间：
2020-06-01
期刊：
影响因子：
1.7
通讯作者：
Bai, Xue
中科院分区：
文献类型：
--
作者：
Wang, LiYun;Zhang, RuiNa;Bai, Xue
干细胞是能够自我更新的永生细胞群;它们对人类发育和衰老至关重要，是再生医学研究的主要焦点。尽管干细胞在体外分化方面取得了相当大的进展，但培养条件需要进一步优化，以最大限度地提高扩增过程中多细胞分化的潜力。本研究的目的是建立一种无饲养层、无血清的人胚胎干细胞（hESC）培养方法，建立hESC增殖的最佳条件，并确定所获得的hESC的生物学特性。采用自制的无血清、无饲养层培养系统培养H9 hESC细胞，观察细胞生长情况。通过免疫荧光和蛋白质印迹法测定hESC中多能性蛋白（0 CT 4、NAN 0 G、S 0X 2、LIN 28、SSEA-3、SSEA-4、TRA-1-60和TRA-1-81）的表达。RT-PCR检测分化后H9细胞巢蛋白、短尾畸形和甲胎蛋白基因mRNA的表达水平。新开发的培养系统产生了典型的hESC集落，其形状为圆形或椭圆形，具有清晰和整齐的边界。H9细胞多能性蛋白表达增加，编码巢蛋白、短尾畸形和甲胎蛋白的基因在H9细胞中表达，表明细胞具有体外分化能力。我们的培养系统包含一组独特的组分，含有动物源性物质，保持了H9细胞的自我更新潜力和多能性，传代8次。该系统的进一步优化有望扩大hESCs的临床应用。
Stem cells are an immortal cell population capable of self-renewal; they are essential for human development and ageing and are a major focus of research in regenerative medicine. Despite considerable progress in differentiation of stem cellsin vitro, culture conditions require further optimization to maximize the potential for multicellular differentiation during expansion. The aim of this study was to develop a feeder-free, serum-free culture method for human embryonic stem cells (hESCs), to establish optimal conditions for hESC proliferation, and to determine the biological characteristics of the resulting hESCs. The H9 hESC line was cultured using a homemade serum-free, feeder-free culture system, and growth was observed. The expression of pluripotency proteins (OCT4, NANOG, SOX2, LIN28, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81) in hESCs was determined by immunofluorescence and western blotting. The mRNA expression levels of genes encoding nestin, brachyury and alpha-fetoprotein in differentiated H9 cells were determined by RT-PCR. The newly developed culture system resulted in classical hESC colonies that were round or elliptical in shape, with clear and neat boundaries. The expression of pluripotency proteins was increased, and the genes encoding nestin, brachyury, and alpha-fetoprotein were expressed in H9 cells, suggesting that the cells maintainedin vitrodifferentiation capacity. Our culture system containing a unique set of components, with animal-derived substances, maintained the self-renewal potential and pluripotency of H9 cells for eight passages. Further optimization of this system may expand the clinical application of hESCs.