SummaryStem 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 cells in 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 α-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 α-fetoprotein were expressed in H9 cells, suggesting that the cells maintained in vitro differentiation 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.

中文翻译：

---

人胚胎干细胞饲养层和无血清培养体系的建立

摘要干细胞是一种能够自我更新的永生细胞群；它们对人类发展和衰老至关重要，是再生医学研究的主要焦点。尽管在干细胞分化方面取得了长足的进步体外，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。本研究的目的是为人类胚胎干细胞 (hESCs) 开发一种无饲养层、无血清培养方法，为 hESCs 增殖建立最佳条件，并确定所得 hESCs 的生物学特性。使用自制的无血清、无饲养层培养系统培养 H9 hESC 系，并观察生长情况。通过免疫荧光和蛋白质印迹测定 hESC 中多能性蛋白（OCT4、NANOG、SOX2、LIN28、SSEA-3、SSEA-4、TRA-1-60 和 TRA-1-81）的表达。通过RT-PCR测定分化的H9细胞中编码巢蛋白、brachyury和甲胎蛋白的基因的mRNA表达水平。新开发的培养系统产生了圆形或椭圆形的经典 hESC 菌落，边界清晰整洁。多能性蛋白表达增加，编码巢蛋白、brachyury和甲胎蛋白的基因在H9细胞中表达，提示细胞维持体外分化能力。我们的培养系统包含一组独特的成分和动物源性物质，可在八代中保持 H9 细胞的自我更新潜力和多能性。该系统的进一步优化可能会扩大hESCs的临床应用。