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目的建立一种安全、有效、经济且适于人孤雌胚胎干细胞(human parthenogenetic embryonic stemcells,hPESCs)体外培养的无饲养层培养体系。方法将常规体外培养的hPESCs分别以mTeSRTMl培养基(对照组)和人包皮成纤维细胞的条件培养基(human foreskin fibroblasts-conditional medium,hFFs-CM)(实验组)扩增培养,倒置显微镜下观察两组无饲养层培养体系下hPESCs的生长状态;采用ALP检测和核型分析研究hPESCs生物学特性;采用RT-PCR检测hPESCs全能性标记物Oct-4的表达情况;通过体外和体内分化实验观察hPESCs向3个胚层分化的潜能。结果两组hPESCs形态规则、不易分化,在形态、扩增速度等方面无明显差异;已成功在体外培养15代,两组均能保持正常女性的二倍体核型46,XX和全能性;RT-PCR检测示两组Oct-4 mRNA均呈阳性表达;体外分化均可形成拟胚体;在裸鼠体内均可形成含有3个胚层组织成分的畸胎瘤。结论 hFFs-CM无饲养层培养体系可长期支持hPESCs的生长并维持其未分化状态,成功建立了一种不仅能维持hPESCs的有效扩增、减少动物源性污染、降低培养成本,还可满足临床大规模应用的hPESCs无饲养层培养体系。
Objective To establish a feeder-free culture system that is safe, effective, economical and suitable for in vitro human parthenogenetic embryonic stem cells (hPESCs). Methods Conventional hPESCs cultured in vitro were expanded and cultured in mTeSRTMl medium (control group) and human foreskin fibroblasts-conditional medium (hFFs-CM) (experimental group), and observed under an inverted microscope The growth of hPESCs in two feeder-free culture systems was studied. The biological characteristics of hPESCs were studied by ALP and karyotype analysis. The expression of Oct-4, a pluripotency marker of hPESCs, was detected by RT-PCR. The potential of hPESCs to differentiate into 3 germ layers. Results The hPESCs in the two groups showed regular morphological changes and were not easily differentiated. There was no significant difference in morphology and amplification rate between the two groups. The hPESCs were successfully cultured in vitro for 15 generations. The hPESCs in both groups maintained the diploid karyotype 46, XX and pluripotency in normal women. The expression of Oct-4 mRNA in both groups was detected by RT-PCR. The embryoid bodies could be formed by in vitro differentiation. Teratomas with three germ layers were formed in nude mice. Conclusion The hFFs-CM feeder-free culture system can support hPESCs growth for a long time and maintain its undifferentiated state. We successfully established a hFFES-CM feeder that can not only effectively maintain the hPESCs proliferation, reduce animal-derived contamination, reduce the cost of culture, Large-scale application of hPESCs feeder-free culture system.