Liang Rui, Wang Zhiqiang, Chen Tianxing, Zhu Jing, Zhu Shu, Li Ying, Yang Long, Zhu Baosheng
Department of Pathology, 'Genetic Diagnosis Center, the First People's Hospital of Yunnan Province, Kunming Yunnan, 650032, PR China.
Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2013 May;27(5):559-64.
To establish a safe, effective, and economic feeder'-free culture system which is suitable for the culture of human parthenogenetic embryonic stem cells (hPESCs) in vitro.
hPESCs were cultured with mTeSR 1 medium (control group) and human foreskin fibroblasts-conditional medium (hFFs-CM) (experimental group). The growth status of hPESCs in both feeder-free culture systems were observed with inverted microscope. Alkaline phosphatase (ALP) analysis and karyotype analysis were used to study the biological characteristics of hPESCs. The expression of hPESCs pluripotent marker Oct-4 was analyzed by RT-PCR. Differentiation experiment in vivo and in vitro was applied to observe the differentiation potential of hPESCs into three germ layers.
hPESCs had regular morphology with difficulty in differentiation in both culture systems. No obvious difference was observed in morphology and expansion speed of hPESCs between 2 groups. After subcultured for 15 passages in vitro, hPESCs in 2 groups could maintain normal female diploid karyotype 46, XX and pluripotency. The expression of Oct-4 mRNA was positive in 2 groups. hPESCs in 2 groups could form embryonic body in differentiation experiment in vitro and could develop into teratomas containing three germ layers in nude mice.
Feeder-free culture system of hFFs-CM can sustain the growth of hPESCs and keep hPESCs undifferentiated state for long. A feeder-free culture system of hPESCs is successfully established, which can support the growth of hPESCs, reduce the contamination from animals, decrease the cost of culture, and satisfy the clinical large-scale application.
建立一种安全、有效且经济的无饲养层培养体系，适合人孤雌生殖胚胎干细胞（hPESCs）的体外培养。
用mTeSR 1培养基培养hPESCs（对照组）和人包皮成纤维细胞条件培养基（hFFs-CM）（实验组）。用倒置显微镜观察hPESCs在两种无饲养层培养体系中的生长状态。采用碱性磷酸酶（ALP）分析和核型分析研究hPESCs的生物学特性。通过RT-PCR分析hPESCs多能性标志物Oct-4的表达。进行体内和体外分化实验以观察hPESCs向三个胚层的分化潜能。
在两种培养体系中，hPESCs形态规则，分化困难。两组hPESCs的形态和扩增速度无明显差异。体外传代15代后，两组hPESCs均可维持正常的女性二倍体核型46, XX及多能性。两组Oct-4 mRNA表达均为阳性。两组hPESCs在体外分化实验中均可形成胚体，在裸鼠体内可发育成含有三个胚层的畸胎瘤。
hFFs-CM无饲养层培养体系可维持hPESCs生长并使其长期保持未分化状态。成功建立了hPESCs无饲养层培养体系，该体系可支持hPESCs生长，减少动物源性污染，降低培养成本，满足临床大规模应用需求。
