人孤雌胚胎干细胞在人包皮成纤维细胞饲养层上的生长状态

人孤雌胚胎干细胞(human parthenogenetic embryonic stem cells,hPESCs)体外培养常需饲养层的支持以保持干细胞特性.通过原代培养获得人包皮成纤维细胞(human foreskin fibroblasts,hFFs)并将其制备成饲养层,使hPESCs在hFFs上进行体外培养及传代.倒置显微镜下观察hPESCs的生长状态,采用碱性磷酸酶(alkalinephosphatase,AKP)检测、核型分析和体内分化实验研究hPESCs的生物学特性及分化潜能,以探索hFFs能否长期支持hPESCs的生长并维持其未分化状态.经原代培养成功获得了hFFs,通过形态学观察和免疫细胞化学染色鉴定符合成纤维细胞的生物学特性;在hFFs上生长的hPESCs克隆形态规则,不易分化;已成功在体外培养20余代,hPESCs仍能够保持基本生物学特性和正常核型,在裸鼠体内可形成含有3个胚层组织成分的畸胎瘤.作为人源性饲养层,hFFs可长期支持hPESCs的生长并维持其未分化状态.

Growth of Human Parthenogenetic Embryonic Stem Cells on Feeder layer Derived from Human Foreskin Fibroblasts

Human parthenogenetic embryonic stem cells(hPESCs) usually need the sustainment of feeder layer to keep stem cells characteristics in vitro culture.Human foreskin fibroblasts(hFFs) were obtained by primary culture and prepared into the feeder layer of hPESCs.hPESCs were cultured and passaged on hFFs feeder layer in vitro.Morphology of cell growth was observed with inverted microscope.Alkaline phosphatase activity analysis,karyotype analysis and differentiation experiments in vivo were used to detect the biological char-acteristics and differentiated potency of hPESCs.Thereby whether hFFs can sustain hPESCs and keep hPESCs undifferentiated for long was explored.hFFs were obtained successfully by primary culture.Then hFFs were verified by morphology observation and immunocytochemical stain.Morphous of hPESCs on hFFs feeder layer is regular and it is not easy for hPESCs to differentiate.After subcultured successfully for more than 20 passages in vitro,hPESCs still maintained biological characteristics and normal karyotype.hPESCs can develop into teratomas containing three germ layers in nude mice.As a kind of feeder layer derived from human,hFFs can sustain the growth of hPESCs and keep hPESCs undifferentiated for long.