猕猴胚胎干细胞的分离培养和鉴定

目的从体外培养成熟囊胚中分离并鉴定猕猴胚胎干细胞(embryonic stem cell,ES cell).方法猕猴卵母细胞经体外成熟培养、体外受精和早期胚胎体外成熟培养后,获得猕猴囊胚.当囊胚由透明带自然孵出后,用细玻璃针剥离囊胚中的内细胞团(inner cell mass,ICM)并与饲养细胞进行共培养.由ICM分离,培养并鉴定胚胎干细胞集落.结果由4只FSH超排猕猴中共取得92个处于GV期的猕猴卵母细胞,选取其中的22个用HECM-10培养基培养后,获得6个高质量的囊胚,由此6个囊胚中分离得到3个内细胞团,并由此最终获得1株猕猴ES细胞,即RS5细胞.RS5细胞具高比例核/质比,核仁多,其细胞集落边缘平整,其内各单个细胞清晰.经约5个月的连续传代后,仍保持了正常二倍体的核型,其染色体数目为42条.碱性磷酸酶细胞组织化学染色为阳性,说明RS5细胞为未分化态的胚胎干细胞.经高密度和长时间培养后,RS5细胞可进一步分化为多种类型细胞.结论 RS5细胞株具有自我更新能力和多分化潜能,属于胚胎干细胞.

Isolation, cultivation, and identification of embryonic stem cells of rhesus monkey

Objective To isolate and identify embryonic stem (ES) cells of rhesus monkey from mature blastocysts cultured in vitro . Methods Rhesus monkey blastocysts were obtained after in vitro maturation of oocytes, fertilization and maturation of early embryos. After the blastocysts were hatched naturally from the zone pellucida, the inner cell mass (ICM) was dislodged from blastocysts using the sealed end of a finely drawn Pasteur pipette and co cultured with the feeder cells. The ICM, resembling embryonic stem cell colony, was isolated, cultured, and identified. Results A total of 92 normal GV oocytes were obtained from 4 FSH prime rhesus monkeys. Six blastocysts with high quality were selected from 22 oocytes after culture in HECM 10 medium. One of the rhesus monkey ES cell lines, i.e. RS5 cell line, was finally isolated from 3 of the inner cell mass isolated from the 6 blastocysts. The RS5 cells presented a high nucleus/cytoplasm ratio, prominent nucleoli, and flatter colonies with individual and distinct cells. After successive passages for 5 months passage, the RS5 cells remained a normal karyotype, i.e. 42 of chromosomes and alkaline phosphatase (AP) positive, which meant the RS5 ES cell colony remained undifferentiated. After high density culture for a longer time, the ES cell could differentiate into multi types of cells. Conclusion The RS5 cell line has the ability to self renew and potential to differentiate. Thus, RS5 cell line belongs to embryonic stem cells.