Isolation, cultivation, and identification of embryonic stem cells of rhesus monkey / 第三军医大学学报

ABSTRACT

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.