Establishment of Human ES Cell on STO Fibroblast Cell Lines

초록

Embryonic stem (ES) cells should be cultured on feeder cells for the maintaining of its undifferentiated state. It is not exactly known what roles the feeder cell has in Co-culture with ES cells. Almost research teams that established human ES cells used the primary mouse embryonic fibroblasts (PMEFs) as a feeder cell for establishing human embryonic germ(EG) cells. There are many evidences that STO fibroblasts are as effective as PMEFs as a substrate for establishing ES sell lines. Therefore, here we used STO fibroblasts as a feeder cell for establishing human ES cell lines. Human embryos, produced by in vitro fertilization (IVF) for clinical purpose, were cultured to the blastocyst after institutional review board (IRB) approval. Inner cell masses (ICMs) were isolated and then cultured on mitotically inactivated STO fibroblast (about 7.5×104 cells/cm). Human ES-like colony appeared after 7 days of cilture. These colonies were mechanically dissociated and replated on new STO fibroblast feeder cells every 7~10 days. Human ES cells formed relatively flat, compact colonies that easily dissociate into single cells. Like undifferentiated primate ES cells and human EC cells, human ES cells expressed stage-specific embryonic antigens 4 (SSEA - 4), and alkaline phosphatase (AP). Human ES line retained a normal 46, XY karyotype after 5 months of culture and has now been passed continuously for mere than 6 months (24 passages). Embryoid bodies (EBs) formation was induced by suspension culture of dissociated ES cells and this Ebs incltded a wide variety of differentiated cell type. These results suggested that STO fibroblast was sufficiently useful to establish human ES cells.