Characterization and culture of human embryonic stem cells

Objective To explore the in vitro maintenance and characterization of human embryonic stem cells(hESCs).Methods hESCs were cultured on feeder layer with ES culture medium,which consists of 20% Knockout Serum Replacement,Knockout DMEM and 10 ng/mL bFGF.Undifferentiated status of hESCs was identified by cell morphology,and the expressions of cell surface marker SSEA-1,SSEA-3 and TRA-1-60.G banding technique was employed for cell karyotype analysis.Pluropotency of cells were analyzed via in vitro embyoid body(EB) formation and in vivo terotoma formation.Results Most of cells showed undifferentiated properties in cell morphology and normal karyotype throughout extended culture periods.They maintained undifferentiated status with positive immunoreactivity to SSEA-3,SSEA-4 and TRA-1-60.in vitro EB formation and in vivo teratoma formation demonstrated the pluripotency of human ES cells.Conclusion The fundamental requirement to hESCs for research and clinical application were their undifferentiated status and pluropotency in culture.Our result demonstrated their potential for these purposes.