Abstract:

BACKGROUND: Containing fetal bovine serum medium is used in the traditional culture and amplification method of human embryonic stem cells (hESCs), and this method relys on feeder layer cell culture and significantly limits culture formula of stem cells in vitro. In addition, the intervention of heterologous serum components significantly increases pathogen contamination and the probability of immune rejection.
OBJECTIVE: To elucidate the feasibility of serum-free medium mTeSR?1 on the hESCs long term cultured in vitro and to establish a platform of induced hESC differentiate into endothelial cells.
METHODS: Serum-free medium mTeSR?1 was applied to culture in vitro and amplificate hESCs line H9 by non- feeder layer cell dependent. After more than 40 times passage in vitro, the growth morphology of hESCs was observed by inverted microscope, and their phenotype was evaluated by immunofluorescence staining method. Moreover, a conditioned medium was utilized to induce hESCs line H9 to differentiate into endothelial cells. The phenotype and function of ESC-derived endothelial cells were assayed by immunofluorescence staining, quantitative RT-PCR, and low-density lipoprotein (LDL) uptaking experiment.
RESULTS AND CONCLUSION: mTeSR?1 medium can support hESCs line H9 long term amplification in vitro by non- feeder layer cell dependent and maintain its potential of undifferentiated stem cells. When the hESCs were cultivated under a conditioned medium with vascular endothelial cell supplementation, the cells were induced differentiation into H9 endothelial-like cells. These cells not only one of important surface markers of endothelia cells (kdr, pecam) and expressed CD31, but also uptake LDL, formed vascular-like structure during the differentiation. The system of culture and induced differentiation experiment provided can support the proliferation and differentiation behavior of ESCs.