Culture And Directed Differentiation Of Murine Embryonic Stem Cells Into Osteoblasts In Vitro

Objective:research systematically into the isolation,culture and direct differentiation to osteoblast of embryonic stem cells.Method: To obtain feeder layer of primary mouse embryo fibroblasts(PMEF), 13.5d pregnant KM mouse was chosen as a donor. After using mitomycin-C treated PMEF, they were used as feeder cells in media of DMEM for isolation ES cell colonies and for coculture with ES-D3 cells. We flushed blastocysts from pregnant uterus at p.c. 4.After 4 to 5 days,nest-like clones of inner cell mass appeared and were dispersed by 0.25% trypsine-0.02%EDTA.Then replate them onto feeder layer culture system (containing leukemia inhibitory factor) in order to passage and expand. Subsequently we identified the expanding cells by alkaline phosphatase staining,karyotype analysis and the formation of embryoid body which are typical characteristics of ESCs.The cells of 5-day embryoid bodies (EBs) derived from murine ES-D3 lineage were inoculated into each well of 6-well culture plate at 5×104/mL in DMEM. During the period from 14 to 21 days of the EBs-derived single cells attachment DMEM was supplemented with 50μg/mL ascorbic acid,50 mmol/Lβ- glycerophosphate,1μmol/L dexamethasone(group A), 50μg/mL ascorbic acid,50 mmol/Lβ- glycerophosphate,1μmol/L dexamethasone,10ng/mL bone morphogenetic protein-2(group B),and without inducer(group C), respectively, and at the 22th day the positive cells were characterized by the formation of discrete mineralized bone nodules that were stained with 1% alizarin red S, and the ratio of obstoblast differentiation were calculated and analyzed by variance analysis and multiple comparison.Result: Fibroblast cells grow very fast and attach in 30minuts, after 24 hour,it will extend to a large extent. ES cells from ICM proliferate to some extent. ES cells attach the bottom of the cultivate bottle. ES cells push away the trophoblast cells. The cells grows very fast and tightly packed in large nest and colonies in which it was difficult to recognize individual cells, each cell was small, round or fusiform, with large nucleus and minimal cytoplasm. We found that these cells aggregated to form colonies with a clear borderline between colonies and feeder layer cells.Colonies were alkaline phosphatase-positive staining and had normal diploid karyotype. What is more, there are more litle clones of ES cells or single ES cell around the big clone. During the period from 14 to 21 days of the EBs-derived single cells attachment DMEM was supplemented with inducers,the shape of ESC have been turning from round to fusiform. These cells were alizarin red-positive staining.Conclusion: 1.PMEF can be separated easily,obtained conveniently and the method of...