Study On The Differentiation Of Embryonic Stem Cells Into Obsteoblsts In Vitro

Pluripotent embryonic stem cells(ES)have the potential to differentiate to all kinds of cells and might represent a useful source for tissue engineering.in this paper we show that differentiation of ES cells toward the obsteoblast lineage can be enhanced by supplementing serum -containing media with AA , βGly and Dex / 1,25-OH₂VD3 or coculture with fetal murine osteoblasts.our work would be useful to study how the obsteoblasts oringate and lay the ground for establishing a stable system that induce ES cells to differentiate into the obsteobasts. The results gained were as follows:1. The embryonic stem cells — ES-D₃ were cultured at least for a month in vitro and stem cell characteristic identification was carried out. After continuous proliferation for more than 10 passages, ES-D3 displayed typical murine ES cell morphology and maintained several crucial ES cell features, including positive staining for AKP as well as over 88.1% of normal XY dipliod karyotype.2. Osteoblast were isolated from fetal mouse cranial bone with method which based on combination of tissue block and mixed enzyme digestion. AKP and Alizarin red staining for the cultured cells were performed to determine the obsteoblast activity and got a positive resultgrowth curve shows that the cells has a regular growth habit.3. After ES-D3 formed embryoid body in suspension culture for 3～4 days, cells were transferred to cell culture dishes to allow attachment and cultured in mineral medium contained DMEM with 10%fetal bovine serum and supplements as 1,25-OH₂VD₃ (5×10^-8 mol/L), βgly (10 mmol/L) and ascorbic acid (50 mg/L) (B group)/dexamethasone (1×10^-8 mol/L), βgly (10 mmol/L) and ascorbic acid (50 mg/L) (C group).on 25d, Mineralized cells from osteogenetic differentiation of ES-D3 can form bone nodules which could be clearly identified by Alizarin Red staining. Experiment group formed more bone nodules than control group.and also, bone nodules of B group amount to 60±5 (P<0.05), bone nodules of C group amounts to 68±9 (P<0.05) .we can draw the conclusion from this results that under the condition metion above 1,25-OHVD3 has an enhanced effect on differentiation of ES-D3 into obsteoblasts.group D, ES-D3 co-culture with fetal murine osteoblasts, bone nodules amount to 47±2 (P<0.05) group A,control group, bone nodules add up to 20±8 (P<0.05) .