[Study on differentiation of embryonic stem cells into osteoblast in vitro inducing by 1,25(OH)2VD3].

Objective: To investigate the effect of 1,25(OH)2VD3 on differentiation of embryonic stem cells (ESCs) into osteoblasts.

Methods: Osteoblasts were isolated and cultured from calvarium of 2-day-old Kunming white mice, embryoid bodies (EBs) were prepared with modified zur Nieden method. EBs were divided into 4 groups according to different mediums: group A, as the control group, in which EBs medium contained no leukemia inhibitory factor; group B, in which EBs medium contained supplements of Vitamin C (VC, 50 microg/mL) and beta-glycerophosphate (beta-GP, 50 mmol/L); group C, in which EBs medium was the same as that of group B and 5 x 10(4) osteoblasts of 3rd passage were seeded into each well; group D, in which the medium contained supplements of VC (50 microg/mL), beta-GP (50 mmol/L) and 1,25(OH)2VD3 (4 x 10(-9) mol/L), and 5 x 10(4) osteoblasts of 3rd passage were seeded into each well. The ALP activity was determined by ALP reagent kit every 5 days. The RQ-PCR was performed to measure the mRNA expressions of osteocalcin (OCN). Alizarin red S staining was performed to count the bone nodules.

Results: The expression of ALP witnessed no obvious change in each group within 5 days after adherence of EBs, but increased gradually after 5 days. The expression of ALP in group D reached the peak at 20 days. Red nodules with clear outline and different sizes were evident by microscope. Alizarin red S staining testified the number of bone nodules in groups A, B, C and D was 20 +/- 8, 18 +/- 5, 31 +/- 1 and 50 +/- 1, respectively, indicating significant differences between groups C, D and groups A, B (P < 0.05), no significant difference between group A and group B (P > 0.05), and a significant difference between group C and group D (P < 0.05). The result of RQ-PCR showed that the mRNA expressions of OCN in groups A, B, C and D was 10.18 +/- 1.17, 20.29 +/- 1.03, 18.84 +/- 4.07 and 32.15 +/- 5.23, respectively, indicating significant differences between groups C, D and groups A, B (P < 0.05), no significant difference between group A and group B (P > 0.05), and a significant difference between group C and group D (P < 0.05).

Conclusion: The combined action of 1,25(OH)2VD3 (4 x 10(-9) mol/L), VC, and beta-GP can effectively promote the differentiation of the ESCs-derived osteoblasts.