Study On Oct-4 Methylation In Induced Differentiation Of Bone Mesenchymal Stem Cells

Objective: Isolated and cultured mouse bone marrow-derived mesenchymal stem cells (mBMSCs) in vitro were induced to the osteoblasts. The Oct-4 expression and methylation status were detected in mBMSCs before and after induction in order to provide the research guideline for the further study of Oct-4 function in proliferation and differentiation of mBMSCs.Methods: mBMSCs had been isolated and cultured by a method of adherent bone marrow-wide screening in vitro. They were also identified by immunocytochemical staining of CD29, CD44, c-Kit and CD34. mBMSCs were induced directionally to differentiate into osteoblasts in vitro by dexamethasone, vitamin C andβ?sodium glycerophosphate. Alkaline phosphatase (AKP) staining and alizarin red staining were used to identify mBMSCs transformation phenotype, with in vitro cultured osteoblasts as positive control. Using RT-PCR and indirect immunofluorescence, levels of Oct-4 expression were detected in mBMSCs before and after induction. The methylation status of Oct-4 gene in mBMSCs was explored by a methylation-specific PCR before and after induction.Results: After 24h inoculation, most of mBMSCs were adherent and spreading out. The adherent cells increased their numbers by proliferation, with spindle-shaped or fibroblast-like morphology after 48 h. Passage cells were accelerated its proliferation and showed vortex-like growth, with a more homogeneous morphology. Immunocytochemical staining and indirect immunofluorescence showed that CD29, CD44, c-Kit had a positive staining, while CD34 was negative expression in 3rd generation of mBMSCs. After 4 day's osteogenic-induction mBMSCs changed their shape, and then cells gradually increased their size, changed their long-spindle shap to polygon. After 10 day's induction, the majority of mBMSCs changed their shape apparently, and both AKP and the alizarin red were positive, similar to that of osteoblasts. The RT-PCR and the indirect immunoinfluorescence results also showed that 3rd generation of mBMSCs had Oct-4 expression. After 10 day's indution mBMSCs had no Oct-4 expression, similar to that of osteoblasts. methylation-specific PCR showed that the promoter's CpG island of Oct-4 gene appeared methylation status in mBMSCs after 10 day's induction, similar to that of osteoblasts. But in non-induction mBMSCs, Oct-4 gene has not been the methylation status.Conclusion:1. Successful to establish the culture system of mice osteoblasts in vitro in this laboratory.2. Successful to isolate and culture mBMSCs, and induce those cells to osteoblasts and observe that Oct-4 expression in committed induction of differentiation was down-regulated.3. Firstly observed that the methylation status of Oct-4 gene in committed induction of differentiation of mBMSCs had been changed, which suggest the down-regulation of Oct-4 expression in mBMSCs after induction might be due to methylation status of this gene.