Acta Academiae Medicinae Sinica

Abstract:

To investigate the methylation Oct4 in orientationinduced differentiation in bone marrow mesenchymal stem cells Methods Mice BMSCs were isolated and purified from bone marrow by adherent culture，and then identified by morphology and immunocytochemistryMouse osteoblastic cells were cultured by bone fragments inoculation，and then identified by alkaline phosphatase（AKP）staining and alizarin red stainingBMSCs were induced to differentiate into osteoblasts in vitro Indirect immunofluorescence staining and reverse transcription polymerase chain reaction（RTPCR）were used to detect the expressions of Oct4 in BMSCs before and after inductionThe methylation status of Oct4 gene in mouse BMSCs was explored by a methylationspecific PCR before and after induction Results The isolated mice BMSCs massively proliferated in vitro and formed cell colones with uniform morphologyPositive expressions of CD29，cKit，and CD44 and negative expression of CD34 were found in the isolated cellsAfter 10 days[DK]’[DK] induction，both AKP and the alizarin red were positive in cells and osteoblastic cells isolated from mice skull bonesThe indirect immunoinfluorescence staining and RTPCR also showed that the Oct4 expression in the directed differentiation of mouse BMSCs was downregulatedThe CpG island of Otc4 gene promoter in mouse BMSCs became methylated during the induced differentiation Conclusions Mice BMSCs and osteoblasts were successfully cultured in vitro in this studyOct4 may be involved in the maintenance of adult stem cell pluripotencyThe downregulated expression of Oct4 gene in mouse BMSCs during the directed differentiation may contribute to the methylation of CpG island in Otc4 gene promoter

Key words: Oct4, bone marrowderived mesenchymal stem cells, differentiation, methylation