| Mesenchymal stem cells(MSCs) are multipotent stem cells that can self-renew and differentiate into osteocytes, adipocytes, chondrocytes,myocytes, and other cells. MSC-based transplantation is a promising therapeutic approach for tissue regeneration and repair. Therefore,understanding the molecular mechanisms underlying MSC differentiation is of paramount importance. Wild type p53- induce phospatase 1(Wip1) is a novel p53-dependent proto-oncogene involved in multiple biological processes including tissue homeostasis, DNA damage, aging,immunoregulation, and tumorigenesis. Recently, Wip1 has been shown to inhibit the proliferation and enhances the migration of bone marrow mesenchymal stem cells. However, the potential role of Wip1 in modulating the biological characteristics of MSC is still unclear. Here, we isolated compact bone derived MSCs from Wip1 knockout(Wip1-/-) mice to explore the effect of Wip1 gene on the biological function of MSCs and its mechanisms.We first isolated MSC from compact bone in Wip1-/- and Wip1 wild type(WT) mice, and then the morphology of MSC was observed by inverted microscope and the cell growth curve was analyzed by CCK8 method. The immunophenotype and cell cycle of the MSC derived from Wip1-/- and Wip1-WT mice was determined by flow cytometry and the induction ability was determined by different induction reagents including oil-red-O and alkaline phosphatase staining. To assess mineralization,deposit calcium in cultures was stained with silver nitrate by the method of von Kossa. The key transcription factor expression of adipogenic and osteogenic differentiation for murine MSCs was detected by quantitative real-time polymerase chain reaction(Q-PCR). T-cell proliferation bycoculture with Wip1 knockout mice MSCs was evaluated by lymphocyte transformation test(LTT) and mixed lymphocyte reaction(MLR).Furthermore, the immunoregulation ability of murine Wip1-/- MSCs in vivo was assessed by mouse tail skin flap allotransplantation.The results showed that the MSCs were isolated from Wip1-WT mice and Wip1-/- mice by cultivating the digested compact bone chips. These cells exhibited the typical fibroblast-like cells morphology; both MSCs were positive for Sca-1, CD29, CD105 and negative for CD31, CD34,CD45, and MHCII. These data suggested Wip1 deficiency had no significant effects on the morphology and cell surface antigen expression.Comparing with Wip1-WT MSCs, the growth ability of Wip1-/- MSCs was significantly decreased by CCK8 method. Flow analysis cell cycle was shown that the proliferation of Wip1-/- MSCs was blocked at G2 period. All of the MSCs had multi-lineage differentiation ability and were able to differentiate into adipocytes and osteoblasts. Wip1 depletion alters the adipogenic and osteogenic differentiation, resulting in a significant decrease in mature adipocyte and osteocyte formation as well as lower mRNA expression of C/EBP?, PPAR? after adipogenic induction and Runx2 and Osteocalcin for osterogenic induction. Functionally, Wip1-/-MSCs could suppress the allogeneic T cells proliferation in a dose-dependent manner. When Wip1-/- MSC coculture with dendritic cells(DCs), it could inhibit DCs to the mature state. Moreover, Wip1-/- MSCs leads to decline immunoregulation in vivo by the allogeneic mouse skin flap transplantation.Collectively, these results suggest that Wip1 is a novel regulator of MSC biological property and immunomodulatory function. This study will be clinically beneficial. |
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