Up-regulated Immunogenicity During Osteogenesis Derived From MSCs And Dedifferentiated-MSCs

Objective: To compare the osteogenic differentiation potential between mesenchymal stem cells(MSCs) and dedifferentiated-MSCs(De-MSCs), and to investigate the alteration of immunogenicity of MSCs and De-MSCs and involved molecular mechanisms during osteogenesis.Methods: MSCs were isolated from human placenta digestied by collagenase. De-MSCs were obtained by withdrawing the osteoblast-inducible factors in cell culture medium. After being identified, MSCs and De-MSCs were used for subsequent experiments.(1) To compare the osteogenic differentiation efficiency between MSCs and De-MSCs, the proliferation capacity of MSCs and De-MSCs during osteogenesis for 7 days was detected by Cell count kit-8(CCK8). Upon osteogenic induction for MSCs and De-MSCs for 7 days, Ob-MSCs and Re-MSCs were acquired respectively and osteogenic related-gene expressions of cells were detected by using real-time quantitative PCR(q-PCR, SYBR Green method) assay. ALP staining of MSCs and De-MSCs were conducted to compare the osteogenic differentiation efficiency.(2) To explore the alteration of immunogenicity of MSCs and De-MSCs during osteogenesis in vitro, the expressions of costimulatory molecules on MSCs,Ob-MSCs,De-MSCs and Re-MSCs were detected by FCM. All the cells were cultured with T cells and the proliferation of T cells was determinated by CCK8 method.(3) Mitomycin C-treated MSCs,Ob-MSCs,De-MSCs and Re-MSCs were implanted subcutaneously into BALB/c mice. After implanted for 5 days, the peripheral blood was obtained from orbital veins of immunized mice, and the activation of immune cell populations in PBMCs was detected by FCM. After implanted for 7 days, the activation of immune cell populations in splenocytes of immunized mice was detected by FCM.(4) The expressions of mi RNAs which contribute to regulate osteogenesis(mi R-20 a and mi R-29b) and immunogenicity(mi R-125 b and mi R-296) of MSCs and De-MSCs were detected by using q-PCR(Taqman probe method) assay.Results:(1) De-MSCs had similar cell morphology, stem cell markers and multilineage differentiation potential compared with MSCs. The results of CCK8 assay demonstrated De-MSCs had higher proliferation efficiency than MSCs during osteogenic differentiation for 7 days. The results of q-PCR showed that the expression levels of osteogenic related genes BMP2, COL1 and Runx2 in Re-MSCs were higher than that in Ob-MSCs. The ALP staining images proved De-MSCs had higher osteogenic potential compared to MSCs.(2) Data of FCM displayed that the positive costimulatory molecules had up-regulated expression and the nagative costimulatory molecules had down-regulated expression during the osteogenesis of MSCs and De-MSCs in vitro, and the phenomenon was more obvious in De-MSCs. The results of mixed lymphocyte reaction(MLR) indicated that MSCs and De-MSCs inhibited T-cell proliferation, while the degree of inhibition was reduced after osteogenic differentiation. T cell proliferation efficiency of Re-MSCs group was higher than that of Ob-MSCs group.(3) The results of PBMCs revealed the effect on activating T cells, macrophages, dendritic cells and B cells in PBMCs of Ob-MSCs and Re-MSCs immunized mice higher than that of MSCs and De-MSCs immunized mice. Meanwhile, the activated immune cell populations of Re-MSCs group were more than that of Ob-MSCs group. Intriguingly, the trend was more obvious in the spleen cells of immunized mice.(4) mi R-20 a and mi R-29 b were associated with osteogenesis, and mi R-125 b and mi R-296 were related with immune regulation. Data of q-PCR showed that mi R-20 a and mi R-125 b expressed down-regulatedly during osteogenic differentiation of MSCs and De-MSCs, and the expression of mi R-29 b and mi R-296 up-regulated during osteogenesis. The degree of down-regulation and up-regulation of mi RNAs in De-MSCs was higher than that in MSCs.Conclusions: Compared with MSCs, De-MSCs had similar cell morphology, stem cell markers and multilineage differentiation potential and had higher osteogenic differentiation potential. MSCs and De-MSCs had low-immunogenicity, but the immunogenicity up-regulated upon the osteogenic differentiation. Meanwhile, the increased degree of De-MSCs was higher than that of MSCs. The up-regulated immunogenicity of MSCs and De-MSCs during osteogenesis was associated with mi RNAs. In summary, De-MSCs could substitute MSCs for appling in tissue engineering and regenerative medicine, but the alteration of immunogenicity during differentiation which had influence on the graft effects should be fully taken into account.