The Effect Of Endothelial Progenitor Cells On Stemness Of Mesenchymal Stem Cells

Objective:To verify the EPCs to promote the self-renewal, in vivo and in vitro, so as to prove that the EPCs may possibly be the niche cell of MSCs.Methods: C57BL/6J mice as a cells source. MSCs and EPCs were isolated and identified by FACS. GFP and PKH26was used to label the MSCs and EPCs respectively. MSCs and MSCs were co-cultured with EPCs to observe the amount of CFU-Fs. Five green fluorescent protein (GFP)-positive (GFP+) clonal strains were collected by cloning cylinder method and transplanted to the femoral marrow cavity of Rag2-/-, in the form of MSCs, MSCs with EPCs and MSCs with PBS.8weeks later, mice were sacrificed and peripheral blood was collected. GFP+MSCs were sorted by FACS, and then further applied to CFU-F assay to form the secondary CFU-Fs. he GFP+MSCs also detected from the liver and lung frozen slides under immunofluorescent microscope.Results:The MSCs and EPCs were successfully isolated and identified, and labeled by GFP and PKH26, respectively. In the vitro study, it was found that the CFU-Fs of MSCs from co-cultured with EPCs system were more than that of the single cultured of MSCs (P <0.05). In the vivo, the Secondary CFU-Fs of the mice injected with the MSCs with EPCs were more than that of the injection of single MSCs. For mice received MSCs alone, few GFP+MSCs were detected. However, much more GFP+MSCs were detected in the animals received MSCs with EPCs. The difference in quantities of GFP+MSCs was statistically significant (P<0.05)Conclusion:Endothelial progenitor cells as a possible component of stem cell niche to promote self-renewal of mesenchymal stem cells Objective:To explore the role of FN and its receptor integrin α5β1in the cohesion of MSCs and EPCs in the niche.Methods: ELISA was used to examine the expression of FN in the culture medium. Immunofluorescence was used to examine the FN expression of the two cell lines. FN-siRNA to knock down the expression of FN and further to observe the phenomenon of MSCs and EPCs. RT-PCR and Western-blot to identify the expression of FN of the two cells interfered by the opposite serum-and factors free medium.Results:Both MSCs and EPCs were secreted FN to to the culture medium in the way of autocrine model. Immunofluorescence results showed that both of the MSCs and EPCs expressed FN in the cytoplasm and cell membrane. Adhesion was observed of the two cell lines. And adhesion was not occurred when the FN was knocked down by FN-siRNA. The RT-PCR results showed that the MSCs cultured LG-DMEM medium up-regulated the FN mRNA expression of EPCs, and the EPCs cultured EBM-2medium up-regulated the integrin α5mRNA expression of the MSCs.Conclusions:The fibronectin (FN) of EPCs activated the integrin α5β1of MSCs, and further mediated the cohesion of the two cells in the niche. Objective: to explore the Wnt/β-catenin signaling pathway that may involves in the proccess of EPCs to promote the self-renewal of MSCs.Methods: CFU-F assay was employed to examine the self-renewal of MSCs in the following conditions:normal culture, cultured with EPCs culture-medium, cultured with Wnt/β-catenin signaling pathway inhibitor XAV939. CFU-Fs was counted. RT-PCR and Western-blot was used to detect the expression of cystosolic β-catenin mRNA and protein.Results:the CFU-Fs of EPCs cultured medium was more than that in the normal cultured medium (P<0.05), and the CFU-Fs in culture medium with XAV939was less than that in the normal. RT-PCR showed that the EPCs cultured medium increased the β-catenin mRNA expression of MSCs. And the western-blot indicated that the cystosolic β-catenin protein expression also enhanced.Conclusion:EPCs may through the activation of Wnt/β-catenin signaling pathway to promote the self-renewal of MSCs.