In Vitro Differentiation Of Mouse Gingival-derived Mesenchymal Stem Cells To Epithelial-like Cells

Objective:Rapid re-epithelization of would surface is the key to reduce scar forming and improve repair effect of large-sized epithelial defect. Under certain condition, bone marrow mesenchymal stem cells (BMSCs) can differentiate into epithelial-like cells, kidney epithelial cells, intestinal epithelial cells, corneal epithelial cells, et al. However the more subculture times of BMSCs are, the more problems arise, including decline of proliferation rate, abnormity of cell morphology, decline of differentiation capability, genetic change, et al, which restrict large scale application of BMSCs. Lately, as a new kind of stem cell, gingiva-derived mesenchymal stem cells (GMSCs) have many special characters that BMSCs don’t have, including easiness to gain, high proliferation rate,stable cell characteristics and ageing slowly. Being injected into mouse tail vein, GMSCs can locate in both epidermal layer and subepidermal layer, which suggests that GMSCs have migration ability and may differentiate into epithelial-like cells through mesenchymal-to-epithelial transition(MET) to repair defect. There is no report about differentiation to epithelial-like cells and migration mechanism of GMSCs, so we decide to induce the differentiation of mouse GMSCs using cytokines, observe the influence of IL-6 or IL-10 to the differentiation and evaluate whether SDF-1 is one kind of effective chemotactic factor for mouse GMSCs.Methods:1. Mouse gingival cells were isolated and cultured using enzymolysis method and the mesenchymal stem cells’markers were detected by flow cytometry including CD11b, CD45, CD34, CD31, CD105, CD140, CD29, CD44 and sca-1.2. After culturing GMSCs for 14 days in inducing culture medium, we observed the cell morphology and detected CK18 and vimentine using immunohistochemical and immunofluorescence staining.3. There were 4 groups including control group, induction group, IL-6 induction group and IL-10 induction group according to different culture medium. CK-18 and vimentine were detected using immunohistochemical staining and immunofluorescence assay and immunofluorescence staining positive cell were counted after culturing GMSCs for 14 days. The proliferation curve of 4 groups were made according to the A value of the cell proliferation assay.4. Corning transwells were used to evaluate mouse GMSCs’migration. The experimental group:100ng/ml SDF-1α+2%FBS+DMEM medium in lower chambers,2%FBS+DMEM medium in upper chambers; the control group: 2%FBS+DMEM medium in both upper and lower chambers. GMSCs were inoculated in upper chambers, then the cells migrating to the lower surface of transwell membrane were counted after 22 hours.Results:1. Mouse GMSCs were successfully cultured. They were typical fibroblast-like morphology, and the cell markers they expressed were the same as the makers of mesenchymal stem cells.2. Induced for 14 days, GMSCs were polygonal like paving-stone, and CK18 and vimentine were positively stained by immunohistochemical and immunofluorescence staining, while CK18 were negative and vimentine were positive in control group.3. CK18 and vimentine were positive in induction group, IL-6 induction group and IL-10 induction group, and there was no obvious difference in staining degree and propotion of positive cells among the 3 groups (P>0.05).CK18 was negative and vimentine was positive in control group. The propotion of CK18 positive cells was obviously different between experimental groups and control group (P<0.01),while The propotion of vimentine positive cells was similar among each group(P>0.05). The cell proliferation assay showed that A value was obviously different between experimental groups and control group (P<0.05) on the lst,3nd,5th,7th,9th day, A value was obviously different between IL-6 induction group and induction group (P<0.05) on the 3nd,5th,7th,9th,llth day, and A value was similar between IL-10 induction group and induction group (P>0.05) on each day except the 3nd day.4. The cells migrating to the lower surface of transwell membrane were obvious more in experimental group compared with control group.Conclusion:We have isolated and cultured mouse GMSCs successfully, and firstly report that mouse GMSCs can express CK18 induced by ATRA and EGF in certain concentration, which suggests that GMSCs have the possibility to differentiate into epithelial-like cells. IL-6 or IL-10 has neither promotion nor inhibition effect to the above-mentioned epithelial differentiation of GMSCs. To some extent, our report has offered theory basis to epithelial defect repair using GMSCs as new seed cells. Otherwise we certify that SDF-1 can induce mouse GMSCs migrating, which can be used to improve GMSCs’homing ability and thus can enhance cells’epithelial defect repair effect.