Study On Differentiation Of Human Bone Marrow Mesenchymal Stem Cells Into Limbal Stem Cells In Vitro

Objective:To explore the feasibility of differentiation of human bone marrow mesenchymal stem cell into ilmbal stem cells in vitro.Methods:Human bone marrow derived mesenchymal stem cells were isolated by gradient density centrifugation and cultured in a total medium with LG-DMEM/F12 containing 10%FBS. cell phenotype were detected by flow cytometry. limbal stem cells were harvested by tissue cultivation after digestion and cultured in a medium with HG-DMEM/F12 containing 20%FBS,cell core antigen were detected by flow cytometry. Subcultured HBMSCs of passage 4th were seeded on the bottom of transwell culture plate at the density of 5 X 104/ml. The HBMSCs were divide into tow groups in the second passage. In the experiment group, Subcultured LSCs of passage 2rd were seeded on the upper tier of transwell culture plate at the density of 5 X 104/ml and cocultured for 10days, using a special medium, which containing 90%LG-DMEM/F12 and 10%FBS,and supplemented with 10μg/ml EGF, 10ng/ml bFGF,1μg/mLPS. On the other hand,none of inductive agents were addde in the control group , BMMSCs were cultured in the medium contained of 90% LG-DMEM/F12 and 10% FBS. Morphological observations were performed with phase contrast microscope.Results:Higher density purity of BHMSCs could be isolated and cultured by combining gradient centrifugation with plastic adherence. The typical morphology of HMSCs was with long spindle shape, little volume and large caryoplasmic ratio. In addition, most of them arrayed in whirlpool. And Subcultured HBMSCs of passage 4th express CD29(96.78%), CD44(96.23%), CD105(91.45%), CD166(81.70%), while CD34(2.34%), CD45(3.92%) and HLA-DR(1.11%) in flow cytometry analysis.LSCs were successfully cultured in vitro. Cells were shown in shape of round, ellipse and polygon. Cell body was lucency ,Cells were arranged in inlay and fuse in film after 2 weeks. Subcultured LSCs of passage 2rd expressP63 (83.35%)in flow cytometry analysis.Part MSCs were shown in ellipse,round and part were fusiform after cocultured for 72 hours. When the induction of P3 MSC in exoteric-microenviroment for 10 days, Cells were polygon and round ellipse in bulk, and only a small part were fusiform. In the control group,cell was with long spindle shape.Flow cytometry analysis show that P63 was weakly positiv (7.16±0.56%)in the experiment group, P63 were negative (0.74±0.49%)in the control group.Conclusions:The HBMSC showed the tendency of induction from HBMSCs into LSCs -like cells in exoteric-microenviroment with LSCs by the intervention of LPS and cytokines...