Study On Culture And Transdifferentiation Into Corneal Epithelial Cells Of Bone Marrow Mesenchymal Stem Cells In Vitro

Aim: To isolate, culture, and multiply mesenchymal stem cells (MSCs) from bone marrow aspiration, then induce MSCs with special culture medium and detect the expression of corneal epithelial cell markers to explore the possibility of its transdifferentiating into corneal epithelial cells in vitro. That will give a new choice for theraping the functional disorder or deficiency of corneal epithelial cells and limbal stem cells to clinical oculist. Methods: Bone marrow aspiration samples were obtained from 7 aborted fetuses 3 to 5 months old and 7 adult human donors 32 to 65 years old without severe hematopietic diseases. They were added slowly to 4ml to 6ml Ficoll solution (density 1.077g/ml) and centrifugated 25 minutes at 2500rpm. Subsequently, the grey layer solution was aspirated out that contains mononuclear cells. Cells were washed with PBS twice. Then cells derived from fetal bone marrow were seeded with a concentration of 5×10⁵ cells/ml and cells derived from adult human bone marrow were seeded with a concentration of 1×10⁶ cells/ml in DMEM/F12 supplemented with 10% fetal bovine serum (FBS) in a hole of 12 holes culture plate. Cultures were maintained at 37℃in a humidified atmosphere containing 5% CO₂. The medium was changed first time after 48 hours, and once every 3 days thereafter. When the cultures reached nearly 90% of confluence, cells were trypsinized by 0.25% trypsin and 0.02% EDTA, collected, resuspended, and passaged with a concentration of 4×10³ cells/cm² in DMEM/F12 supplemented with 10% FBS in a 25cm2 culture flask. The medium was changed every 3 days. Cell surface antigens which contain CD29, CD44, CD166, CD105, CD34, CD45, HLA-DR were detected with flow cytometry. The passage 5 MSCs were seeded with a concentration of 2×104 cells/ml in a hole of 12 holes culture plate containing cover glass. The following 5 different culture media were added to every two holes: a. DMEM/F12, 10 % FBS; b. DMEM/F12, 10 % FBS, 5μg/ L EGF; c. DMEM/F12, 10% FBS, 10μg/ L EGF; d. DMEM/F12, 10% FBS, 15μg/ L EGF; e. DMEM/F12,10% FBS, 50% conditional medium, 10μg/ L EGF. The culture media were changed every third day. After 30 days, cells were stained for cytokeratin AE1 and cytokeratin AE5 with immunohistochemistry. Results: A small percentage of isolated cells were adherent to the flasks and grow as typically fibroblastic shape 48 hours after plating. Primary cells derived from fetal bone marrow reached 90% of confluence in 10 to 14 days. Passaged cells derived from fetal bone marrow reached 90% of confluence in 7 to 10 days. To contrast, Primary cells derived from adult human bone marrow reached 90% of confluence in 20 to 25 days. Passaged cells derived from adult human bone marrow reached 90% of confluence in 10 to 15 days. Its reproductive activity was very good. Cells can keep a rapid proliferation until 10th passage. The isolated cultured adherent cells derived from both fetal bone marrow and adult human bone marrow typically expressed CD29, CD44, CD166 and CD105, while CD34, CD45, HLA-DR of them were negative. Results of cytokeratin AE1 immunohisto-chemistry stain show: the first group was negative, the second group was light positive, the third group and the fifth group was moderately positive, the forth group cells were strongly positive with brown plasma. Results of cytokeratin AE5 immunohistochemistry stain show cells of every group were lightly stained in cytoplasm. Conclusions: We succeeded in isolation, cultivation and multiplication human bone marrow MSCs. Content of MSCs in fetal bone marrow is more abundant than that in adult human bone marrow, and reproductive activity ofMSCs derived from fetal bone marrow is stronger than that of MSCs derived from adult human bone marrow. EGF can cause MSCs synthetize cytokeratin, so it is possible that MSCs can be induced to transdifferentiate into corneal epithelial cells with optimal culture conditions in vitro. EGF with the concentration of 5μg/ L to 15μg/ L can all induce MSCs to transdifferentiate into corneal epithelial cells, and the effectiveness of its induction enhanced gradually in the wake of its concentration increased. 50% conditional media prepared with cultured medium of adult corneal limbal epithelial cell cannot induce MSCs to transdifferentiate into corneal epithelial cells.