Preliminary Study On Human Bone Marrow Mesenchymal Stem Cells Differentiation Into Corneal Epithelial-like Cells

Objective To establish a method of culturing human bone marrow mesenchymal stem cells(MSC) in vitro,and observe their biologic features.To investigate the differentiation potential of transdifferentiation of MSC into corneal epithelial cells,and to discuss the plasticity that make MSC be the seed cell in corneal tissue engineering.Methods MSC were isolated and purified by whole bone marrow adherence method and passaged in vitro.The cells were identified by flow cytometry.The passaged MSC were planted on fresh corneal stroma.The expression of CK12 on MSC,which is a marker for corneal epithelial cells,was identified by immunofluorescent staining.We used also in vitro and in vivo methods to make MSC multilayer.When MSC formed a monolayer,using the air-lifting cultivation that formed by Millicell culture plate inserts to make them stratification. Four weeks later,after fixed and dehydrated,the MSC were observed under the light microscope after HE staining and immunohistochemistry staining.In vivo stratification,we used methods of superficial lamellar keratoplasty.Automated corneal shaper was used to make corneal stroma of 8.5mm diameter and 160μm thickness,MSC were planted on the corneal stroma.Then we transplanted the fresh corneal stroma with MSC onto the New Zealand white rabbits corneal surface that removed superficial lamellar of 160μm thickness. We have transplanted three pig lamellar corneal grafts to eyes of three rabbits.Four weeks after transplantation,corneas were collected,detected by HE staining and immunohistochemistry staining.Results MSC can be cultured and expanded in vitro,which showed great potential of proliferation.The result of flow cytometry:the positive percentage of CD45 was 0.06%,and CD34 was 0.41%,CD44 was 86.43%,CD29 was 85.72%,CD105 was 90.72%,which showed that MSC express CD44,CD29,CD105 but not CD45 and CD34.These results indicated that the cultured cells were MSC.After three weeks induction,part of MSC expressed CK12.In vitro stratification,HE staining showed that there was still monolayer after using the air-lifting cultivation.But cultured in vivo,four weeks after transplantation, MSC were stratificated and became corneal-like stratificating epithelial cells.We have transplanted three eyes of three rabbits,there were two corneas kept transparent for four weeks and one cornea kept transparent for one week. Conclusion The data suggested that MSC have the potential to differentiate into corneal epithelial cells.In vivo culture can make MSC multilayer.MSC could be the option of seed cells to reconstruct the corneal epithelium by tissue engineering technology.