Differentiation of rabbit bone marrow mesenchymal stem cells into corneal epithelial cells in vivo and ex vivo

PURPOSE: To examine whether bone marrow mesenchymal stem cells (MSCs) could be differentiated into corneal epithelial cells in vivo and ex vivo. METHODS: In vivo, BrdU labeled rabbit MSCs (Rb-MSCs) were suspended in the fibrin gels and transplanted onto the surface of the damaged rabbit corneas. Histology and molecular phenotype were studied on postoperative day 28. In vitro, labeled Rb-MSCs were cultured for three days in two different systems: (1) Group A: Rb-MSCs were co-cultured with rabbit limbal stem cells (Rb-LSCs) by the Transwell culture system. A suspension of Rb-LSCs was added to the upper membrane surface, and the inserts were positioned in the culture wells, which were incubated with Rb-MSCs; (2) Group B: Supernatant medium that had first been used to culture Rb-LSCs and then filtered with a 0.45 μm filter was used to culture Rb-MSCs. For both groups, immunofluorescence and flow cytometric analysis were used to examine the expression of cytokeratin 3 (CK3) in differentiated Rb-MSCs. RESULTS: In vivo, the data showed that following transplantation of Rb-MSCs, the rabbit’s damaged corneal surface was successfully reconstructed and that some Rb-MSCs participated in the healing of the injured corneal epithelium and expressed CK3. In vitro, the data showed that Rb-MSCs rapidly differentiated into cells with a morphological and molecular phenotype of corneal epithelial-like cells. For both groups, the differentiated Rb-MSCs were positive for corneal epithelial-specific marker CK3. In Group A, flow cytometry analysis showed that at day one, only 3.46±1.9% of cells expressed CK3. This increased to 7.24±3.80% at day two and decreased slightly (5.50±3.33%) at day three. The proportion of CK3 in Group B was 4.09±1.84% at day one, rising to 9.31±5.92% after 24 h, but falling (4.37±2.61%) at day three. The mean differences are significant between each group and the negative control, but was not significant between Group A and Group B. CONCLUSIONS: MSCs could differentiate into corneal epithelial-like cells in vivo and ex vivo.