Objectives: Considering that there is a shortage of eye donor, the aim of corneal tissue engineering is to develop substitutes for the replacement of damaged tissues. Limbal stem cells have been considered as a source for continual renewal of corneal epithelium; however, it has limitation on the ocular surface disorders with limbal stem cell dysfunction. In addition, there is no reliable source for regeneration of corneal endothelium. This study is to investigate the ability of differentiation of bone marrow-derived mesenchymal stem cells (BMMSC) into corneal cells in vitro and their possible roles in corneal repair.
Methods: Bone marrow was collected from the femoral and tibial bones of WISTAR Albino rats. The MSCs were isolated and cultured with Ficoll Hypaque gradient method. The purification of MSCs was achieved by removal of nonadherent cells during subsequent changes of medium. Bone marrow derived cells were characterised for the presence of mesenchymal marker (CD29) and absence of hematopoietic marker (CD34) before performing experiments. MSC to epithelium and endothelium differentiation experiments were performed. The epithelial cells differentiated from BMMSCs were characterised by morphology and immunophenotyping using K3, ZO-1. The flow cytometry and Western blot were also used to confirm the cell differentiation. Epithelial and endothelial cell proliferation assays using various MSC mixed-cultures and MSC conditional medium were also conducted.
Results: Primary cultures of BMMSCs were obtained successfully. Immunohistochemistry study and flow cytometry analysis indicated that BMMSCs constantly expressed CD29 (mesenchymal marker). They were negative for CD34 (hematopoietic marker). In the induction of multi-lineage differentiation experiments, corneal epithelial cells were successfully differentiated from BMMSCs. Those cells were Cytokeratin 3 (corneal epithelial marker) and ZO-1 positive and CD 31 (endothelial marker) negative. The original MSC marker of CD29 was switched to negative on those differentiated epithelial cells. The endothelial-like cells were observed from the MSC cultures induced with corneal endothelial growth factor. The cells were ZO-1 positive. However they could not be characterised due to the lack of available specific corneal endothelial markers. SMCs showed promotion effects on corneal cell proliferation.
Conclusion: The present study demonstrates that bone marrow-derived mesenchymal stem cells have the ability to differentiate into corneal epithelial and endothelial-like cells. The study opens up the possibility of using these cells in corneal tissue engineering and regeneration in the treatment of corneal surface disorders. This project is supported by Corneal Stem Cell Research Grant of Save Sight Institute and Dr McClellan’s Corneal Research Donation Fund.