Evaluation of mesenchymal stem cells as feeder cells for the cultivation of corneal epithelial cell sheets in dogs

Corneal epithelial cell sheet transplantation is a promising therapy for reconstructing the ocular surface in canines with severe corneal disease. Cultivation of these sheets typically requires feeder cells that support the proliferation and maintenance of immature epithelial cells by secreting growth factors. However, using conventional murine 3T3 fibroblasts as feeder cells raises ethical and safety concerns. This study investigated canine adipose-derived mesenchymal stem cells (cADMSCs) and bone marrow mesenchymal stem cells (cBMMSCs) as alternative feeder cells. Both MSCs were cultured alone or with canine corneal epithelial cells. Changes in epidermal growth factor, keratinocyte growth factor, and hepatocyte growth factor (HGF) expression in both MSC types were evaluated by quantitative polymerase chain reaction. HGF expression was further assessed by western blot and enzyme-linked immunosorbent assay. Canine corneal epithelial cell sheets were cultivated with cADMSCs, cBMMSCs, or 3T3 fibroblasts, or without feeder cells, and their structure and quality were evaluated histopathologically. Results showed that cADMSCs and cBMMSCs originally expressed soluble factors, and HGF mRNA expression significantly upregulated when co-cultured with epithelial cells. However, no significant differences in sheet structure or quality were observed among feeder types. In conclusion, cADMSCs and cBMMSCs are viable alternatives to 3T3 fibroblasts for cultivating canine corneal epithelial cell sheets. Canine corneal epithelial cells interact with MSCs and promote the secretion of soluble factors from MSCs to promote epithelial cell growth. Moreover, canine corneal epithelial cells may maintain their proliferative capacity and immature state independent of feeder cells.