Abstract
Purpose :
Limbal epithelial stem/progenitor cells (LEPC) and supporting cell populations have been shown to form structural units in the human limbal stem cell niche. Here, we explored the role of supporting niche cells for LEPC function governing corneal epithelial homeostasis.
Methods :
Pure cultures of LEPC and associated niche cells, i.e., melanocytes and mesenchymal stromal cells, were established by stepwise purification protocols after their isolation from LEPC clusters by collagenase digestion. Cell cultures were characterized by flow cytometry and immunocytochemistry and used as feeder layers in direct and indirect LEPC coculture experiments with embryonic 3T3 fibroblasts and epidermal melanocytes as controls. Cultured cells were analyzed by light and electron microscopy, immunocytochemistry, qPCR and Western blotting. Colony forming efficiency, cell migration, proliferation, and apoptosis of LEPC were analyzed by appropriate assays.
Results :
Cultivated limbal melanocytes expressed Melan-A, MITF, TYRP1, HMB45 and c-kit, whereas mesenchymal stromal cells expressed CD90, CD73, CD105, CD44, nestin and Sox2. LEPC cocultivated with both niche cell populations showed similar colony forming efficiency but increased colony growth compared to cocultivation with 3T3 fibroblasts. Whereas melanocytes infiltrated the LEPC clones to enwrap stem/progenitor cells with cytoplasmic processes, stromal cells stayed at the clone periphery. Both niche cell populations suppressed expression of differentiation markers (K3, K12, involucrin) and upregulated expression of progenitor markers (K15, N-Cadherin, ABCG2) in LEPC, both in direct and indirect cocultures. Their effect on K3 suppression was obviously mediated by downregulation of transcription factors Sp1 and Pax6, and was significantly enhanced over that of 3T3 fibroblasts. Furthermore, cocultivation with melanocytes protected LEPC from UV-induced apoptosis and supported LEPC migration and proliferation in 2D-culture and an organ culture wound healing model. Skin melanocytes used as controls failed to show similar effects as limbal melanocytes on LEPC phenotype and function.
Conclusions :
These findings support the concept of supporting cells governing LEPC homeostasis in the limbal niche. Based on their unique properties, limbal melanocytes might be utilized to maintain stem cell phenotype and function during cultivation and transplantation for treatment of limbal stem cell disease.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.