Abstract
Purpose :
Limbal epithelial stem/progenitor cells (LEPC) and melanocytes have been shown to form structural units in the human limbal stem cell niche (Polisetti et al. 2016). Here, we explored the specific role of melanocytes for LEPC function and niche homeostasis.
Methods :
Primary cultures of limbal melanocytes were established and characterized by flow cytometry and immunocytochemistry. Melanocytes were used as feeder layers in direct and indirect LEPC co-culture experiments using 3T3 fibroblasts and limbal mesenchymal stromal cells (MSC) as controls. Co-cultures were analyzed by light and electron microscopy, immunolabeling, qPCR and Western blotting. Colony forming efficiency, cell migration, proliferation, and monocyte adhesion were analyzed by appropriate assays. Limbal epithelial cell sheets were generated on fibrin-based hydrogels with or without melanocytes. The effect of melanocytes on proliferation of T cells and human umbilical vein endothelial cells was investigated using mixed lymphocyte reaction (MLR) and proliferation/tube formation assays.
Results :
LEPC co-cultivated with melanocytes showed increased colony growth compared to co-cultivation with 3T3 fibroblasts and MSC. Melanocytes infiltrated the epithelial clones to closely enwrap LEPC with their cell processes and to transfer melanosome-rich packages via phagocytosis. Melanocytes also suppressed mRNA and protein expression of differentiation markers (K3, K12) and upregulated expression of progenitor markers (K15, N-Cadherin, ABCG2) in LEPC, both in direct and indirect co-cultures. Melanocytes also supported LEPC migration and proliferation in 2D- and 3D-organ culture models, and supported epithelial sheet formation on fibrin gels. Stimulation with IFN-γ significantly increased the adhesion of monocytic cells to melanocytes via upregulation of ICAM-1. Most importantly, melanocytes markedly suppressed proliferation of activated T cells and vascular endothelial cells through direct and indirect effects, which exceeded those of MSC.
Conclusions :
These findings support the concept that melanocytes, apart from melanogenesis and melanosome transfer, control LEPC homeostasis in the limbal niche and contribute to the integrity of the limbal barrier. Based on their effective immunomodulatory and anti-angiogenic properties, limbal melanocytes have great potential for LEPC ex vivo-expansion and therapeutic application.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.