Abstract
Limbal melanocytes (LM) are located in the basal epithelial layer of the corneoscleral limbus and interact with adjacent limbal epithelial progenitor cells. The exploration of their biological role in the maintenance of the limbal stem cell niche has been limited by the difficulty of LM isolation and cultivation. Here, we report on a facile protocol for the efficient isolation and enrichment of pure populations of human LMs by fluorescence-activated cell sorting (FACS) using antibodies raised against the cell surface marker CD117 (c-Kit). The enriched LMs retain self-renewal capacity and sustained melanin production, and are suitable to study the potential of LMs in stem cell-based corneal tissue engineering.
Highlights
Limbal melanocytes (LM) are embedded in the basal limbal epithelium and interact with adjacent limbal epithelial progenitor cells (LEPC)
Various methods have been suggested to obtain pure melanocyte cultures from epidermal and limbal epithelia, the most common ones rely on differential cytotoxic effects of G418 to prevent rapid overgrowth by epithelial cells and fibroblasts[5,11,12,13,14]
CD117 expression was observed in LMs, and CD117/stem cell factor (SCF) signaling was shown to play an important role in the limbal stem cell n iche[5,17]
Summary
Limbal melanocytes (LM) are embedded in the basal limbal epithelium and interact with adjacent limbal epithelial progenitor cells (LEPC). Various methods have been suggested to obtain pure melanocyte cultures from epidermal and limbal epithelia, the most common ones rely on differential cytotoxic effects of G418 (geneticin) to prevent rapid overgrowth by epithelial cells and fibroblasts[5,11,12,13,14] Most of these cytotoxic procedures provide pure melanocyte populations, subtle toxic side effects on the phenotype and functional properties of the surviving cells remain a possibility[14]. To avoid these limitations, Hayashi and coworkers used fluorescence-activated cell sorting (FACS) for isolation of LMs based on N-cadherin e xpression[7].
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