Isolation and enrichment of melanocytes from human corneal limbus using CD117 (c-Kit) as selection marker

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.

Efficient Isolation and Functional Characterization of Niche Cells from Human Corneal Limbus

“…Earlier studies have shown that clusters derived from collagenase digestion included more epithelial progenitor cells, LMSCs, and melanocytes than dispase-isolated cell sheets [ 3 , 15 ]. Moreover, it has also been reported that cluster-derived cell suspensions contained more CD117 + /Melan-A + cells [ 24 ] and that stromal cells located immediately subjacent to limbal basal epithelial cells support LEPC better than stromal cells located in deeper stromal layers [ 18 , 19 ]. Hence, in the present study, we used the cluster-derived cells for efficient individual cell type-specific isolation of LEPC, LM and LMSC.…”

“…Recently, we have reported on a novel protocol to obtain LM in a very short period of time and to avoid any toxic effects exerted by commonly used selection agents [ 24 ]. However, the sorted CD117 + LM populations still contained small amounts of contaminating stromal cells in most of the cultures (6/7 cultures) and required a second CD117-based FACS sort [ 24 ].…”

“…Recently, we have reported on a novel protocol to obtain LM in a very short period of time and to avoid any toxic effects exerted by commonly used selection agents [ 24 ]. However, the sorted CD117 + LM populations still contained small amounts of contaminating stromal cells in most of the cultures (6/7 cultures) and required a second CD117-based FACS sort [ 24 ]. The refined strategy of using CD117 in combination with CD90 provided primary cultures of CD117 + /CD90 − cells which did not show any fibroblast-like or vimentin + melan-A − cells ( Figure 2 C), suggesting a pure population of LM.…”

“…Very few studies on LM isolation have been reported and most of them rely on differential cytotoxic effects of G418 (geneticin) to prevent rapid overgrowth by epithelial cells and fibroblasts [ 5 , 23 ]. Recently, we have developed a protocol for isolation of melanocytes using CD117 as a selection marker [ 24 ]. However, this protocol also requires two rounds of flow sorting to eliminate contaminating stromal cells in order to get a pure LM population.…”

“…Therefore, the aim of this study was to establish a technique for instantaneous retrieval of pure LEPC, LMSC, and LM populations from organ cultured corneal samples by means of fluorescence-activated cell sorting (FACS) using CD117 (as a surface marker of melanocytes) [ 24 ] and CD90 (as a surface marker of stromal cells) [ 21 ] as selection markers. The essence of this new approach is that stromal cells are simultaneously extracted from the total limbal cell population, which minimizes the risk of stromal contamination.…”