Purchase this article with an account.
Marc Dziasko, Stephen J Tuft, Julie T Daniels; Limbal melanocytes support limbal epithelial stem cells in 2D co-culture and RAFT collagen tissue equivalents. Invest. Ophthalmol. Vis. Sci. 2014;55(13):2198.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Human limbal epithelial stem cells (LESCs) are essential for the maintenance of the ocular surface. The distribution of LESC around the circumference of the limbus is not uniform. A higher density of LESC is found within limbal crypts (LCs). We have recently observed that the LCs, are also populated with limbal melanocytes. More than a protective barrier against UV light, it has been proposed that limbal melanocytes might also interact with LESCs in the niche. The aim of this study was 1. to assess the function of limbal melanocytes in limbal epithelial cell (LEC) support and 2. incorporate limbal melanocytes into a rudimentary 3D model of the native niche.
LEC and limbal melanocytes were isolated from human cadaveric donors, co-cultured and maintained in corneal epithelial culture medium (CECM) supplemented with 0.5% FBS. LEC morphology and expression of putative +ve and -ve LESC markers were evaluated by immunocytochemistry. The proliferative capacity of LEC pre-expanded on limbal melanocytes was assessed by colony forming efficiency (CFE) assays. For 3D co-cultures, LECs and limbal melanocytes were seeded on top of RAFT collagen tissue equivalents. Distribution of melanocytes within the 3D construct, expression of LESC markers and the morphology of the epithelium were evaluated by immunohistochemistry and transmission electron microscopy.
LECs in co-culture with limbal melanocytes exhibited morphological stem cell characteristics and expressed putative LESCs markers such as p63α, Bmi1 and CK15. CFE assays revealed that LECs pre-expanded on limbal melanocytes maintained high proliferative potential. In RAFT cultures, limbal melanocytes co-localized with basal undifferentiated epithelial cells as observed in the native limbal stem cell niche.
In the present study we show that limbal melanocytes can be isolated from the limbus and expanded in vitro. Moreover, these cells were able to support LECs that maintained stem cell characteristics suggesting a functional involvement of limbal melanocytes in the native stem cell niche. Finally, 3D culture based on RAFT allowed us to develop a model for studying melanocyte and LEC interactions that could also be suitable for transplantation.
This PDF is available to Subscribers Only