Cellular cross-talk in the limbal epithelial stem cell niche revealed by high-resolution microscopy

Abstract

Purpose: Maintenance of the ocular surface by limbal epithelial stem cells (LESCs) is essential to preserve corneal transparency and therefore vision. Previously our group has identified within the limbus (the transition zone between the peripheral cornea and bulbar conjunctiva) a candidate for the LESC niche: the limbal crypts (LCs). The aim of this study was to demonstrate if LCs act as a reservoir for LESCs using a functional test and to identify any putative crosstalk between LESCs and stromal cells in this specific area.

Methods: In vitro single cell clonal analysis was performed to compare the growth potential of epithelial cells isolated from LC-rich and non-crypt regions of the human limbus. Transmission electron microscopy in conjunction with serial block-face scanning electron microscopy (SBSEM), manual segmentation and 3D reconstruction were used to image and characterize putative LESCs and their surrounding environment.

Results: A greater number of stem cell holoclone colonies were founded from epithelial cells isolated from LC-rich regions of the superior and inferior limbus compared with epithelial cells isolated from non-crypt regions of the temporal and nasal limbus. This provides functional evidence, in support of our earlier putative LESC marker expression and cell morphology observations, that the distribution of LESCs around the limbus is not uniform and that LESCs tend to co-locate with LCs. Furthermore, imaging LCs by transmission electron microscopy revealed a population of epithelial cells of small size and poorly differentiated “stem-like” phenotype in close proximity to an underlying stromal cell population, suggesting possible cell-cell interaction. These observations were confirmed by serial block face SEM and highlighted, for the first time, interruptions of the basement membrane with direct contact between putative LESCs and neighbouring stromal cells.

Conclusions: These observations directly support the emerging concept that cellular crosstalk maintains cell stemness at the corneal limbus as seen in other epithelial stem cell niches.

Keywords: 447 cell-cell communication • 482 cornea: epithelium • 480 cornea: basic science