Three-dimensional reconstruction of the mouse limbal epithelial stem cell niche by immunofluorescence tomography

Abstract

Purpose: Immunofluorescence tomography is a recently developed method to three-dimensionally reconstruct large tissue volumes with multiple immuno-labels at high-resolution. Here, we used immunofluorescence tomography to characterize the limbal epithelial stem cell niche in the H2B-GFP/K5tTA transgenic mouse and human limbus.

Methods: After 8wks pulse (no doxycycline) and 8wks chase (2g/kg doxycycline diet), H2B-GFP/K5TtA mice retain GFP in slow-cycling epithelial cells only, which are termed label-retaining cells (LRCs) or limbal epithelial stem cells (LESCs). Mice were sacrificed after 8wks chase by carbon dioxide asphyxiation before corneas were excised and fixed overnight in 2% paraformaldehyde along with human cadaver limbus samples. Fixed tissues were embedded in butyl-methyl methacrylate (BMMA) by UV polymerization at 4^oC to maintain antigenicity. Serial tissue sections were then cut at 2µm and sequential immunostaining was carried out using the Ted Pella BioWave and primary antibodies against Integrin β4, BLIMP1, vimentin, collagen IV, DKKL1, keratin 19 and keratin 5, β-catenin, SOX9 and α-smooth muscle actin. Imaging of GFP fluorescence and secondary antibody (rabbit AlexaFluor 546) was carried out using a Leica DMi6000B and semi-automated image registration and three-dimensional reconstruction was performed using Amira software.

Results: Three-dimensional reconstructions were generated of the limbal region from over 300sections (600µm) per tissue and with a voxel size of 0.46µmx0.46µmx2µm. After 8wks doxycycline chase, GFP⁺ cells are primarily localized to the basal layer of the limbal epithelium but can infrequently exist in the anterior limbal stroma. GFP⁺ LRCs in the H2B-GFP/K5tTA mouse limbal epithelium express SOX9, keratin 5, β-catenin and BLIMP1. In human limbus three-dimensional reconstructions, weak keratin 5 expression can be observed in cells of the anterior stroma whereas a small population of limbal epithelial cells express the mesenchymal marker, vimentin.

Conclusions: Taken together, these findings suggest that epithelial-mesenchymal transition (EMT) may be a feature of limbal epithelial cells in mice and humans and, from observations of GFP⁺ cells in the H2B-GFP/K5TtA mouse limbal stroma, may be a characteristic of LRCs/LESCs themselves, however, further evidence is required to validate this hypothesis.