Patterned Localization of Neurotrophic Factors and Receptors at the Human Limbal Stem Cell Niche

Abstract

Purpose:: Neurotrophic factors play a vital role in survival and self renewal of neuron and non-neuron stem cells. This study was to explore patterned expression and potential role of neurotrophic factors and their receptors at the human limbal epithelial stem cell niche.

Methods:: Fresh human corneoscleral tissues were prepared for cryosections containing the corneo-limbal junction and limbal Palisades of Vogt. Immunofluorescent staining and laser scanning confocal microscopy were performed with primary antibodies against 6 neurotrophic factors and 5 receptors: 1) neurotrophin family: nerve growth factor (NGF), brain-derived neurotrophic factors (BDNF), neurotrophin 3 (NT-3), neurotrophin 4 (NT-4), and their receptors, TrkA, TrkB and TrkC; 2) glial cell-derived neurotrophic factor (GDNF) and receptor GFRα-1; 3) ciliary neurotrophic factor (CNTF) and receptor CNTFR.

Results:: The immunoreactivity of NGF and GDNF were exclusively and sparsely localized in the basal layer of limbal epithelium where stem cells reside, but they were detected in whole corneal and suprabasal limbal epithelium and stroma. NGF receptor TrkA and GDNF receptor GFRα-1 shared a similar pattern to their ligands, with extended expression to a few suprabasal limbal cells and scattered basal cells of peripheral cornea. BDNF was expressed by all epithelial layers and stroma cells with uniquely strong staining in the limbal basal epithelial layer. NT-3 and NT-4 were positive in all layers of corneal and superficial layers of limbal epithelium, but negative in the basal layer of limbal epithelium. NT-3, but not NT-4, was weakly positive in a few stroma cells. TrkB and TrkC antibodies evenly stained all layers of epithelium and stroma cells. All these factors and receptors were expressed in limbal epithelial cultures. Interestingly, NGF, GDNF, BDNF and TrkA and GFRα-1 were strongly expressed by basal cells in stratified limbal epithelium cultivated on corneal stroma. CNTF and CNTFR staining was not detected.

Conclusions:: NGF, GDNF, TrkA and GFRα-1 were exclusive localized to a subpopulation of basal limbal epithelial cells; BDNF expression was relatively higher in the limbal basal layer; and NT-3 and NT-4 were negative in the limbal basal layer. These three expression patterns of neurotrophic factors and receptors may play a differential role in the human limbal stem cell niche.

Keywords: cornea: epithelium • neuropeptides • immunohistochemistry