Expression of glial cell-derived neurotrophic factor and its receptor in the stem-cell-containing human limbal epithelium

Abstract

Aim-To evaluate the expression pattern of glial cell line-derived neurotrophic factor (GDNF) with its receptors GDNF family receptor alpha-1 (GFRα-1) and Ret in the human corneal and limbal tissues, as well as in the primary human limbal epithelial cultures (PHLEC).Methods-Expression of GDNF and its receptors, and the co-localisation with stem cell associated and differentiation markers were evaluated by immunofluorescent staining, western blot analysis and real-time PCR in the fresh human corneoscleral tissues, as well as in the PHLEC. Single cell colony-forming and wound-healing assays were also evaluated in PHLEC.Results-GDNF and GFRα-1 were found to be expressed by a subset of basal cells and colocalised with ATP-binding cassette, subfamily G (WHITE), member 2 (ABCG2) and p63, but not with cytokeratin 3 in the human limbal basal epithelium. In PHLEC, they were expressed by a small population of cells in the less differentiated stage. The GDNF and GFRα-1-positive subpopulations were enriched for the expression of ABCG2 and p63 (p<0.01). Recombinant human GDNF promoted the proliferation and wound healing of epithelial cells in the PHLEC. In contrast, Ret was abundantly located in the human corneal epithelium except for the basal cells of the limbal epithelium.Conclusion-These findings indicate that GDNF and GFRα-1 may represent a property for the phenotype of human corneal epithelial precursor cells. GDNF may signal independently of Ret through GFRα-1 in the stem cell-containing limbal epithelium.Glial cell line-derived neurotrophic factor (GDNF) is a member of the GDNF family of neurotrophic factors, which also includes neurturin, artemin and persephin. 1 GDNF exerts its effects through a multicomponent receptor system consisting of the GDNF family receptor alpha-1 (GFRα-1), Ret receptor tyrosine kinase (Ret) and neural cell adhesion molecule (NCAM).2 3 GDNF has been shown to support the growth, maintenance and differentiation of a wide variety of neuronal systems. In recent years, GDNF was identified as an essential growth factor maintaining mouse spermatogonial stem cells,4 and it was found to increase motility and survival of cultured mesenchymal stem cells and ameliorate Corneal epithelial homeostasis is governed by a small subpopulation of corneal epithelial SCs located in the basal layer of the limbus.6 -8 However, no direct method has been found to identify the corneal epithelial SCs to date because of the lack of a unique molecular marker. In recent years, the functional role and the possible signal-transduction pathways induced by GDNF in the corneal epithelium have been investigated.9 10 GDNF may play an important role in corneal regeneration and wound healing. In the present study, we evaluated the expression pattern of GDNF with its receptors in the human corneal and limbal epithelia, as well as in the primary limbal epithelial cell cultures (PHLEC), with the intention of exploring the potential role of GDNF in the human stem cell niche. MATERIALS AND METHODS Materials and reagentsProgenit...

Molecular signatures and biological pathway profiles of human corneal epithelial progenitor cells

“…The microarray data allowed us to analyze all these known markers at one time. Interestingly, the expression patterns of 15 known genes, proposed as stem cell associated (ABCG2, p63, integrin α9, integrin β1, EGFR, enolase-α1, K15, TrkA, N-cadherin and C/EBP-δ) or differentiation associated (connexin 43, E-cadherin, K12, K3 and involucrin) markers, were consistent to the limbal stem cell phenotype as previous reports (Barbaro et al, 2007; Chen et al, 2004; Figueira et al, 2007; Hayashi et al, 2007; Qi et al, 2008a; Qi et al, 2008b). Among them, 9 genes were verified again by quantitative real-time PCR using samples from separate adhesion experiments.…”

“…We have extensively evaluated these molecular markers and characterized that the basal cells at limbal epithelium are small primitive cells expressing three patterns of molecular markers (Chen et al, 2004): (1) exclusively positive for p63, ABCG2 and integrin α9 by a subset of basal cells; (2) relatively higher expression of integrin β1, EGFR, K19 and enolase-α1 by most basal cells, and (3) lack of expression of E-cadherin, connexin 43, involucrin, K3 and K12. Very recently, we have observed that two neurotrophic factors, nerve growth factor (NGF) and glial cell-derived neurotrophic factor (GDNF), and their corresponding receptors TrkA and GFRα-1 were exclusively localized to a subpopulation of basal limbal epithelial cells (Qi et al, 2008a; Qi et al, 2008b). Furthermore, keratin 15 (Figueira et al, 2007), N-cadherin (Hayashi et al, 2007) and CCAAT enhancer binding protein δ (C/EBP-δ, Barbaro et al, 2007) have been proposed as markers to identify limbal stem cells.…”

The β-catenin/Tcf4/survivin signaling maintains a less differentiated phenotype and high proliferative capacity of human corneal epithelial progenitor cells

“…A non-template control was included to evaluate DNA contamination. The results were analyzed by the comparative threshold cycle (CT) method and normalized by a housekeeping gene GAPDH [47, 48]. …”

Congenital Aniridia and the Ocular Surface

“…38,39 Nerve growth factor and its receptor Trk-A have been implicated as potential markers for human corneal epithelial stem cells. 38,40 In mouse models, heterozygous Pax6 +/-adult mice exhibited decreased epithelial innervation and nerve patterns were disrupted. 41 In humans, IVCM has been used to quantify subbasal epithelial nerves in LSCD, where a markedly reduced nerve density has likewise been found in a case series without aniridia patients 35 and in a series including two aniridia patients.…”