Nerve growth factor and its receptor TrkA serve as potential markers for human corneal epithelial progenitor cells

Abstract

Nerve growth factor (NGF), a member of the neurotrophin family, has been identified as an essential growth factor supporting stem cell self-renewal outside the nervous system and was previously shown to stimulate corneal epithelial proliferation both in vivo and in vitro. In this study, we evaluated the expression of NGF and its corresponding receptors in the human corneal and limbal tissues, as well as in primary limbal epithelial cultures by immunofluorescent staining and relatively quantitative real-time polymerase chain reaction. We found that NGF was uniquely expressed in the human limbal basal epithelium, together with its two corresponding receptors: the high-affinity receptor TrkA and the low-affinity receptor p75NTR. TrkA was shown to preferentially localize to limbal basal epithelial cells. NGF and TrkA were also found co-localized with stem cell-associated molecular markers (drug-resistance transporter ABCG2 and p63), but not with the differentiation marker cytokeratin 3 in the human limbal basal epithelial layer. In cultured limbal epithelial cells, NGF and TrkA were found to be preferentially expressed by a small population of limbal epithelial cells. The NGF and TrkA immuno-positive subpopulations were enriched for certain properties (including ABCG2 and p63 expression) of putative limbal epithelial stem cells (P<0.01, compared with the entire cell population). Levels of NGF and TrkA transcripts were found to be much more abundant in limbal than in corneal tissues, and in young cultured cells in the proliferative stage than in airlifted stratified cultures containing differentiated cells. The co-expression of NGF with its two corresponding receptors in limbal basal epithelial cells, but not in the cornea, suggests that NGF may function as a critical autocrine or paracrine factor supporting stem cell self-renewal in the limbal stem cell niche. The spatial expression of NGF and TrkA by small clusters of basal cells interspersed between negative cell patches suggests that they are potential markers for human corneal epithelial progenitor cells.

Figures

Figure 1

Immunofluorescent staining of NGF, TrkA and p75 (green) in human corneoscleral tissue sections with propidium iodide nuclear counterstaining (red). S-Limbus, the horizontal cross-section cut through the superior limbus showed the papilla-like limbal epithelial columns; Limbus, the meridional sections cut from the limbus through the central cornea displayed a traditional view of the limbus. Arrow heads (▲) mark small clusters of immuno-positive basal cells; arrows (↑) mark negative cell patches. Scale bars, 20 μm.

Figure 2

Expression of NGF and TrkA mRNA in the human limbal epithelia (A) and in primary cell cultures at 40% confluent, 90% confluent and air-lifted stages (B) evaluated by relative quantitative real-time polymerase chain reaction. Levels of NGF and TrkA mRNA transcripts were much higher in the limbal than in the corneal epithelia (A. n = 8; Student’s t-test, compared with corneal epithelium); both transcripts decreased more than half in 90% confluent cultures and in the airlifted stratified limbal epithelial cultures (B. n = 5; Dunnett’s test, compared with 40% confluent cells). NGF mRNA was barely detectable in the airlifted stratified limbal epithelial cultures. **, P < 0.01.

Figure 3

Dual immunofluorescent staining of NGF and TrkA (green) with ABCG2, p63 and K3 (red) in human limbal tissue frozen sections. NGF and TrkA were co-expressed by ABCG2 (membrane) and p63 (nuclei) positive cells in the limbal basal layer. All NGF and TrkA positive cells were K3 negative (red). Scale bars, 20 μm.

Figure 4

Single immunofluorescent staining of NGF and TrkA (A), double staining of NGF and TrkA with p63 (B), ABCG2 (C) in human limbal epithelial cultures established from limbal explants with Hoechst 33342 nuclear counterstaining (blue). NGF and TrkA were preferentially expressed in the cytoplasm or membrane of small population of corneal epithelial cells (A, red). The NGF and TrkA immuno-positive subpopulations (B and C1, red) expressed higher levels of p63 (B, green) and ABCG2 (C2, green). Scale bars, 25 μm. These images are representative of the data summarized in Table 2.