Abstract
Nerve growth factor (NGF) has demonstrated great benefit in the treatment of neurotrophic corneal ulcers. There is evidence for multiple modes of action in promoting corneal healing, but only indirect evidence exists for NGF's effects on limbal stem cells (LSCs). Understanding the role of NGF in LSC biology will improve our understanding of paracrine regulation of the limbal niche and the design of stem cell‐based therapies for conditions such as LSC deficiency. In this article, we studied the regulation of NGF signaling components during LSC differentiation and the role of NGF in LSC proliferation and maintenance of the stem cell phenotype. LSC differentiation was induced by prolonged (40 day) culture which resulted in a significant increase in cell size, decrease in colony‐forming efficiency and expression of putative LSC markers. A protein microarray measuring expression of 248 signaling proteins indicated the low affinity NGF receptor p75NTR to be the most downregulated protein upon differentiation. Further confirmation by Western blotting and real‐time quantitative polymerase chain reaction indicated that NGF and p75NTR are expressed in early LSC cultures and downregulated upon differentiation. LSC cultures grown in the presence of anti‐NGF antibody showed decreased colony‐forming efficiency, DNA replication and expression of putative LSC markers ABCG2 and C/EBPδ. Supplementation of LSC culture medium with NGF extended the life span of LSC cultures in vitro and increased the expression of putative LSC markers ΔNp63α and ABCG2. Taken together, our data indicate that NGF signaling is a key promoter of LSC proliferation, colony‐forming efficiency, and a maintainer of the LSC phenotype. stem cells 2019;37:139–149
Highlights
The cornea is the transparent structure and main refractive surface of the eye [1]
Gene expression analysis showed an increase in the expression of corneal differentiation markers and a decrease in putative limbal stem cells (LSCs) markers phenotype with time from culture initiation (Fig. 2B)
Our previous work has shown that replication of the limbal niche in vitro results in directed differentiation of human ESC to corneal progenitors, suggesting a role for paracrine signaling in promoting the LSC phenotype [30]
Summary
The cornea is the transparent structure and main refractive surface of the eye [1]. It is composed of three main layers: an outer stratified epithelium, stroma, and an inner single-cell layered endothelium. It can be degraded by direct insults such as infection, trauma, and chemical burns. Nerve growth factor (NGF) is a member of the neurotrophin family, and is constitutively expressed in the basal limbus with lower expression in the basal corneal
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