The role of nerve growth factor in limbal stem cell biology and corneal healing

Session Details

Session Title: Cornea Miscellaneous

Session Date/Time: Monday 24/09/2018 | 16:30-18:00

Paper Time: 16:42

Venue: Room A3, Podium 3

First Author: : A.Ghareeb UK

Co Author(s): :    S. Kolli   S. Bojic   F. Figueiredo   M. Lako           

Abstract Details

Purpose:

Nerve growth factor (NGF) has demonstrated great benefit in the treatment of neurotrophic corneal ulcers and potential benefit in the treatment of several other ocular conditions. There is evidence for several modes of action in promoting corneal healing, however 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 the LSC niche and the development of stem cell-based therapeutics. We study the changes in cell signalling which take place upon LSC differentiation and the effects of NGF-blocking on primary human LSCs.

Setting:

Centre for Life, Institute of Genetic Medicine, Newcastle University.

Methods:

Primary human limbal cultures derived from cadaveric corneoscleral rims were cultured on a layer of murine 3T3 fibroblasts for 40 days to allow differentiation. Differential expression of signalling proteins between days 10 and 40 was measured by protein microarray, with subsequent bioinformatic enrichment and network analysis. Expression of NGF and its receptors TrkA and p75NTR was also measured by Western Blot. The effect of addition of anti-NGF antibody on cell morphology, colony-forming efficiency and gene expression of putative differentiation markers was measured. Flow cytometry-based assays of proliferation and apoptosis were also used to study the effect of NGF blocking.

Results:

Of 248 signalling proteins studied, NGF receptor p75NTR underwent the greatest fold-change in expression between the LSC and differentiated phenotypes, with expression decreasing 2.77-fold upon differentiation. NGF signalling exists at the top of a hierarchical network, controlling cell cycle, toll-like receptor, senescence regulatory pathways and VEGF signalling. Expression of NGF and TrkA also decreases upon differentiation. Primary human limbal cultures grown in the presence of anti-NGF antibodies show significant decreases in colony-forming efficiency (p=0.039), proliferation (p=0.042) and expression of putative stem cell markers ABCG2 and C/EBPδ (p=0.03, p=0.04, respectively).

Conclusions:

NGF maintains the LSC phenotype by promoting self-renewal. NGF signalling potentially coordinates the expression of several biological processes involved in corneal healing, making it a central paracrine signalling factor in the response to corneal epithelial injury. NGF, TrkA and p75NTR are markers of the stem cell phenotype.

Financial Disclosure:

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