Cell and Molecular Biology of Cornea Epithelial Stem Cells

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DESCRIPTION (provided by applicant): The cornea, our "window to the world," is subject to damage caused by injury, infection and other diseases. Approaches are being developed to treat these corneas by transplantation of cornea epithelial stem cells. A major medical challenge lies in being able to isolate sufficient quantities of these cells for culture, expansion, and transplantation. We still lack a basic understanding of their proliferation, migratory behaviors and precise markers to identify these cells. We also do not know what factors regulate these behaviors, which is essential for enhancing the therapeutic potential of cornea epithelial stem cells. Recent evidence suggests that cutaneous nerves may be a key component of the epithelial stem cell niche. The frog Xenopus is an established vertebrate model with distinct advantages for studies of cell and molecular biology. The development and morphology of the mature frog cornea is essentially identical to that of humans. We developed tools that make Xenopus an excellent system to study the biology of these cells. Our central hypotheses are that limbal stem cells and their proliferative progeny exhibit specific cleavage patterns and express specific combinations of genes that will allow one to distinguish these cells. Further, these cells respond to cues released by damage to undergo increased proliferation, being able to migrate to wound sites, and finally that nerves serving the cornea represent a critical component of the epithelial stem cell niche that regulates the behavior of these cells. This proposal has three specific aims to address these hypotheses: I) determine the normal proliferative patterns and gene expression profiles to distinguish these stem cells and their progeny, and examine the role of pluripotency genes in regulating the behavior of these cells. II) Determine how these cells respond to damage and whether they undergo active migration during homeostasis and repair. III). Determine if cutaneous nerves of the cornea play a key role in supporting these proliferative cells. To address these aims we developed new tools that permit direct, continuous visualization of these cells in live animals over prolonged periods of time. We have also developed Xenopus models to study cornea wound repair and limbal stem cell deficiency (LSCD). Finally, we can ligate the corneal nerves with tremendous precision to test their role in supporting these cells. This research is innovative in its use of high-throughpu transgenic approaches in Xenopus to permit real-time observations of live cornea epithelial stem cells in which we can characterize specific patterns of gene expression (markers) that identify these cells. This study will also determine if corneal nerves represent a critical component of the stem cell niche that supports these cells. Tens of thousands of patients develop neurotrophic keratitis, forms of corneal dystrophy, LSCD, and other eye conditions annually in the U.S. that require corneal transplants (about 40,000/year). The proposed research is significant, as it will develop tools and approaches that can be used to treat these diseases and injuries of the cornea.