PROJECT SUMMARY/ABSTRACT
Corneal diseases have a significant impact on the quality of life of patients and also constitute a major problem
for health care system. Corneal diseases such as limbal stem cell deficiency (LSCD) and diabetic keratopathy,
associated with limbal epithelial stem cell (LESC) dysfunction/loss, may lead to epithelial defects,
conjunctivalization, recurrent epithelial erosions, abnormal wound repair and altered vision. Therefore,
mechanisms of LESC function in normal and diseased corneal homeostasis and wound healing process could
be key to understanding various corneal diseases. The important part of the stem cell niche functioning is
interaction between stem cells and their neighboring stromal cells. LESC are in close contact with the cells of
the underlying limbal stroma and the vasculature surrounding limbal crypts. It is well established that there is a
crosstalk between stromal cells, or keratocytes, and limbal epithelial cells (LECs) through their secreted
factors, which are essential for the stem cell maintenance. Any damage to LESC or limbal stromal niche due to
the external insults or diseases such as diabetes may lead to pathological state of altered vision and, in severe
cases of LESC loss, results in limbal stem cell deficiency and blindness. Therapeutic potentials of extracellular
vesicles (EVs) released from various cell type into extracellular space have been widely reported. EVs are
nano-sized vesicles containing mRNA, miRNA, DNA, and protein cargo and mediating physiological
intercellular crosstalk. They play important roles in intercellular communication and in stem cell maintenance
and activation. We propose to investigate limbal EV contribution to corneal regeneration and wound healing,
and examine possible differences in EV cargos released from normal and diabetic limbal stromal cells (LSCs)
and LECs. We show that normal LSC-derived EVs (LSC-EVs) significantly promote epithelial healing in
wounded organ-cultured human corneas. In addition, we found that there is a difference in cargos of EVs
derived from normal and diabetic LSC. We hypothesize that limbal resident cell-derived EVs contribute to
limbal epithelial and stromal homeostasis. We predict that the unique complement of EV cargos mediates
corneal regeneration and wound healing in normal and diseased conditions. The goal of this proposal is to
investigate the major mechanisms of action of both human LSC- and LEC-EVs, which are taken up by
neighboring cells, in stem cell maintenance or survival and wound repair in cell cultures and in normal and
diabetic human organ-cultured corneas. Techniques to be used include, EV isolation, miRNA isolation, next
generation sequencing, proteomics, transmission electron and confocal microscopy, and trans-well co-culture
system. Relevance to Public Health: investigation of molecular changes in diabetic corneas is essential for
understanding of underlying causes and identification of therapeutic targets for diseased cornea and wound
healing process in different layers of the cornea.