Stem cells are crucial for both homeostasis and wound healing in self renewing tissues such as corneal epithelium. Corneal epithelial stem cells reside in the basal layer of corneal peripheral region called limbus. Loss of limbal epithelial stem cells because of eye diseases or severe injury leads to visual loss, which is called Limbal stem cell deficiency (LSCD). Transplantation of cultured limbal epithelial sheets has been used to treat LSCD with some success. The failure of transplantation of these limbal epithelial cultures is significantly associated with the lack of limbal stem cells. Therefore, it is of clinical significance to identify regulators that could be pharmacologically targeted to enhance stem cell numbers. However, it is unclear what affects the preservation of limbal stem cells in culture. Recently, we identified a small RNA called microRNA-107 (miR-107), preferentially expressed in the stem cell-enriched limbal epithelium. Our preliminary study showed that increasing miR-107 in limbal epithelial cultures raised the number of stem cell-like cells. This suggests that miR-107 may have a positive role in enhancing stem cell self-renewal. Therefore, we propose that miR-107 can preserve more stem cells in limbal epithelial cultures via facilitating stem cell self-renewal. To test this hypothesis, we will perform gain- and loss-of-function studies for miR-107 to determine whether such microRNA regulates the stem cell self-renewal in limbal epithelium by holoclone assays in submerged cultures and label-retaining cell assays in 3D organotypic rafts. Since Akt2 has been shown to play an important role in stem cell self-renewal, we will also investigate whether Akt2 is a direct target of miR-107 by luciferase assays and whether knock down of Akt2 will increase the stem cell numbers in submerged or 3D organotypic raft cultures by holoclone assays and label-retaining cell assays. Proper numbers of stem cells in limbal epithelial cultures is critical for the successful transplantation in LSCD patients. This study will help explore the previously unconsidered roles of miR-107 in maintaining the stem cell self-renewal, which will form the foundation for development of innovative approaches to generate stem cell-enriched limbal epithelial cultures. Establishment of such stem cell-enriched cultures has a potential to significantly improve the outcome of transplantation in LSCD.
|Effective start/end date
|7/1/14 → 6/30/15
- Eversight (Check 76095)
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