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Jiexi Zeng, Yan Zhang, Grace May Chuang, Christine Frances Wildsoet; Culture of Guinea Pig Limbal Epithelial Stem Cells in Three Different Media. Invest. Ophthalmol. Vis. Sci. 2017;58(8):1116. doi: https://doi.org/.
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Limbal epithelial stem cells (LESCs) are a tissue-specific stem cells located in the limbal palisades of Vogt at the corneo-scleral junction of the eye. It has been previously shown that LESCs can be converted to both corneal epithelial cells and corneal keratocytes. Our interest is in whether LESCs can be used as a source of scleral fibroblasts, for treatment of high myopia, the first step being to isolate LESCs from our guinea pig model of myopia.
Limbal tissue, harvested from 1-4 year-old guinea pigs, was dissected to isolate superficial layers, which were then subjected to a two-step enzyme digestion using Dispase II and Trypsin-EDTA. Cell suspensions were expanded ex vivo on fibronectin-coated cell culture plates in one of three different media: Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS), serum-free keratinocyte medium (KSFM), or KSFM with 10% FBS. Immunolabelling with p63a, a nuclear transcription factor and ABCG2, another putative marker of stem-ness, was used to distinguish LESCs from other cell types. Effects on cell morphology proliferation and viability were also evaluated.
Guinea pig LESCs were successfully isolated and cultured in three different media. After one week of culture, there was no significant difference in total cell numbers, density and viability related to the culture medium used. However, after another week (day 14), the yield of LESCs was much higher with KSFM compared to DMEM+FBS and KSFM+FBS. With the KSFM medium, most cells exhibited epithelial cell-like morphology, with positive staining of nuclei for p63α and cell membranes/bodies for ABCG2.
A simple, reproducible method for isolating, culturing and characterizing guinea pig LESCs was developed. Our next step will be to attempt conversion into scleral fibroblasts for transplantation into a guinea pig myopia model.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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