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Djida Ghoubay, Kate Grieve, celine De Souza, Raphaël Martos, OLIVIER THOUVENIN, Vincent M Borderie; New method for isolating and growing both corneal stromal and epithelial limbal stem cells. Invest. Ophthalmol. Vis. Sci. 2017;58(8):1384.
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© ARVO (1962-2015); The Authors (2016-present)
To develop a new method to isolate and grow both corneal stromal and epithelial limbal stem cells from small human limbal biopsies.
Superficial limbal explants were retrieved from human donor corneo-scleral rims. They were first assessed with full field optical coherence microscopy and confocal microscopy. Corneal stromal stem cells (SSC) and limbal epithelial stem cells (LSC) were isolated by digestion with collagenase A. Isolated cells were cultured either with Essential 8 medium (E8), E8 medium supplemented with EGF (E8+) or Green’s medium on a layer of 3T3 feeders (Green’s). Cells were characterized by immunostaining for p63 alpha, Pax6, Keratocan, CK3 and nestin, colony forming efficiency, sphere formation, number of population doubling before senescence and differentiation potential assessed by culture with specific media.
Pre-culture assessment of limbal explants showed presence of limbal stem cells in the limbal crypts and stromal stem cells in the limbal stroma. Limbal stem cells characterized by holoclones and p63alpha expression were obtained with E8+ and Green’s culture conditions. They featured 21 population doublings and only corneal epithelial differentiation. Stromal stem cells characterized by sphere formation, pax6, keratocan and nestin expression were obtained with E8 and E8+ culture conditions. They featured 47 population doublings and keratocyte, fibroblast, myofibroblast, neuron, adipocyte, chondrocyte, osteocyte and melanocyte differentiations, with no epithelial differentiation.
Both corneal stromal and limbal epithelial human stem cells can be obtained from small superficial biopsies. Depending on the culture condition one or both cell types can be grown separately. E8+ medium helps us to obtain the two types of stem cells in the same culture dish. This finding demonstrates that both stem cell types are located in the same niche close to each other
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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