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T. Kawakita, A. Hornia, S. Shimmura, K. Higa, H. Miyashita, 1, K. Tsubota, J. Shimazaki, S.C. G. Tseng; Corneal Epithelial Sheet Equivalent Generated From a Single Murine Corneal/Limbal Epithelial Progenitor Cell . Invest. Ophthalmol. Vis. Sci. 2006;47(13):1111.
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We previously reported that murine corneal/limbal epithelial cells rapidly senesce when cultured on plastic in a low [Ca2+] serum–free KSFM at a high density and passaged every one week. We speculate that such a failure owes to predominant expansion of trans–amplifying cells (TAC), and can be overcome if a low seeding density and a prolonged cultivation time is used.
Murine corneal/limbal epithelial sheets were isolated by similar diapase digestion, trypsinized, seeded at 500, 5000 and 50,000 cells per cm2 on plastic in KSFM, and passage every 3 to 4 weeks. After successful expansion over 20 passages, single cell limiting dilution in 96 wells was performed to generate clonal growth curve, doubling time and selection. Differentiation was promoted by increasing extracellular [Ca2+] to 0.9 mM and/or 5% FBS and analyzed by immunostaining and western blotting against p63, Pax 6 and Keratin 12. Karyotyping was performed by cytogenetic analysis. A single cell was expanded into a monolayer on denuded amniotic membrane and air–lifted.
A low cell density (<500 cells per cm2) and a prolonged culture time (beyond the life span of TAC) allowed clonal expansion of small epithelial progenitor cells which could be selected into three clones and continuously maintained beyond 45 passages. The doubling time was 31.6 ± 0.5 h during the first 10 days, and cells had uniformly positive nuclear p63 staining with ΔNp63 expression but negative K12 staining, and had normal karyotyping. Nevertheless, they became large squamous cells with negative nuclear p63 but positive K12 by elevating [Ca2+] and adding FBS or increasing the seeding density. Late clonally expanded small cells showed weak Pax 6 expression. A single cell could be expanded to a monolayer on amniotic membrane and promoted into stratification.
Murine corneal/limbal epithelial progenitor cells can be preferentially preserved and continuously expanded in KSFM by a low seeding density and a prolonged culturing time. These progenitor cells can be clonally expanded and rendered proper differentiation and stratification.
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