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Sophie Deng, Martin Nakatsu, SHEYLA GONZALEZ, Hua Mei; Expansion of Human Corneal Epithelial Stem/Progenitor Cells in Feeder-Free Explant Cultures. Invest. Ophthalmol. Vis. Sci. 2013;54(15):999.
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© ARVO (1962-2015); The Authors (2016-present)
To present a feeder-free culturing method of human corneal epithelial stem/progenitor cells in vitro.
Primary limbal epithelial cell (LEC) sheets isolated from human sclerocorneal tissues were trypsinized to produce single LECs and co-cultured with growth arrested 3T3-J2 feeder cells for 14 days. Additionally, the outgrowth of LECs from limbal explant pieces was also cultured for 14 days in the presence or absence of 3T3-J2 feeder. The phenotype of the cultured LECs was assessed by their mRNA expression level of putative stem cell markers and differentiation marker by qRT-PCR and immunocytochemistry. The percentage of p63 bright cells in each culture was assessed, and the cell proliferation was evaluated by the Ki67 expression and cell number.
We observed no significant difference in cultured LEC morphology among each culturing method. The LEC growth rate increased over 9-fold in NF explant cultures compared to 3T3-J2 explant cultures and the adjusted growth rate between NF cultures and 3T3-J2 single LEC cultures had similar yields (p>0.05). Gene expression of putative limbal stem cell markers, ABCG2 and ΔNp63 were elevated among NF cultures compared to the gold standard and explants on 3T3-J2. We observed a 7.6-fold and 2.2-fold increase in ABCG2 and ΔNp63 expression respectively when comparing NF explant cultures to the gold standard, while there was only a 4.4-fold and 1.4-fold increase in ABCG2 and ΔNp63 expression respectively when comparing 3T3-J2 explant cultures to the gold standard. However, we did observe an increase in K12 expression in NF explant cultures when compared to the gold standard (6.2-fold). There was a large increase in Ki67 proliferation (4.0-fold) when compared to the gold standard. There was no difference in Ki67 expression between 3T3-J2 explant cultures and the gold standard. Finally, examination of p63α expression in each condition reviewed no discernable differences in the percentage of p63α bright cells between the gold standard (9.0%) and NF explant cultures (10.0%), but we did see a decrease in the 3T3-J2 explant cultures (6%).
3T3 feeder cells may not be necessary for the growth of stem/progenitor cell population in the primary explant culture. The explants themselves may already contain niche factors that are required for the viability of corneal stem cells.
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