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SHEYLA GONZALEZ, Martin Nakatsu, Hua Mei, Sophie Deng; Comparison of Different Limbal Epithelial Stem Cell Isolation Methods to Improve the Epithelial Sheet Quality for Transplantation. Invest. Ophthalmol. Vis. Sci. 2013;54(15):989. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
To investigate different preparation methods of human limbal stem cells (LSCs) for cultivation using dispase II, collagenase A and the explant culture.
Limbal tissues were incubated with dispase II at 37°C for 2 hours (h) or with collagenase A at 37°C or 4°C for 2h or overnight to obtain limbal epithelial cell sheets. Half of the cell sheets were further incubated with trypsin to achieve single cell suspension. The explant culture was done using a 2x2 mm limbal biopsy. Limbal epithelial cells and limbal tissues were cultured on growth-arrested 3T3-J2 mouse cells in SHEM5 medium. The phenotype assessment included stem/progenitor morphology and expression level of LSC putative markers including ABCG2, p63α, N-cadherin and cytokeratin (K) 14, the differentiation marker K12, and the stromal cell markers vimentin (Vim) and α-smooth muscle actin by qRT-PCR and immunohistochemistry.
No LSC growth on 3T3-J2 cells was achieved when limbal tissues were incubated with collagenase overnight at 37°C. Among the other collagenase treatments tested, the most efficient was incubation at 37°C for 2h in the incubator. This isolation method yielded the highest K14+Vim- progenitor cell population (86.2%) and a moderate K14-Vim+ stromal cell number (9.7%). The LSCs had the best stem/progenitor-like morphology and the highest level of putative LSC markers. Collagenase but not dispase isolated the entire epithelial sheet with the underneath stromal cells. When the phenotype of cultured LSCs was compared among all three culture methods, all LSCs displayed a stem/progenitor-like morphology. However, LSCs cultured from collagenase digestion had the highest mRNA level of putative LSC markers and the highest percentage of p63α bright cells (21.7% vs. 6% from explant and 12.7% from dispase, P<0.05). Cultures from collagenase isolation and explant yielded a higher total number of p63α bright cells (P=0.3) that was 4-fold higher than that from dispase isolation, due to a higher cell growth rate in explant. When LSCs were cultured as cell sheets, the mRNA level of putative LSC markers was higher than that from as single cell suspension.
Limbal stromal cells from the collagenase treatment and in the explant culture support a more efficient expansion of LSCs in vitro on 3T3 feeder cells. The presence of native LSC niche appears to help maintaining the LSC phenotype.
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