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R. D. Megaw, C. Osei-Bempong, B. Shaharuddin, S. Ahmad; Sequential Collagen IV and Laminin Attachment Enriches for Human Corneal Epithelial Progenitors. Invest. Ophthalmol. Vis. Sci. 2010;51(13):3762.
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© ARVO (1962-2015); The Authors (2016-present)
Collagen IV and laminin are basement membrane components in the limbal stem cell (LSC) niche. Collagen IV attachment has been shown to enrich for corneal epithelial progenitors i.e. LSCs and transient amplifying cells. Laminin is also produced by LSCs and has been implicated as an important factor in their niche. The purpose of this study was to elucidate whether combined collagen IV and laminin attachment enriches for the corneal epithelial progenitors.
Limbal epithelial cells were isolated from human limbal tissue, which had been donated for research and obtained from the UK Eye Bank Service, and cultured for 10 days until confluence on mitotically inactivated 3T3 fibroblasts. Culture wells were coated with collagen IV and laminin separately. After the 10 day culture, epithelial cells were trypsinised and re-suspended in limbal epithelial medium. The cell suspension was then placed in either collagen IV or laminin wells at 37°C for 2 hours. Attached cells were removed by trypsinisation. Cells which attached to collagen IV were then placed in laminin wells at 37°C for 2 hours (and vice versa for those which attached to laminin). After 2 hours, attached cells were trypsinised. Cell samples were analysed for colony forming efficiencies (CFEs) and by real time PCR for putative corneal epithelial progenitor markers p63 and ABCG2. Experiments were performed in triplicate. Data was statistically analysed using ANOVA.
The 5 samples analysed in this study were cultured limbal epithelial cells (LECs), cells attaching to collagen IV alone (C), cells attaching to laminin alone (L), cells attaching to collagen IV and then laminin (CL), and cells attaching to laminin and then collagen IV (LC). CFE of LECs was the lowest (4.2%) followed by those for L and C (10-14%), and highest for CL and LC (18-25%). These results were statistically significant (p<0.01). Real time PCR data showed that p63 and ABCG2 expression was lowest in LECs, followed by C and then L, with the highest expression being in CL and LC (p<0.05).
In conclusion, this study shows that although both collagen IV and laminin can be used to enrich for corneal epithelial progenitors by attachment, when used sequentially, the enrichment is further enhanced. This is corroborated by both the CFE and real time PCR data.
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