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Yi-Hsin Chen, I-Jong Wang, Tai-Horng Young; Time-elapse Colony Formation Characteristic Of Bovine Limbal Epithelial Cells On Different Feeder Layers. Invest. Ophthalmol. Vis. Sci. 2011;52(14):324.
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To investigate the time-elapse colony formation characteristic of bovine limbal epithelial cells on embryonic fibroblast, corneal fibroblast, and dermal fibroblast.
Bovine corneal limbal epithelial cells (LEC) were harvested on mitomycin-treated embryonic fibrobast (NIH-3T3), corneal fibroblast, dermal fibroblast, and in supplement hormonal essential media (SHEM). Colony formation efficiency was examined on day 7. Time-elapse phase contrast microscope was used to observe the dynamic colony formation from single cells. Limbal epithelial stem cell marker, such as p63, ABCG2, and K14, corneal epithelial marker K3, proliferative association marker Ki67 examined on day 3.5, day 5 and day 7.
LECs were adhered to three kinds of feeder layers at 48 hr. Single cells were observed to proliferate at 72 hr. LECs were then formed 8-cell colony within additional 12hrs on three kinds of feeder layers. Colony formation efficiency, however, were 11.7% (1.5%), 6.3% (0.3%), and 2.1% (0.4%) on 3T3, corneal fibroblast, and dermal fibroblast, respectively (mean (st)). On day 3.5, LECs on all feeder layers were all express limbal epithelial stem cell markers and Ki67 but not corneal epithelial marker K3. On day 5, the portion of nucleus p63-positive cells were distributed in the outer region of the colony on three kinds of feeder cells. The location of K14 and ABCG2 were also located at the outer region of colony. The ratio of nucleus p63-positive cells in colony, however, was significantly decreased on corneal fibroblast and dermal fibroblast feeder layers. Ki67 positive cells were randomly distributed in the colony on three feeder cells and still K3 negative. On day 7, p63 trans-located from nucleus to cytoplasm was observed in colonies on dermal fibroblast feeder layers, whereas the p63 was still located in nucleus portion and randomly distributed in colonies on 3T3 feeders. The lost of ABCG2 expression were observed on dermal fibroblast feeder layer. On 3T3 feeders, however, ABCG2-positive cells were randomly located in the colony. On corneal fibroblast feeders, nucleus p63-positive cells, Ki67 positive cells and ABCG2-positive cells mostly located at the junction of colony and feeders.
Low efficiency of LECs colony formation on dermal fibroblast is related to p63 trans-located from nucleus to cytoplasma.
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