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Hua Mei, Martin N. Nakatsu, Thuy T. Truong, Patrick Pham, Grace Lee, Sophie X. Deng; Wnt Signaling Pathway Regulates the Differentiation of Human Corneal Epithelial Stem/Progenitor Cells via the Frizzled 7 Receptor. Invest. Ophthalmol. Vis. Sci. 2012;53(14):5246.
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© ARVO (1962-2015); The Authors (2016-present)
Frizzled 7 (Fz7), one of the Wnt receptors, is preferentially expressed at the human limbal basal epithelium. This study aimed to investigate the possible function of Fz7 in the regulation of human corneal epithelial stem/progenitor cells (CESCs).
Freshly human limbal epithelial cells in single cell suspension were isolated from sclerocorneal tissue using Dispase II digestion followed by trypsin/EDTA treatment. The limbal epithelial cells were transduced with lentivirus that contained plasmid construct of Fz7 shRNA-GFP (Fz7KD) or scrambled control shRNA-GFP. The transfected cells were cultured on growth-arrested 3T3 feeder cells for 3 to 4 weeks (P0). The GFP-positive cells were selected through either flow cytometry or scraping away the GFP-negative cells/colonies. The phenotype of the cells at P0 was evaluated by the expression level of putative stem cell marker and differentiation marker. The GFP-positive cells from P0 were passaged twice (P1 and P2) to examine the colony-forming efficiency (CFE).
After expansion of the limbal epithelial cells on 3T3 feeder layers, the expression level of putative stem cell markers, ABCG2, ΔNp63 and N-cadherin decreased 77%, 44%, and 73%, respectively (all p<0.0001), whereas the differentiation marker keratin (K) 12 was reduced by 99.5% after culture as expected because of selection of progenitor cells in the culture (p<0.0001). Interestingly, the expression of Fz7 was also significantly decreased by 82% (p<0.0001). After the primary limbal epithelial cells were transduced with the lentiviral Fz7KD or control scramble shRNA, the Fz7 mRNA level was knocked down by 37% in the Fz7KD compared to that in the control (p<0.0001). The reduced expression of Fz7 was confirmed by Western blot at the protein level. In the Fz7KD population, expression of ABCG2, ΔNp63α and K14 was decreased by 25%, 20%, and 20%, respectively (all p<0.05), however expression of N-cadherin and K12 did not differ from the control population. The Fz7KD cells produced 18% and 44% fewer CFE in P1 and P2, respectively compared to the control.
Loss of stem/progenitor cell phenotype is associated with the loss of Fz7 expression. The results suggest that Wnt signaling maintain the undifferentiated state of CESCs via the Fz7 receptor.
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