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Hua Mei, Martin N. Nakatsu, Madelena Ng, Thuy T. Truong, Grace Lee, Sophie X. Deng; Differential Expression Of Frizzled 7 In Human Corneal Epithelial Stem Cells. Invest. Ophthalmol. Vis. Sci. 2011;52(14):5114.
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Wnt signalling pathway has been shown to play an essential role in regulating epithelial stem cell maintenance/differentiation. Frizzled 7 (Fz7), the receptor of Wnt ligand, is investigated in human corneal epithelial stem/progenitor cells.
RNAs extracted from the corneal and limbal epithelia of 3 donors were DNase treated and reverse-transcribed into cDNA. Quantitative real-time PCR was performed to examine the expression pattern of Fz7 in human cornea and limbus. Immunohistochemistry was performed to examine the protein expression of the Fz7 in human cornea and limbus. Limbal stem/progenitor cells were further enriched from the primarily isolated limbal epithelial cells based on their absence of SSEA4 expression. Expression of Fz7 in the enriched primary limbal stem/progenitor cells was examined through quantitative real-time PCR. Limbal epithelial cells were cultured on mouse 3T3 feeder layer for 14 days and then isolated from the feeder layer using collagenase. Expression of stem cell markers were evaluated using qRT-PCR and immunohistochemistry.
Expression of Fz7 was consistently at least 4-fold higher in the limbus than in the cornea in all 3 donors at the mRNA level. Immunohistochemistry of the human sclerocorneal tissue revealed that Fz7 was predominantly expressed in the basal epithelium of human limbus. SSEA4- limbal epithelial cells showed a higher expression of the putative limbal stem cell markers, ABCG2, ΔNp63α, and keratin 14 than the SSEA4+ population, indicating a less differentiated state. Expression of Fz7 was also 1.4-fold higher in the SSEA4- cells. After expansion of the limbal epithelial cells on 3T3 feeder layer, expression of Fz7 decreased significantly. Expression of ABCG2 and ΔNp63α also decreased.
Fz7 is preferentially expressed in limbal stem/progenitor cells. Current expansion method of limbal epithelial cells does not favor stem cell self-renewal. Wnt signaling via Fz7 might be important in maintaining limbal stem cell phenotype.
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