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J. Li, H. Wu, R. W. Beuerman; The Effect of Wnt Signaling on Human Limbal Epithelial Cell Proliferation and Differentiation. Invest. Ophthalmol. Vis. Sci. 2009;50(13):2048.
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© ARVO (1962-2015); The Authors (2016-present)
To understand the role of Wnt pathway on the proliferation and differentiation of cultured human limbal progenitor cells.
Human limbal epithelial cells were enzymatically isolated from 10-day postmortem corneal scleral tissues (donor age 46-76 years old). Limbal and corneal fibroblasts were grown by "explant" methods. Passage 1-2 cells were used in this study. Human fetal dermal epithelial cells (HDF) were purchased from Invitrogen. Limbal epithelial cells were initially propagated in SHEM and MMC-treated 3T3 cells. Wnt pathway PCR array was purchased from SuperArray. Gene expression was determined by Taqman real time PCR analysis. The 3T3 cell conditioned medium (3T3-CM) was concentrated 100-fold by a Millipore centrifugal filter device with a 10,000 nominal molecular weight limit. COS-7 cells were transfected with TOP/FOP FLASH luciferase reporter plasmid. The luciferase activity in COS-7 cells was measured by Bright-GloTM Luciferase assay system.
Limbal epithelial cells showed highest colony forming efficiency and cell proliferation when co-cultured with MMC-treated 3T3 cells, followed by MMC-treated HDF, limbal fibroblasts and corneal fibroblasts. Wnt2b expression was 2.42 fold higher; Wnt1 and Wnt7A expression was 3.45 and 6.62 fold lower in limbal fibroblasts compared to corneal fibroblasts of the same donor. When compared to HDF, limbal fibroblasts showed a 2.5 to 5.0-fold increase in Wnt5, Wnt2 and Wnt16 expression. Serially passaged limbal epithelial cells showed increased expression of multiple Wnt genes. Five microliters of 3T3-CM inhibited basal, Wnt3a stimulated, and GSK-3X stimulated TCF-luciferase activity by 57%, 61% and 83% in COS-7 cells, respectively, and the inhibition increased in a volume-dependent manner. When stimulated with 100 ng/mL purified DKK1 protein, a Wnt signaling inhibitor, human limbal epithelial cells showed marginally increased colony forming efficiency but decreased cell proliferation. On the other hand, Wnt3a at 100 ng/mL increased limbal cell proliferation by 15% compared to PBS-treated controls. DKK1 also increased p53 and p63 gene expression and inhibiting Ck3 and Ck12 gene expression.
Our study suggested that the Wnt signaling pathway regulates the balance between proliferation and differentiation of human limbal epithelial cells. The inhibition of Wnt signaling is an important mechanism responsible for 3T3 cell promoted maintenance of stem cell like features of human limbal epithelial cell.
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