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Hua Mei, Chi Zhang, Elfren Ray Baclagon, Jie J Zheng, Sophie Xiaohui Deng; Small Wnt Inhibitors Modulate the Expansion of Limbal Stem/Progenitor Cells in vitro. Invest. Ophthalmol. Vis. Sci. 201657(12):.
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© ARVO (1962-2015); The Authors (2016-present)
To study the possible function of small molecules of Wnt inhibitors in regulating the in vitro expansion of limbal stem/progenitor cells (LSCs).
Two Wnt inhibitors (MFH, ND) were tested in this study. Single cell suspension of limbal epithelial cells were cultured on 3T3-J2 cells in the SHEM medium containing 1, 2 or 5μM of small Wnt molecules for up to 2 weeks. Cells cultured in medium containing corresponding concentrations of DMSO served as the control. Medium were refreshed every 2-3 days and single cells were collected at the end of culture. Cells were analyzed for morphology, proliferation rate, and percentages of keratin (K) 14-positive, K12-positive, p63α-bright cells and small cells (cell diameter ≤ 12μm).
At 1μM, MFH did not affect cell proliferation or cell morphology but significantly reduced the percentage of small cells by 56% (p<0.05). At 2μM, MFH increased cell proliferation by 19% (p<0.05) and induce vacuoles formation. At 5μM, MFH decreased the proliferation by 51% (p<0.05) and majority of cells showed big cytoplasmic or secreted vacuoles. MFH had no effect on percentage of small cells at 2 or 5 μM and no effect on percentage of K14-positive, K12-positive, or p63α-bright cells at all tested concentrations. Cells cultured with ND had comparable epithelial morphology to the control at all concentrations but showed different proliferation rate and stem-cell phenotype. At 1μM, ND decreased the percentage of small cells by 63% (p<0.05) and did not alter the proliferation rate or expressions of markers. ND increased the percentage of K14-positive cells by 4% at 2μM (p<0.05) and did not affect the proliferation rate or percentages of p63α-bright, K12-positive, or small cells. At 5μM, ND decreased cell proliferation rate by 18% (p<0.05) but increased the percentage of p63α-bright cells by 2 folds (p<0.05). It had no effect on percentages of K14-positive, K12-positive or small cells at 5μM.
MFH differentially regulated cell proliferation and change the morphology at different concentrations without affecting the expression of K12, K14 and p63α. ND enhanced the population of the undifferentiated LSCs at higher concentrations although it decreased the proliferation rate. Small Wnt molecules targeting different components of Wnt signaling pathway might exert different effects on cultured LSCs within a narrow window of concentration.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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