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Justyna Kanska, Martin N Nakatsu, Jie Zheng, Sophie Xiaohui Deng; Effect of Wnt signaling small molecule modulators on the cultured human LSCs.. Invest. Ophthalmol. Vis. Sci. 2014;55(13):6042.
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© ARVO (1962-2015); The Authors (2016-present)
To investigate the effect of Wnt small molecule modulators on human limbal stem/progenitor cells (LSCs) in vitro.
Primary human limbal epithelial cells were isolated with Dispase II and cultured on the 3T3-J2 monolayer with or without (control) small molecules that either activate or inhibit Wnt signaling pathway. These Wnt compounds were designed to specifically bind to the LRP5/6 receptor to either prevent Dickkopf binding (activator, IIIc3) or Wnt ligands binding (inhibitor, IC15). LSCs were cultured for 14-21 days, the cell numbers and colony formation efficiency (CFE) were measured, as well as the phenotype of the cultured cells, including cell morphology, expression of the putative stem cell markers at the mRNA and protein level, was analyzed.
IIIc3 at higher concentration decreased CFE but increased cell growth by 75% and upregulated the expression of ABCG2 in comparison to that of the control. In contrast, the presence of IC15 decreased CEF and cell growth. It also resulted in the downregulation of ABCG2. No significant difference was observed in the expression of Keratin (K) 14, Ki67 and ΔNp63 at the mRNA level in all cultures. Interestingly, both Wnt activator and inhibitor resulted in a significant decrease in N-cadherin expression. Furthermore, our immunocytochemical data shows that neither of the Wnt small molecules significantly affected the percentage of K14+ cells. The percentage of K12+ cells was decreased in cultures supplemented with both IIIc3 and IC15. The cell morphology was not affected by either of the small molecules.
Our preliminary data suggests that modulation of Wnt signaling could increase the efficiency of LSCs growth in vitro without promoting cell differentiation.
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