Wnt Signaling Is Required for the Maintenance of Human Limbal Stem/Progenitor Cells In Vitro

Abstract

PurposeA chemical approach to examine the role of Wnt signaling in maintaining the stemness and/or proliferation of limbal stem/progenitor cells (LSCs).MethodsLSCs were isolated from human donor eyes and cultured as single cells for 12 to 14 days with the following small molecules: IIIC3, an antagonist of the Wnt signaling inhibitor Dickkopf (DKK), and IC15, a Wnt signaling inhibitor. Proliferation of LSCs in the presence of IIIC3 and IC15 was determined by the number of cells and colonies established. Maintenance of stemness was determined by p63α, cytokeratin (K)12, and K14 expression.ResultsActivation of Wnt, through IIIC3-mediated DKK inhibition, resulted in similar colony forming efficiency (CFE) as in the untreated LSCs, but significantly increased the number of cultivated cells 7.21% with 5 μM. Inhibition of Wnt with IC15 significantly reduced the CFE (P ≤ 0.01) and the number of cultivated cells by 16% to 29%. Percentage of cells expressing high levels of p63α (p63αbright) and quantity of small cells (≤12 μm), which contain the LSCs, increased 4.71% and 11.26% (both P < 0.05), respectively, with 5 μM IIIC3. All concentrations of IIIC3 and IC15 retained the K14 undifferentiated marker (97%), while differentiation, as detected by expression of K12, was found in up to 2% of cells in 1 μM IIIC3, 1 μM IC15, or 5 μM IIIC3.ConclusionsWnt signaling is required in LSC proliferation and maintenance of an undifferentiated state. The current study is a proof of concept that the Wnt pathway could be modulated in LSCs to enhance or decrease the efficiency of human LSC expansion.

KIT ligand produced by limbal niche cells under control of SOX10 maintains limbal epithelial stem cell survival by activating the KIT/AKT signalling pathway

“…Growth factors are key components for the development and function of the limbal niche and accumulating evidence suggested that they play important roles in regulating LESCs survival and development 22,38,41‐46 . In addition, as shown above, LNCs act in a paracrine fashion to support LESCs in vitro, and so, we decided to analyse whether SOX10 acts through growth factors to influence the survival of LESCs in LNCs.…”

“…that they play important roles in regulating LESCs survival and development. 22,38,[41][42][43][44][45][46] In addition, as shown above, LNCs act in a paracrine fashion to support LESCs in vitro, and so, we decided to analyse whether SOX10 acts through growth factors to influence the survival of LESCs in LNCs. To identify which factors might be involved, we compared the expression of a series of growth factor genes in si-Sox10 transfected LNCs vs si-C or mock-transfected LNCs.…”

Modulation of Wnt/BMP pathways during corneal differentiation of hPSC maintains ABCG2-positive LSC population that demonstrates increased regenerative potential

“…There are also studies suggesting similar regulation of quiescence and stem cell activation/renewal by the Wnt/BMP signaling pathways in the limbus. In general, Wnt signaling and suppression of BMP are required for LSC proliferation and maintenance of an uncommitted state [42, 43]. However, the outcome appears to be dependent on the culture system used, as the same exogenous signals have been reported to have opposite effects in explant versus isolated limbal cultures [44].…”

The Canonical Wnt Pathway as a Key Regulator in Liver Development, Differentiation and Homeostatic Renewal

“…Wnt/β-catenin signalling plays a key role in embryogenesis, as evidenced by loss of function gene knockout studies targeting components of the Wnt/β-catenin pathway including LRP5/6 [ 5 ], Wnts [ 6 ] and FZDs [ 7 ] (for review see [ 8 ]). The Wnt/β-catenin signalling pathways plays a central role in regulating proliferation and differentiation during development as well as in stem cell renewal [ 9 , 10 ]. These properties make it an attractive prospect for those seeking druggable targets in the treatment of cancer, in which Wnt/β-catenin signalling is often implicated [ 3 ].…”

“…Wnt/β-catenin signalling confers a proliferative capability in the liver throughout development and adulthood by promoting transcription of genes associated with re-entry into the cell cycle including CyclinD1 and c-Myc [ 67 , 107 , 108 ]. Resident stem cells from many tissues, for example intestine, express components of the Wnt/β-catenin signalling machinery LGR5 and Axin2, and Wnt/β-catenin signalling has been shown to maintain self-renewal in stem cells [ 10 , 109 , 110 ]. Evidence from tumorigenesis studies support the idea that active β-catenin signalling promotes hepatocyte proliferation [ 91 , 111 , 112 ].…”