Stein Eye Institute, University of California, Los Angeles, California, United States.
Invest Ophthalmol Vis Sci. 2019 Jan 2;60(1):107-112. doi: 10.1167/iovs.18-25740.
A chemical approach to examine the role of Wnt signaling in maintaining the stemness and/or proliferation of limbal stem/progenitor cells (LSCs).
LSCs 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.
Activation 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.
Wnt 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.
采用化学方法研究 Wnt 信号在维持角膜缘干细胞/祖细胞（LSCs）的干性和/或增殖中的作用。
从人供体眼中分离 LSCs，作为单细胞培养 12 至 14 天，使用以下小分子：IIIC3，Wnt 信号抑制剂 Dickkopf（DKK）的拮抗剂，和 IC15，一种 Wnt 信号抑制剂。通过细胞数量和建立的集落来确定 LSCs 在 IIIC3 和 IC15 存在下的增殖情况。干性的维持通过 p63α、细胞角蛋白（K）12 和 K14 的表达来确定。
通过 IIIC3 介导的 DKK 抑制激活 Wnt，导致与未处理的 LSCs 相似的集落形成效率（CFE），但在 5 μM 时显著增加了 7.21%的培养细胞数量。用 IC15 抑制 Wnt 显著降低了 CFE（P≤0.01）和培养细胞数量 16%至 29%。高表达 p63α（p63αbright）的细胞百分比和包含 LSCs 的小细胞（≤12 μm）的数量分别增加了 4.71%和 11.26%（均 P<0.05），而 5 μM IIIC3。所有浓度的 IIIC3 和 IC15 保留了未分化的 K14 标记（97%），而分化，如 K12 的表达所检测到的，在 1 μM IIIC3、1 μM IC15 或 5 μM IIIC3 中高达 2%的细胞中发现。
Wnt 信号在 LSC 增殖和维持未分化状态中是必需的。本研究证明了 Wnt 途径可以在 LSCs 中被调节，以提高或降低人 LSCs 扩增的效率。
