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

Abstract

Purpose: A chemical approach to examine the role of Wnt signaling in maintaining the stemness and/or proliferation of limbal stem/progenitor cells (LSCs).

Methods: 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.

Results: 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.

Conclusions: 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.

Figure 1

Effect of IIIC3 and IC15 on limbal stem/progenitor cell (LSC) proliferation. (A) Treatment of LSCs with IIIC3 and IC15 did not change the cuboidal morphology. (B–E) Different concentrations of IIIC3 and IC15 affect colony forming efficiency (CFE) and cell proliferation of LSCs. IIIC3 treatment generated larger colonies while IC15 treatment generated colonies of smaller size (B). The CFEs obtained from IIIC3 treatment were similar to those for untreated controls except at the highest concentration of 20 μM. CFE was significantly reduced with all concentrations of IC15 (C). Cell population doubling calculated as Log₂ (no. cells harvested/no. cells seeded) (D). Cell numbers were significantly increased with 5 μM IIIC3, but decreased in a dose-dependent manner for IC15 treatment, and 20 μM IIIC3 (E). *P < 0.05.

Figure 2

LSCs continue to actively divide upon small-molecule treatment. (A) Immunostaining of Ki67 in LSCs treated with IIIC3 and IC15. (B) Ki67 expression is significantly increased in LSCs treated with 20 μM IIIC3. Percentage of Ki67⁺ cells showed no significant differences between all tested concentrations of IIIC3 and IC15. Scale bar: 20 μm. *P < 0.05.

Figure 3

Maintenance of limbal stem/progenitor cell (LSC) undifferentiated phenotype is IIIC3 concentration dependent. (A) Expression of p63α is retained in LSCs upon IIIC3 treatment but reduced with all tested concentrations of IC15. (B, C) Quantification of p63^bright cells showed a significant increase with 5 μM IIIC3. IC15 treatment reduced the percentage and the total number of p63^bright cells (D). The percentage of cells maintaining a cell size smaller than 12 μm was significantly increased with 5 μM IIIC3. There was a trend of decrease with IC15 treatment. *P < 0.05, **P < 0.001.

Figure 4

Expression of K14 is maintained by LSCs treated with IIIC3 or IC15. (A) Immunostaining of K14, the LSC marker, and K12, the differentiation marker. (B) Most cells retain K14 expression. (C) K12 is detected in a small population of LSCs, and is increased within a narrow range, 1 to 5 μM IIIC3 and 1 μM IC15. *P < 0.05.