Wnt signaling pathway plays an important role in the regulation of human limbal stem/progenitor cells (LSCs). To examine the possible function of Frizzled (Fz) receptors in LSCs, the expression of ten Fz receptors was profiled in the limbus and cornea. Only Fz7 had preferential expression in the basal limbal epithelium which contains the LSCs. The expression of Fz7 was co-localized with the putative LSC markers including p63α, N-cadherin and keratin (K) 14 and was minimum in cells expressing the corneal maturation marker K12. The expression of Fz7 was higher in the enriched LSCs population and decreased in cultured LSCs when there was a loss of progenitor phenotype. When the Fz7 was knocked down (FzKD) using shRNA in primary LSCs, the expression of putative LSCs markers ABCG2, ΔNp63α and K14 was decreased significantly. The colony forming efficiency of the Fz7KD LSCs was significantly decreased in the subsequent passage 1 and 2 compared to the control. Our finding suggests that Wnt signaling is one of the factors of LSC niche maintenance and Fz7 helps to maintain the undifferentiated state of LSCs.

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.

The current standard method to culture human limbal stem/progenitor cells (LSCs) in vitro is to culture limbal epithelial cells directly on a layer of murine 3T3 feeder cells (standard method). The direct contact between human cells and murine feeder cells poses the potential risk of incomplete removal of feeder cells after culture and cross-contamination in clinical applications. We present here a novel three-dimensional (3D) sandwich method in which LSCs and feeder cells were separately cultured on opposite sides of a porous membrane. Limbal epithelial cells in the form of single-cell suspensions, cell clusters, and tissue explants were subjected to standard culture or to a 3D sandwich culture method. The 3D sandwich method consistently yielded LSCs derived from cell clusters and tissue explants. The expanded LSCs exhibited a small, compact, cuboidal stem-cell morphology and stem cell phenotypes comparable to those of LSCs derived from the standard culture method. Limbal epithelial cell clusters cultured with the sandwich method had a significantly higher proliferation rate than did those cultured with the standard method. The 3D sandwich method did not favor the propagation of single LSCs. In summary, the 3D sandwich method permits complete separation between cultured cells and feeder cells, while providing an even and maximal proximity between them. This alternative method permits culturing of LSCs without the risk of feeder cell contamination.