Abstract
Purpose: :
To investigate the possible role of Wnt6 signaling in human corneal epithelial stem/progenitor cells (CESCs).
Methods: :
3T3-J2 feeder cells were transduced with retrovirus that contained the neomycin-resistant LNCX vector expressing the Wnt6 gene. Infected 3T3-J2s were selected against G418 and the surviving 3T3-J2 colonies were expanded. Overexpression of Wnt6 in 3T3-J2 cells (Wnt6-3T3) was confirmed by quantitative RT-PCR (qRT-PCR) at the RNA level and western blot at the protein level. Primary human limbal epithelial cells isolated from sclerocorneal tissues were grown on the Wnt6-3T3 and control 3T3 cells for 14 to 21 days. Colony-forming efficiency (CFE) was obtained. Total RNA was isolated from the CESC colonies. The phenotype of the cultured corneal epithelial cells was assessed by their expression level of putative stem cell markers and differentiation marker by qRT-PCR. The proliferation rate was evaluated by the expression level of Ki67.
Results: :
Wnt6 protein was overexpressed in the stable Wnt6-3T3 cells. The total CFE of the CESCs in the Wnt6-3T3 and control 3T3 cultures did not differ significantly. The expression of putative stem cell markers, ABCG2 and ΔNp63 were maintained and N-cadherin expression was 3.3-fold higher (p=0.05) in the Wnt6-3T3 cultures compared to that in the control. The proliferation marker, Ki67 was upregulated in the Wnt6-3T3 cultures, but did not reach a significant difference compared to that of the control (P=0.26). More importantly, a 58% decrease (P=0.01) in the expression of the differentiation marker, keratin 12 was observed in the Wnt6-3T3 cultures to the control.
Conclusions: :
These results suggest that Wnt6 might play an important role in the regulation of human CESC differentiation in vitro.
Keywords: cornea: epithelium • signal transduction