PROJECT SUMMARY Limbal epithelial stem/progenitor cells (LSCs) in the human corneal epithelium are the front line of defense to maintain transparency of the cornea. Loss or dysfunction of the LSCs leads to limbal stem cell deficiency (LSCD), which causes pain, inflammation, and loss of corneal transparency in patients. Cultivated LSCs from a patient’s healthy LSCs can be transplanted into the diseased cornea to replenish the LSCs, but maintaining sufficient LSCs and preventing LSC differentiation in culture to allow for a successful transplant remain a major challenge in the field. A canonical Wnt mimic small molecule and the expression of the Wnt co-receptor and putative LSC marker Fzd7 have separately been shown to improve the cultivation of LSCs while preventing differentiation. The goal of this proposal is to investigate a new small molecule PF- 04802367 which activates the β-catenin pathway by inhibiting GSK-3β to activate canonical Wnt signaling. The kinetics of Wnt activation will be investigated using a TopFlash assay first. The function and population of cultivated LSCs following PF-04802367 treatment will be evaluated as described using biomarkers in the parent grant. The proposed project will inform further development of Wnt mimic small molecule PF-04802367 to maximize the population of undifferentiated LSCs in culture. The knowledge gained from the proposed project will allow continued improvement of patient-specific LSCD treatment.