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. We have previously shown that the Wnt co-receptor Frizzled 7 (Fzd7) was preferentially expressed by the LSCs and its expression was required to maintain the stem cell phenotype of LSCs. Additionally, canonical and noncanonical Wnt activation operate in an integrated manner in the LSCs instead of a mutually exclusive manner as we previously thought. In this proposal, the effect of Wnt inhibition using a novel Wnt small molecule inhibitor that functions as a Fzd7 blocking peptide (Fzd7-P) will be investigated first using a TopFlash assay. The function and population of cultivated LSCs following Fzd7-P treatment in a dose dependent manner will be evaluated as described using biomarkers in the parent grant. The proposed project will further shed light on the mechanism of Wnt signaling in the regulation of LSCs. The results could inform further development of Wnt small molecules 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.