Activation of Wnt signaling using PF-04802367 in human limbal epithelial stem cell self-renewal (Chloe Moore)

项目摘要 人角膜上皮中的角膜缘上皮干/祖细胞（LSC）是角膜上皮细胞移植的前沿。 维持角膜的透明度。LSC的丧失或功能障碍导致 角膜缘干细胞缺乏症（LSCD），导致疼痛、炎症和角膜上皮细胞丢失。 患者的透明度。从患者的健康LSC培养的LSC可以移植 进入患病的角膜以补充LSC，但维持足够的LSC并防止 在培养中LSC分化以允许成功移植仍然是本领域的主要挑战。 领域典型的Wnt模拟小分子和Wnt共受体的表达， 推定的LSC标志物Fzd 7已经分别显示出改善LSC的培养， 防止分化。该提案的目标是研究一种新的小分子PF- 04802367，通过抑制GSK-3β激活经典Wnt来激活β-连环蛋白途径 发信号。首先将使用TopFlash测定法研究Wnt激活的动力学。的 将评价PF-04802367处理后培养的LSC的功能和种群 如使用父母授权中的生物标记所描述的。该项目将进一步说明 开发Wnt模拟小分子PF-04802367，以最大限度地增加 未分化的LSC。从拟议项目中获得的知识将使 持续改善患者特异性LSCD治疗。

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.