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

项目总结 角膜缘上皮干/祖细胞(LSCs)是人角膜上皮细胞的一线 以保持角膜的透明度。LSC的丢失或功能障碍会导致 角膜缘干细胞缺乏症(LSCD)，导致疼痛、炎症和角膜丢失 患者的透明度。从患者健康的LSCs中培养的LSCs可以移植 进入病变的角膜以补充LSCs，但保持足够的LSCs并防止 在培养中进行LSC分化以使移植成功仍然是一个主要的挑战 菲尔德。一种规范的WNT模拟小分子及其共受体和WNT的表达 可能的LSC标记FZD7分别被证明可以改善LSCs的培养，而 阻止分化。这项计划的目标是研究一种新的小分子PF- 04802367通过抑制β-3β激活规范Wnt来激活GSK-连环蛋白途径 发信号。WNT的活化动力学将首先使用TopFlash实验进行研究。这个 将评估PF-04802367处理后培养的LSCs的功能和数量 如在母基金中使用生物标记物所描述的。拟议的项目将进一步通知 WNT模拟小分子PF-04802367的开发以实现最大限度的种群数量 培养中未分化的LSCs。从拟议的项目中获得的知识将使 持续改进针对患者的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.