Activation of Wnt signaling to improve in vitro human limbal stem cell maintenance

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可以移植到 病变角膜补充LSCs，但维持足够的LSCs，预防LSC 为了使移植成功，在培养上的差异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。这项提议的目标是使用一种新型的杂化小分子， 被称为FZD7-ND，它特异性地结合FZD7和Wnt共同受体LRP5/6来激活 规范的WNT信号。研究了FZD7-ND对WNT的活化动力学。 TopFlash首先进行检测。FZD7-ND后培养的LSCs的功能和数量 治疗将按照父母拨款中所述的生物标记物进行评估。建议数 该项目将推动WNT模拟小分子FZD7-ND的进一步开发，以最大限度地提高 培养中未分化的LSCs的数量。从提议的项目中获得的知识 将允许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 use a novel hybrid small molecule, termed Fzd7-ND, that specifically binds Fzd7 and the Wnt co-receptor LRP5/6, to activate canonical Wnt signaling. The kinetics of Fzd7-ND of Wnt activation will be investigated using a TopFlash assay first. The function and population of cultivated LSCs following Fzd7-ND treatment will be evaluated as described using biomarkers in the parent grant. The proposed project will inform further development of Wnt mimic small molecule Fzd7-ND 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.