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

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。来自患者的健康LSC的培养的LSC可以被移植到患者体内。 患病角膜补充LSC，但维持足够的LSC并防止LSC 培养中的分化以允许成功的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。该提案的目标是使用一种新的混合小分子， 称为Fzd 7-ND，其特异性结合Fzd 7和Wnt共受体LRP 5/6，以激活 典型的Wnt信号传导。Fzd 7-ND的Wnt活化动力学将使用以下方法研究： 首先进行TopFlash检测。Fzd 7-ND后培养的LSC的功能和数量 治疗将如使用母基金中的生物标志物所述进行评估。拟议 该项目将为Wnt模拟小分子Fzd 7-ND的进一步开发提供信息，以最大限度地提高 培养物中未分化的LSC群体。从拟议项目中获得的知识 将允许持续改进患者特异性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 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.