Activation of Wnt signaling using LY-2090314 in human limbal epithelial stem cell maintenance (Faith Ampadu)

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。 领域我们先前已经证明，使用Wnt模拟物激活Wnt信号传导， 增强LSC在培养中的扩增。此外，典型和非典型Wnt激活 在LSC中以综合方式运作，而不是像我们 以前认为。在该提议中，使用Wnt小分子LY-100活化Wnt的效果被证明是有效的。 将首先使用TopFlash试验研究抑制GSK-3β的2090314。的功能和 LY-2090314以剂量依赖性方式处理后培养的LSC群将 使用父母补助金中的生物标志物进行评估。拟议项目将 进一步阐明了Wnt信号在LSCs调控中的作用机制。结果 可以为Wnt小分子的进一步开发提供信息，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. We have previously shown that activation of Wnt signaling using a Wnt mimic could enhance the LSC expansion in culture. 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 activation using a Wnt small molecule LY- 2090314 that inhibits GSK-3β will be investigated first using a TopFlash assay. The function and population of cultivated LSCs following LY-2090314 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.