Effect of inhibition of Wnt signaling using small molecules in human limbal epithelial stem cell differentiation

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。 领域我们先前已经表明，Wnt共受体Frizzled 7（Fzd 7）优先被激活。 它的表达是维持LSC干细胞表型所必需的。 LSC。此外，规范和非规范Wnt激活以集成方式操作 而不是像我们以前认为的那样相互排斥。在这一提议中， 使用作为Fzd 7功能的新型Wnt小分子抑制剂的Wnt抑制作用 封闭肽（Fzd 7-P）将首先使用TopFlash测定进行研究。的功能和 Fzd 7-P以剂量依赖性方式处理后培养的LSC群体将 如使用父母补助中的生物标志物所述进行评估。拟议项目将进一步 阐明了Wnt信号在LSCs调控中的作用机制。结果可能 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 the Wnt co-receptor Frizzled 7 (Fzd7) was preferentially expressed by the LSCs and its expression was required to maintain the stem cell phenotype of LSCs. 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 inhibition using a novel Wnt small molecule inhibitor that functions as a Fzd7 blocking peptide (Fzd7-P) will be investigated first using a TopFlash assay. The function and population of cultivated LSCs following Fzd7-P 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.