Development of cell-free approaches to the treatment of limbal stem cell deficiency

项目总结(见说明)： 角膜缘干细胞(LSCs)可以形成整个角膜上皮，并且已知存在于交界区。 位于角膜和结膜之间，称为角膜缘。LSC的丢失或LSC龛的破坏可导致角膜缘 干细胞缺乏症(LSCD)--世界上一种常见的失明原因。在自体移植时 摘除对侧眼角膜缘组织可治愈单侧LSCD患者、双侧LSCD患者 没有自体角膜缘组织可用。这些患者通常需要同种异体角膜缘移植。 嫁接；然而，他们的成功是高度不稳定的。此外，世界范围内的角膜捐赠者短缺造成了严重的 同种异体LSCs治疗双侧LSCD患者面临的挑战。因此，最重要的是 该项目的目标是通过发现LSC的新机制来开发无细胞LSCD疗法 维护和再生。我们实验室发现了一个三磷酸腺苷结合盒(ABC)超家族成员B5 (ABCB5)作为一种新的LSC标记。从人类捐献者分离的ABCB5阳性的LSCs能够长期 临床前LSCD模型中的角膜修复。临床试验目前正在进行中，以解决治疗 这一干细胞群体在人类患者中的潜力。我们最新的研究旨在探索细胞 用单细胞RNA测序技术研究ABCB5阳性LSC内的层级结构发现了一个新的LSC亚群 由于角膜特异性基因的低表达水平，可以与其他LSC簇区分开来。在这里我们 假设这个亚群拥有最原始的干细胞特征，最高 再生潜力。进一步的深入分析表明，这些细胞优先表达这些分子 参与成纤维细胞生长因子、骨形态发生蛋白和Axl信号级联反应。我们假设这些分子途径对于 维持未分化的LSC表型，可用于去新生LSC的诱导和修复 在双边LSCD背景下的LSC生态位。这项建议的两个目标将是：机械地剖析角色 Fgf7、BMP2和Axl在小鼠和人基因工程LSC维持中的作用 模型(目标1)，并将测试靶向这些通路治疗LSCD的治疗潜力 临床前小鼠疾病模型(目标2)。这项研究的成功完成将进一步推动我们的 对LSC的发展、维护和调节的理解对临床翻译具有重要意义

PROJECT SUMMARY (See instructions): Limbal stem cells (LSCs) give rise to the entire corneal epithelium and are known to reside in the border area between the cornea and conjunctiva called limbus. Loss of LSCs or destruction of the LSC niche can result in Limbal Stem Cell Deficiency (LSCD) – a common cause of vision loss in the world. While transplantation of the autologous limbal tissues removed from the contralateral eye can cure patients with unilateral LSCD, bilateral LSCD patients have no autologous limbal tissues available. These patients often require transplantation of allogeneic donor limbal grafts; however, their success is highly variable. Moreover, the worldwide corneal donor shortage poses significant challenges for the availability of allogeneic LSCs for the treatment of bilateral LSCD patients. Thus, the overarching goal of this project is to develop cell-free LSCD therapies through the discovery of novel mechanisms of LSC maintenance and regeneration. Our lab has discovered an ATP-binding cassette (ABC) superfamily member B5 (ABCB5) as a novel LSC marker. ABCB5-positive LSCs isolated from human donors were capable of the long-term corneal restoration in pre-clinical LSCD models. Clinical trials are currently on the way to address the therapeutic potential of this stem cell population in human patients. Our most recent studies aimed to explore the cellular hierarchy within ABCB5-positive LSCs using single-cell RNA-sequencing revealed a novel LSC subpopulation that could be differentiated from the other LSC clusters by low expression levels of the cornea-specific genes. Here we hypothesized that this subpopulation possesses the most primitive stem cell characteristics with the highest regenerative potential. Further in-depth analyses revealed that these cells preferentially expressed the molecules involved in FGF, BMP, and AXL signaling cascades. We posit that these molecular pathways are essential for the maintenance of the undifferentiated LSC phenotype and can be employed for de-novo LSC induction and restoration of the LSC niche in the setting of bilateral LSCD. The two Aims of this proposal will: mechanistically dissect the role of FGF7, BMP2 and AXL in the LSC maintenance using murine and human genetically engineered experimental models (Aim 1) and will test the therapeutic potential of targeting these pathways for the treatment of LSCD in pre-clinical murine disease models (Aim 2). Successful completion of this study will further advance our understanding of LSC development, maintenance, and regulation with significant implications for clinical translation