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

标题：无细胞方法治疗角膜缘干细胞缺乏症的发展 项目总结/摘要 角膜缘干细胞（LSC）产生整个角膜上皮，并且已知存在于边缘区域 在角膜和结膜之间的区域称为利姆布斯。LSC的丧失或LSC生态位的破坏可导致 角膜缘干细胞缺乏症（LSCD）-世界上视力丧失的常见原因。移植的同时， 从对侧眼切除自体角膜缘组织可以治愈单侧LSCD，双侧LSCD， LSCD患者没有可用的自体角膜缘组织。这些患者通常需要移植 同种异体供体角膜缘移植;然而，它们的成功是高度可变的。此外，全球角膜 供体短缺对同种异体LSC用于治疗双侧LSC的可用性构成了重大挑战。 LSCD患者。因此，该项目的总体目标是通过以下方法开发无细胞LSCD疗法： 发现LSC维持和再生的新机制。我们的实验室发现了一种ATP结合蛋白 盒（ABC）超家族成员B5（ABCB 5）作为新的LSC标记。ABCB 5阳性LSC分离自 人供体能够在临床前LSCD模型中进行长期角膜修复。临床试验 目前正在研究这种干细胞群在人类患者中的治疗潜力。我们 最近的研究旨在使用单细胞RNA- 测序揭示了一种新的LSC亚群，其可以通过低表达水平与其他LSC簇区分开。 角膜特异性基因的表达水平。在这里，我们假设这个亚群拥有 最原始的干细胞特征，具有最高的再生潜力。进一步深入分析 揭示了这些细胞优先表达与FGF、BMP和AXL信号有关的分子 瀑布我们认为，这些分子途径是维持未分化的 LSC表型，并可用于从头LSC诱导和LSC生态位的恢复， 设置双侧LSCD。本提案的两个目的是：机械地剖析FGF 7、BMP 2 和AXL在LSC维持中的作用（Aim 1)并将测试靶向这些途径治疗LSCD的临床前治疗潜力。 鼠疾病模型（Aim 2）。这项研究的成功完成将进一步促进我们对 LSC的开发、维护和监管对临床翻译具有重要意义。 基于PI在再生医学方面的培训，PI将获得角膜干细胞方面的丰富经验 Frank博士是LSC生物学和ABC领域的世界级领导者， 他是哈佛干细胞研究所的成员。此外，这项研究将提供重要的 在他成为独立调查员的道路上提供职业指导。

Title: Development of cell-free approaches to the treatment of limbal stem cell deficiency PROJECT SUMMARY/ABSTRACT 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. Building on the PI’s training in regenerative medicine, the PI will gain extensive experience in corneal stem cell biology under the mentorship of Dr. Frank, a world-class leader in the fields of LSC biology and ABC transporters, and a member of Harvard Stem Cell Institute. Furthermore, this research will provide an important career guidance on his path of becoming an independent investigator.