Ocular surface applications of Descemet's membrane

项目摘要/摘要 角膜缘干细胞(LSC)缺乏是一种致盲疾病，估计占角膜面积的15-20%。 世界范围内的失明。LSC缺乏症是由LSC过度丧失引起的，LSC是一组多能细胞， 再生透明的角膜上皮。化学损伤和自身免疫性疾病导致的LSC丢失 导致角膜结膜、侵蚀和融化。治疗选择有限；然而，培养 角膜缘上皮细胞移植(CLET)是一种很有前途的治疗方法。CLET体外扩张角膜缘的实验研究 干细胞/祖细胞(LSPC)被移植到患病的眼睛上，以取代天然的LSC并再生 角膜上皮细胞。据报道，CLET在短期内取得了成功；然而，长期结果是 受限于移植的LSPC的丢失和LSC缺陷的复发。这其中的一个原因是缺乏 一个支持性的利基市场。在生理条件下，角膜缘的微环境，称为角膜缘 利基，维持原生LSC的多能性和增殖潜力。然而，在LSC缺乏症中，角膜缘 利基市场经常遭到破坏。鉴定可作为生态位替代品的合成和生物底物 支持移植的LSPC仍然是一项持续的挑战，也是长期成功的未解决障碍 在眼表的再生疗法中。虽然人羊膜(HAM)是主要的 用于CLET的底物，由于其不透明、快速降解和缺乏，作为长期利基替代品而受到限制 角膜缘特有的蛋白质。相比之下，Descemet膜(DM)是位于后部的基底膜 角膜表面透明，不易被胶原酶消化。此外，胎儿前束带层 富含角膜缘特异性基底膜蛋白，包括IV型胶原α1、α2亚型、玻璃体连结蛋白、 和BM40/SPARC。本研究的目的是比较供者和IPSC来源的干细胞的干性和存活率。 DM与HAM上的LSPC。在目标1中，我们将进行体外培养的LSPC的表型和功能比较 应用生物标记物表达和LSC缺乏症器官培养模型对DM与HAM的研究在目标2中，我们将 使用LSC缺乏的小鼠模型，对DM和HAM上培养的LSPC进行体内比较。在AIM 3，我们将比较IPSC来源的LSPC中生物标记物的表达和再生角膜上皮的能力 在DM和HAM上培养。该项目有可能为我们在CLET中选择衬底提供信息，并改进 我们治疗LSC缺乏症的方法。培训计划将为申请者提供以下方面的技术能力 LSPC的特征，LSC缺乏的动物模型的使用，IPSC的操作；以及 口头和书面交流的专业技能，以促进独立调查人员的发展。 培训将在明尼苏达大学(UMN)高度协作和资源充足的研究中进行 环境。申请者将由IPSC应用领域的领先者Deborah Ferrington博士指导 老年性黄斑变性的技术，角膜缘治疗干预的先驱阿里·贾利安博士 干细胞缺乏症和UMN干细胞研究所创新设施主任詹姆斯·达顿博士。

PROJECT SUMMARY/ABSTRACT Limbal stem cell (LSC) deficiency is a blinding disease that accounts for an estimated 15-20% of corneal blindness worldwide. LSC deficiency is caused by excessive loss of LSC, a population of pluripotent cells that regenerate the transparent corneal epithelium. Loss of LSC due to chemical injuries and autoimmune disease results in corneal conjunctivalization, erosions, and melting. Treatment options are limited; however, cultured limbal epithelial cell transplantation (CLET) is a promising emerging therapy. In CLET ex vivo expanded limbal stem/progenitor cells (LSPC) are transplanted onto diseased eyes to replace the native LSC and regenerate the corneal epithelium. Short-term success has been reported with CLET; however, long-term outcomes have been limited by loss of transplanted LSPC and recurrence of LSC deficiency over time. One reason for this is the lack of a supportive niche. Under physiological conditions, the microenvironment of the limbus, known as the limbal niche, sustains the pluripotency and proliferative potential of native LSC. However, in LSC deficiency, the limbal niche is often damaged. Identification of synthetic and biological substrates that can function as niche substitutes to support transplanted LSPC remains an ongoing challenge and an unaddressed barrier to long-term success in regenerative therapies for the ocular surface. Although human amniotic membrane (HAM) is the primary substrate used for CLET, it is limited as a long-term niche substitute by its opacity, rapid degradation, and lack of limbus-specific proteins. In contrast Descemet’s Membrane (DM), is a basement membrane on the posterior surface of the cornea is clear and resistant to collagenase digestion. Furthermore, the anterior fetal banded layer of DM is rich in limbus-specific basement membrane proteins, including collagen IV α1, α2 subtypes, vitronectin, and BM40/SPARC. The goal of this study is to compare the stemness and survival of donor and iPSC-derived LSPC on DM vs HAM. In aim 1, we will perform in vitro phenotypic and functional comparisons of LSPC cultured on DM vs HAM using biomarker expression and an organ culture model of LSC deficiency. In aim 2, we will perform an in vivo comparison of cultured LSPC on DM vs HAM using a mouse model of LSC deficiency. In aim 3, we will compare biomarker expression and capacity to regenerate corneal epithelium in iPSC-derived LSPC cultured on DM vs HAM. This project has the potential to inform our choice of substrate in CLET and improve our therapies for LSC deficiency. The training plan will provide the applicant with technical competencies in the characterization of LSPC, use of animal models of LSC deficiency, and manipulation of iPSC; as well as professional skills in oral and written communication to facilitate development as an independent investigator. Training will take place in University of Minnesota’s (UMN) highly collaborative and well-resourced research environment. The applicant will be mentored by Dr. Deborah Ferrington, a leader in applications of iPSC technology in age-related macular degeneration, Dr. Ali Djalilian, a pioneer in therapeutic interventions for limbal stem cell deficiency, and Dr. James Dutton, director of the UMN Stem Cell Institute, Innovation Facilities.