Ocular surface applications of Descemet's membrane

后弹力层在眼表的应用

• 批准号: 10673188
• 负责人: Joshua H Hou
• 金额: $ 24.21万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-01 至 2027-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10673188
• 关键词: ABCG2 gene; Address; Age related macular degeneration; Air; Animal Model; Anterior; Autoimmune Diseases; Basal Cell; Basement membrane; Biological; Biological Assay; Biological Markers; Blinded; Blindness; Cell Transplantation; Cells; Characteristics; Chemical Burns; Chemical Injury; Clinic; Collagen Type IV; Communication; Competence; Cornea; Defect; Descemet' s membrane; Development; Digestion; Disease; Effectiveness; Engineering; Environment; Epithelial Cells; Extracellular Matrix; Extracellular Matrix Proteins; Eye diseases; Future; Goals; Human; Immune; Immunohistochemistry; In Vitro; Lifting; Mechanics; Membrane; Membrane Proteins; Mentored Clinical Scientist Development Program; Mentors; Minnesota; Modeling; Natural regeneration; Operative Surgical Procedures; Optics; Oral; Organ Culture Techniques; Outcome; Pain; Patient-Focused Outcomes; Patients; Phenotype; Physiologic pulse; Physiological; Population; Proliferating; Proteins; Protocols documentation; Publishing; Quantitative Reverse Transcriptase PCR; Rattus; Recurrence; Rehabilitation therapy; Reporting; Research; Research Personnel; Resistance; Resources; Stem cell transplant; Surface; Testing; Therapeutic Intervention; Time; Tissue Donors; Training; Transplantation; Universities; Vitronectin; Work; Writing; cell regeneration; clinical application; collagenase; comparative; comparative efficacy; conjunctiva; corneal epithelium; corneal regeneration; fetal; graft failure; improved; in vivo; induced pluripotent stem cell; induced pluripotent stem cell technology; innovation; invention; limbal; melting; mouse model; novel; ocular surface; personalized medicine; pluripotency; programs; reconstruction; regenerative therapy; research study; skills; stem cell biomarkers; stem cell function; stem cells; stemness; success; therapy outcome; tissue processing

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.

项目概要/摘要 角膜缘干细胞 (LSC) 缺乏症是一种致盲性疾病，约占角膜的 15-20% 全世界失明。 LSC 缺乏是由 LSC 过度丢失引起的，LSC 是一组多能细胞， 再生透明的角膜上皮。由于化学损伤和自身免疫性疾病导致 LSC 丧失 导致角膜结膜化、糜烂和融化。治疗选择有限；然而，有文化的 角膜缘上皮细胞移植（CLET）是一种有前途的新兴疗法。 CLET 离体扩张角膜缘 干/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。据报告，CLET 取得了短期成功；然而，长期结果是 随着时间的推移，移植的 LSPC 丢失和 LSC 缺陷复发受到限制。造成这种情况的原因之一是缺乏 一个支持性的利基市场。生理条件下，角膜缘的微环境，称为角膜缘。 利基，维持天然 LSC 的多能性和增殖潜力。然而，在 LSC 缺乏时，角膜缘 利基经常被破坏。鉴定可作为利基替代品的合成和生物底物 支持移植的 LSPC 仍然是一个持续的挑战，也是长期成功的未解决的障碍 眼表再生疗法。虽然人类羊膜 (HAM) 是主要的 用于 CLET 的基质，由于其不透明、快速降解和缺乏，其作为长期利基替代品受到限制。 角膜缘特异性蛋白质。相比之下，后弹力层 (DM) 是后部的基底膜 角膜表面清澈，能抵抗胶原酶消化。此外，胎儿前带层 DM 富含角膜缘特异性基底膜蛋白，包括 IV 型胶原蛋白 α1、α2 亚型、玻连蛋白、 和 BM40/SPARC。本研究的目的是比较供体和 iPSC 来源的干性和存活率 DM vs HAM 的 LSPC。在目标 1 中，我们将对培养的 LSPC 进行体外表型和功能比较 使用生物标志物表达和 LSC 缺陷的器官培养模型对 DM 与 HAM 进行比较。在目标 2 中，我们将 使用 LSC 缺陷小鼠模型对 DM 和 HAM 上培养的 LSPC 进行体内比较。瞄准目标 3，我们将比较 iPSC 衍生的 LSPC 中生物标志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 中选择基材提供信息并改进 我们针对 LSC 缺乏症的疗法。培训计划将为申请人提供以下方面的技术能力 LSPC 的表征、LSC 缺陷动物模型的使用以及 iPSC 的操作；也 口头和书面沟通的专业技能，以促进作为独立调查员的发展。 培训将在明尼苏达大学 (UMN) 高度协作且资源丰富的研究中心进行 环境。申请人将得到iPSC应用领域领军人物Deborah Ferrington博士的指导 年龄相关性黄斑变性技术，角膜缘治疗干预先驱 Ali Djalilian 博士 干细胞缺乏症，以及 UMN 干细胞研究所创新设施主任 James Dutton 博士。