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.

项目总结/摘要 角膜缘干细胞（LSC）缺乏症是一种致盲性疾病，估计占15-20%的角膜 全世界失明LSC缺陷是由LSC的过度损失引起的，LSC是一群多能细胞， 使透明角膜上皮再生。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，培养 角膜缘上皮细胞移植（CLET）是一种有前途的新兴治疗方法。在CLET离体扩张的角膜缘中， 将干/祖细胞（LSPC）移植到患病的眼睛上以替代天然的LSC并再生眼睛。 角膜上皮据报道，CLET的短期成功;然而，长期结果还有待观察。 其受到移植LSPC损失和随时间推移LSC缺陷复发的限制。其中一个原因是缺乏 一个支持性的利基。在生理条件下，被称为角膜缘的利姆布斯的微环境 生态位，维持天然LSC的多能性和增殖潜力。然而，在LSC缺乏症中， niche经常被损坏。可作为生态位替代品的合成和生物基质的鉴定 支持移植的LSPC仍然是一个持续的挑战，也是长期成功的一个未解决的障碍 用于眼表的再生治疗。虽然人羊膜（HAM）是主要的 作为用于CLET的基质，由于其不透明、快速降解和缺乏 角膜缘特异性蛋白质与此相反，后弹力膜（DM）是位于后部的基底膜。 角膜的表面是透明的并且抵抗胶原酶消化。此外，胎儿前带状层 DM的角膜缘富含特异性基底膜蛋白，包括IV型胶原α1、α2亚型，玻连蛋白， BM40/100。本研究的目的是比较供体和iPSC衍生的干细胞的干细胞性和存活率。 DM与HAM的LSPC。在目的1中，我们将对体外培养的LSPC进行表型和功能比较， 使用生物标志物表达和LSC缺陷的器官培养模型对DM与HAM的影响。在目标2中，我们 使用LSC缺陷的小鼠模型，对DM与HAM上培养的LSPC进行体内比较。在aim中 3，我们将比较iPSC衍生的LSPC中的生物标志物表达和再生角膜上皮的能力。 在DM与HAM上培养。该项目有可能告知我们在CLET中选择底物并改善 我们治疗LSC缺乏症的方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缺陷的动物模型的使用和iPSC的操作;以及 具备口头和书面沟通的专业技能，以促进独立调查员的发展。 培训将在明尼苏达大学（UMN）高度合作和资源充足的研究中进行 环境申请人将由iPSC应用领域的领导者Deborah Ferrington博士指导 年龄相关性黄斑变性的治疗技术，Ali Djalilian博士，角膜缘治疗干预的先驱 干细胞缺乏症，和詹姆斯达顿博士，UMN干细胞研究所，创新设施主任。