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.

项目摘要/摘要 边缘干细胞（LSC）缺乏症是一种盲目的疾病，估计占角膜的15-20％ 全球盲目。 LSC缺乏症是由LSC过度丢失引起的，LSC是多能细胞的人群 再生透明的角膜上皮。由于化学损伤和自身免疫性疾病而导致LSC的丧失 导致角膜整合，侵蚀和融化。治疗选择有限；但是，培养 边缘上皮细胞移植（Clet）是一种有希望的新兴疗法。在Clet ex ex Vivo中扩展了缘 将茎/祖细胞（LSPC）移植到解散的眼睛上以替代天然LSC并再生 角膜上皮。据报道，Clet已报告了短期成功。但是，长期结局已经 受移植LSPC的丧失和LSC缺乏症的复发的限制。原因之一是缺乏 支持的利基市场。在生理条件下，边缘的微环境，称为缘 利基，维持天然LSC的多能性和增殖潜力。但是，在LSC缺乏症中 利基经常受到损害。可以用作利基替代品的合成和生物基底物的鉴定 支持移植的LSPC仍然是一个持续的挑战，并且是长期成功的未解决的障碍 在眼表面的再生疗法中。尽管人羊膜（HAM）是主要的 它用于固定的底物，它被其不透明，快速降解和缺乏的长期替代品限制 小边缘特异性蛋白质。相反 角膜的表面清晰且对胶原酶消化有抵抗力。此外，前胎儿带状层 DM的富含边缘特异性基底膜蛋白，包括胶原蛋白IVα1，α2亚型，玻璃体素， 和BM40/sparc。这项研究的目的是比较供体和IPSC衍生的供体和生存 DM与火腿上的LSPC。在AIM 1中，我们将在LSPC培养的LSPC进行体外表型和功能比较 在DM与HAM上使用生物标志物表达和LSC缺乏的器官培养模型。在AIM 2中，我们将 使用LSC缺乏的小鼠模型对DM与HAM上培养的LSPC进行体内比较。目标 3，我们将比较生物标志物的表达和能力以再生IPSC衍生的LSPC中的角膜上皮 在DM vs HAM上进行培养。该项目有可能告知我们在Clet中选择的底物并改进 我们对LSC缺乏症的疗法。培训计划将为申请人提供技术能力 LSPC的表征，LSC缺乏症的动物模型的使用以及IPSC的操纵；也 口头和书面沟通方面的专业技能，以促进作为独立研究者的发展。 培训将在明尼苏达大学（UMN）的高度协作和资源良好的研究中进行 环境。该申请将由IPSC申请的领导者Deborah Ferrington博士考虑 与年龄相关的黄斑变性技术Ali Djalilian博士，一位治疗干预措施的先驱 干细胞缺乏症和创新设施UMN干细胞研究所主任詹姆斯·达顿（James Dutton）博士。