Mechanisms of Retinogenesis in Human Stem Cells

人类干细胞视网膜发生机制

• 批准号: 8321572
• 负责人: David M Gamm
• 金额: $ 35.64万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-30 至 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8321572
• 关键词: Adopted; Affect; Animal Model; Biological Models; Cell Culture Techniques; Cell Cycle Regulation; Cell Proliferation; Cell Therapy; Cells; Characteristics; Complement; Cultured Cells; Defect; Development; Disease; Distal; Dysplasia; Event; Exhibits; Eye; Eye Development; Eye diseases; Face; Fibroblast Growth Factor; Foundations; Functional disorder; Generations; Genes; Health; Homeobox; Human; Human Development; Impairment; Individual; Inherited; Investigation; Knowledge; Ligands; Maintenance; Mediating; Methods; Microphthalmos; Mission; Modeling; Molecular; Mutation; Neural Retina; Optic vesicle; Pathway interactions; Patients; Pattern; Pluripotent Stem Cells; Population; Process; Production; Proliferating; Proteins; Quality of life; Regulation; Regulator Genes; Repression; Research; Retina; Retinal; Retinal Diseases; Role; Seminal; Signal Pathway; Signal Transduction; Source; Staging; Stem cells; Structure; Structure of retinal pigment epithelium; Surface Ectoderm; Suspension Culture; Testing; Time; Tissues; Vertebrates; Vision; Visual system structure; cell type; cellular development; clinically relevant; design; human embryonic stem cell; human stem cells; improved; in vitro Model; induced pluripotent stem cell; inhibitor/antagonist; novel; postnatal; public health relevance; relating to nervous system; research study; response; retinal progenitor cell; retinogenesis; theories

DESCRIPTION (provided by applicant): Inherited and acquired diseases of the neural retina (NR) and/or retinal pigment epithelium (RPE) are a significant issue in human health and quality of life. Stepwise retinal differentiation of human pluripotent stem cells (hPSCs) can provide a model system to study human retinal development and supply cells for the potential treatment of debilitating retinal diseases. We have shown that two types of hPSCs, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), can differentiate along the retinal lineage in a manner that closely parallels normal human retinogenesis. However, little is known about the factors and events that influence key steps in the differentiation of these cell types from hPSCs. Our long-term mission is to define molecular mechanisms of retinal cell fate determination from hPSCs and apply this knowledge to the study and treatment of human developmental and degenerative retinal disorders. An important step in vertebrate retinogenesis occurs during the optic vesicle (OV) stage, when cells make the seminal decision to develop either as a neural retinal progenitor cell (NRPC) or an RPE cell. VSX2 is the earliest known marker of NRPCs and is hypothesized to pattern the naove OV into the NR and RPE domains by repressing expression of the early RPE-associated gene MITF. Disruption of Vsx2 expression in animal models by various means causes severe defects of the eye and retina, and humans with mutations in this gene exhibit microphthalmia and malformed retinas. Despite the critical roles attributed to VSX2 during retinal development, there is scant information available on its mechanisms of action and regulation in humans. Our unique capability to culture human cell populations from the earliest stages of retinogenesis and to isolate OV- like structures provides a pertinent in vitro model system to study VSX2 function in human retinal cell development. The primary objectives of this proposal are to a) determine the purpose and necessity of VSX2 in the initial production of retinal cell types from hPSCs and b) identify endogenous hPSC signaling mechanisms that control VSX2 expression during differentiation. Experiments are designed to investigate the overall hypothesis that VSX2 expression in hPSC-derived retinal cultures leads to maintenance of a proliferating pool of NRPCs at the expense of RPE. To test this theory, we will pursue the following specific aims: 1. Determine the gene regulatory roles and mechanisms of VSX2 during the early production and proliferation of NRPCs in differentiating hPSCs. 2. Define the ligands and pathways that mediate the reciprocal effects of FGF and TGF2 signaling in the regulation of early VSX2 expression in differentiating hPSCs. 3. Determine the effects of a naturally occurring, human VSX2 mutation on the establishment and expansion of NRPC vs. RPE cell populations using patient-derived hiPSCs. PUBLIC HEALTH RELEVANCE: The purpose of this proposal is to advance understanding of the development of the human retina. By using our stepwise method to coax stem cells to become a full range of retinal cell types in a culture dish, we can model how the building blocks of the human retina are produced, beginning at their most primitive stages. Such a model will have great value in the study of basic mechanisms of retinal development and the causes of inherited eye diseases, and will complement our efforts to develop cell-based therapies for degenerative retinal disorders.

描述（由申请人提供）：神经视网膜（NR）和/或视网膜色素上皮（RPE）的遗传性和获得性疾病是人类健康和生活质量的重要问题。人多能干细胞（hPSC）的逐步视网膜分化可以提供研究人视网膜发育的模型系统，并为衰弱性视网膜疾病的潜在治疗提供细胞。我们已经表明，两种类型的hPSC，人胚胎干细胞（hESC）和人诱导多能干细胞（hiPSC），可以分化沿着视网膜谱系的方式，密切平行于正常的人视网膜发生。然而，对影响这些细胞类型从hPSC分化的关键步骤的因素和事件知之甚少。我们的长期使命是确定hPSC决定视网膜细胞命运的分子机制，并将这些知识应用于人类发育和退行性视网膜疾病的研究和治疗。脊椎动物视网膜发生中的重要步骤发生在视泡（OV）阶段，此时细胞做出决定，发育为神经视网膜祖细胞（NRPC）或RPE细胞。VSX 2是已知最早的NRPC标记物，并被假设通过抑制早期RPE相关基因MITF的表达将noveOV模式化为NR和RPE结构域。通过各种手段破坏动物模型中的Vsx 2表达会导致眼睛和视网膜的严重缺陷，并且该基因突变的人表现出小眼球和畸形视网膜。尽管VSX 2在视网膜发育过程中起着关键作用，但关于其在人类中的作用和调节机制的信息很少。我们从视网膜发生的最早阶段培养人细胞群和分离OV样结构的独特能力提供了相关的体外模型系统来研究VSX 2在人视网膜细胞发育中的功能。该提议的主要目的是a）确定VSX 2在从hPSC初始产生视网膜细胞类型中的目的和必要性，和B）鉴定在分化期间控制VSX 2表达的内源性hPSC信号传导机制。设计实验以研究hPSC衍生的视网膜培养物中VSX 2表达导致以RPE为代价维持NRPC的增殖池的总体假设。为了检验这一理论，我们将追求以下具体目标：1。确定VSX 2在分化的hPSC中NRPC的早期产生和增殖过程中的基因调控作用和机制。 2.确定在分化的hPSC中调节早期VSX 2表达的FGF和TGF 2信号传导的相互作用的配体和途径。 3.使用患者来源的hiPSC确定天然存在的人VSX 2突变对NRPC与RPE细胞群的建立和扩增的影响。 公共卫生相关性：该提案的目的是促进对人类视网膜发育的理解。通过使用我们逐步诱导干细胞在培养皿中成为全系列视网膜细胞类型的方法，我们可以模拟人类视网膜的构建模块是如何产生的，从最原始的阶段开始。这样的模型将在视网膜发育的基本机制和遗传性眼病的原因的研究中具有很大的价值，并将补充我们为退行性视网膜疾病开发基于细胞的疗法的努力。