Mechanisms of Retinogenesis in Human Stem Cells

人类干细胞视网膜发生机制

• 批准号: 8146172
• 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/8146172
• 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(申请人提供)：遗传性和获得性神经视网膜(NR)和/或视网膜色素上皮(RPE)疾病是人类健康和生活质量的重要问题。人多能干细胞(HPSCs)的视网膜分步分化为研究人类视网膜发育提供了一个模型系统，为视网膜衰弱疾病的治疗提供了细胞来源。我们已经证明，两种类型的hPSCs，人胚胎干细胞(HESCs)和人诱导多能干细胞(HiPSCs)，可以沿着视网膜谱系分化，其方式与正常人类视网膜发生非常相似。然而，影响这些细胞类型从hPSC分化的关键步骤的因素和事件却知之甚少。我们的长期任务是确定hPSC决定视网膜细胞命运的分子机制，并将这一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的一个重要步骤发生在视囊泡(OV)阶段，此时细胞做出发育为视网膜神经前体细胞(NRPC)或RPE细胞的开创性决定。VSX2是已知的最早的NRPC标记，它被认为通过抑制早期RPE相关基因MITF的表达，将NAOVE OV分为NR和RPE结构域。在动物模型中，通过各种方法破坏Vsx2的表达会导致眼和视网膜的严重缺陷，而该基因突变的人类会出现小眼球和视网膜畸形。尽管VSX2被认为在视网膜发育过程中起着关键作用，但关于它在人类中的作用机制和调节机制的信息很少。我们独特的能力从视网膜发生的最早阶段培养人的细胞群体，并分离OV样结构，为研究VSX2在人视网膜细胞发育中的功能提供了一个有针对性的体外模型系统。该建议的主要目的是：a)确定VSX2在hPSC分化为视网膜细胞的初始阶段的目的和必要性；b)确定在分化过程中控制VSX2表达的内源性hPSC信号机制。实验旨在研究这样的假设，即在hPSC来源的视网膜培养中VSX2的表达导致以牺牲RPE为代价的NRPC增殖池的维持。为了验证这一理论，我们将追求以下具体目标：1.确定VSX2在NRPC早期产生和增殖过程中对hPSC分化的基因调控作用和机制。2.确定在hPSCs分化过程中调节VSX2早期表达的配体和途径，以介导成纤维细胞生长因子和TGF2信号的相互作用。3.使用患者来源的HiPSCs，确定自然发生的人类VSX2突变对NRPC和RPE细胞群体建立和扩大的影响。 与公共健康相关：这项提议的目的是促进对人类视网膜发育的了解。通过使用我们的循序渐进的方法，在培养皿中诱导干细胞成为各种视网膜细胞类型，我们可以模拟人类视网膜的构建块是如何产生的，从它们最原始的阶段开始。这样的模型将在研究视网膜发育的基本机制和遗传性眼病的原因方面具有重要价值，并将补充我们开发基于细胞的视网膜退行性疾病治疗的努力。