Mechanisms of Retinogenesis in Human Stem Cells

人类干细胞视网膜发生机制

• 批准号: 8025375
• 负责人: David M Gamm
• 金额: $ 37.13万
• 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/8025375
• 关键词: Mechanisms; Retinogenesis; Human; Stem; Cells

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。人类多能干细胞（hPSCs）的逐步视网膜分化可以为研究人类视网膜发育提供模型系统，并为衰弱性视网膜疾病的潜在治疗提供细胞。我们已经证明，两种类型的人胚胎干细胞（hESCs）和人诱导多能干细胞（hiPSCs）可以沿着视网膜谱系以与正常人类视网膜发生密切相似的方式分化。然而，对于影响这些细胞类型从hPSCs分化的关键步骤的因素和事件知之甚少。我们的长期任务是确定hscs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。脊椎动物视网膜形成的一个重要步骤发生在视神经泡（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。VSX2是已知最早的NRPCs标记物，据推测，通过抑制早期RPE相关基因MITF的表达，VSX2可以将nave定向到NR和RPE结构域。通过各种方式破坏Vsx2在动物模型中的表达会导致严重的眼睛和视网膜缺陷，该基因突变的人会出现小眼症和视网膜畸形。尽管VSX2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。我们在视网膜形成早期培养人类细胞群和分离OV样结构的独特能力，为研究VSX2在人类视网膜细胞发育中的功能提供了一个相关的体外模型系统。本研究的主要目的是：a)确定VSX2在hPSC初始生成视网膜细胞类型中的目的和必要性；b)确定分化过程中控制VSX2表达的内源性hPSC信号传导机制。实验旨在研究在hpsc衍生的视网膜培养物中表达VSX2导致NRPCs增殖池维持而牺牲RPE的总体假设。为了验证这一理论，我们将追求以下具体目标：1。确定VSX2在分化hPSCs中NRPCs早期产生和增殖中的基因调控作用和机制。2. 明确介导FGF和TGF2信号相互作用调控早期VSX2在分化的hPSCs中的表达的配体和途径。3. 利用患者源性hiPSCs确定自然发生的人VSX2突变对NRPC和RPE细胞群的建立和扩增的影响。