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（由申请人提供）：神经视网膜（NR）和/或视网膜色素上皮（RPE）的遗传和获得的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一个模型系统，以研究人类视网膜发育和供应细胞的潜在治疗视网膜疾病的潜在治疗。我们已经表明，两种类型的HPSC，人类胚胎干细胞（HESC）和人类诱导的多能干细胞（HIPSC）可以以与正常人视网膜生成相似的方式沿着视网膜谱系区分。但是，对影响这些细胞类型与HPSC分化的关键步骤的因素和事件知之甚少。我们的长期任务是从HPSC中定义视网膜细胞命运测定的分子机制，并将这些知识应用于人类发育和退化性视网膜疾病的研究和处理。脊椎动物视网膜生成的重要步骤是在视力囊泡（OV）阶段发生的，当细胞做出开创性决定为神经视网膜祖细胞（NRPC）或RPE细胞的决定。 VSX2是最早的NRPC标记物，假设通过抑制早期与RPE相关的基因MITF的表达来将Naove OV模拟到NR和RPE结构域中。动物模型中VSX2表达的破坏会导致眼睛和视网膜的严重缺陷，并且该基因中具有突变的人类表现出微观粒细胞和畸形的视网膜。尽管在视网膜发育过程中归因于VSX2的关键作用，但关于其人类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类细胞群体的独特能力并分离出类似结构的结构为研究人类视网膜细胞发育中的VSX2功能提供了相关的体外模型系统。该提案的主要目标是a）确定VSX2在HPSC的视网膜细胞类型初始产生中的目的和必要性，b）识别内源性HPSC信号传导机制，以控制VSX2在分化过程中的表达。实验旨在研究总体假设，即HPSC衍生的视网膜培养物中的VSX2表达会导致维持增殖的NRPC库，而牺牲了RPE。为了检验该理论，我们将追求以下特定目的：1。确定NRPC在分化HPSC中的早期生产和NRPC增殖过程中VSX2的基因调节作用和机制。 2。定义介导FGF和TGF2信号在分化HPSC中早期VSX2表达的调节中介导的配体和途径。 3。确定使用患者衍生的HIPSC的NRPC与RPE细胞群体建立和扩展的自然发生的人类VSX2突变的影响。 公共卫生相关性：该提案的目的是提高对人视网膜发展的理解。通过使用我们的逐步方法哄骗干细胞成为培养皿中的整个视网膜细胞类型，我们可以建模如何产生人类视网膜的构件，从最原始的阶段开始。这种模型将在研究视网膜发育的基本机制和遗传性眼部疾病的原因方面具有很大的价值，并将补充我们为开发基于细胞的退化性视网膜疾病疗法的努力。