Mechanisms of Retinogenesis in Human Stem Cells

人类干细胞视网膜发生机制

• 批准号: 8727557
• 负责人: David M Gamm
• 金额: $ 34.93万
• 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 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8727557
• 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.

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一个重大问题。人类多能干细胞（hPSC）的逐步视网膜分化可以提供一个模型系统来研究人类视网膜发育并为潜在的视网膜衰弱疾病治疗提供细胞。我们已经证明，两种类型的 hPSC，即人类胚胎干细胞 (hESC) 和人类诱导多能干细胞 (hiPSC)，可以以与正常人类视网膜发生非常相似的方式沿着视网膜谱系分化。然而，人们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。我们的长期使命是确定 hPSC 视网膜细胞命运决定的分子机制，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的一个重要步骤发生在视泡 (OV) 阶段，此时细胞做出决定是发育为神经视网膜祖细胞 (NRPC) 还是 RPE 细胞。 VSX2 是最早已知的 NRPC 标记，推测通过抑制早期 RPE 相关基因 MITF 的表达，将 naove OV 模式化为 NR 和 RPE 结构域。通过各种方式破坏动物模型中的 Vsx2 表达会导致眼睛和视网膜的严重缺陷，而携带该基因突变的人类会出现小眼症和视网膜畸形。尽管 VSX2 在视网膜发育过程中发挥着关键作用，但有关其在人类中的作用和调节机制的信息却很少。我们具有从视网膜发生的最早阶段培养人类细胞群并分离 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相关的体外模型系统。该提案的主要目标是 a) 确定 VSX2 在 hPSC 初始生产视网膜细胞类型中的目的和必要性，以及 b) 确定在分化过程中控制 VSX2 表达的内源 hPSC 信号传导机制。实验旨在调查以下总体假设：hPSC 来源的视网膜培养物中 VSX2 的表达导致 NRPC 增殖池的维持，但以 RPE 为代价。为了检验这一理论，我们将追求以下具体目标： 1. 确定 VSX2 在 NRPC 分化 hPSC 的早期产生和增殖过程中的基因调控作用和机制。 2. 定义介导 FGF 和 TGF2 信号在分化 hPSC 早期 VSX2 表达调节中相互作用的配体和途径。 3. 使用患者来源的 hiPSC 确定自然发生的人类 VSX2 突变对 NRPC 与 RPE 细胞群的建立和扩增的影响。