Defining the molecular mechanisms underlying human RPE plasticity

定义人类 RPE 可塑性的分子机制

• 批准号: 8411125
• 负责人: SALLY TEMPLE
• 金额: $ 41.91万
• 依托单位: REGENERATIVE RESEARCH FOUNDATION
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-02-01 至 2017-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8411125
• 关键词: Abnormal Cell; Address; Adipocytes; Adult; Affect; Age related macular degeneration; Attenuated; Cattle; Cell Differentiation process; Cell physiology; Cells; ChIP-seq; Chondrocytes; Chromatin; Clinical Trials; Cues; Data; Development; Disease; Distal; Elements; Enhancers; Ensure; Epigenetic Process; Epiretinal Membrane; Epithelial; Epithelium; Etiology; Event; Exhibits; Exogenous Factors; Exposure to; Eye; Eye diseases; Gene Expression; Gene Expression Regulation; Generations; Genes; Histones; Human; Macular degeneration; Massachusetts; Mesenchymal; Mesenchymal Stem Cells; Metaplasia; Methods; Molecular; Molecular Conformation; Osteogenesis; Pain; Pathology; Pathway interactions; Pattern; Phenotype; Pigmentation physiologic function; Pigments; Positioning Attribute; Process; Proliferating; Proliferative Vitreoretinopathy; Regulatory Element; Reporting; Retina; Retinal Diseases; Signal Pathway; Signal Transduction; Source; Staging; Staining method; Stains; Stem cells; Structure of retinal pigment epithelium; Testing; Therapeutic; Transcriptional Regulation; Transforming Growth Factor beta; Transplantation Conditioning; Universities; Up-Regulation; Vision; Western Blotting; abstracting; bone; cell type; combat; epigenome; fetal; genome-wide; human embryonic stem cell; inhibitor/antagonist; knock-down; loss of function; medical schools; member; novel strategies; osteogenic; prevent; promoter; small hairpin RNA; small molecule; transcription factor; transcriptome sequencing

Project Summary/Abstract In this proposal we seek to understand how human primary retinal pigment epithelial cells (RPE) differentiate into cells of the mesenchymal lineage. We have shown that RPE derived from a variety of sources, including human adult, fetal, ESC-derived, ARPE-19 and bovine, acquire mesenchymal phenotypes. Moreover, primary adult human RPE that were clonally expanded from a single cell produced adipocytes, chondrocytes and bone when placed in the respective differentiation media. This topic is important because RPE, while normally a stable epithelium, can undergo proliferative and metaplastic changes detrimental to vision. RPE metaplasia into mesenchymal-related cells has been long -associated with retinal disease, yet the mechanism underlying these changes remains a mystery. In aim 1 we will study how this plasticity is encoded at the level of the epigenome. Recent studies have identified epigenetic signatures at promoters and enhancers that indicate active genes and poised genes (those that could be activated if given appropriate cues). We will ask whether genes associated with mesenchymal lineages are in a poised conformation in human RPE cells, and whether they transit to an active form upon differentiation into osteo-, chondro- and adipocyte pathways. In aim 2 we will determine exogenous factors stimulating this differentiation, by testing factors known to act on mesenchymal stem cells (MSCs) during development towards osteogenic, chondrocyte and adipocyte fates, including members of the TGF-beta superfamily, and by use of small molecule inhibitors, which will help identify therapeutics to attenuate this process. One of the prominent abnormal cell fates associated with RPE metaplasia is ossification, hence it is particularly important to understand how RPE cells undergo osteogenesis. In aim 3 we will focus on the steps RPE move through when transitioning towards this fate, evaluating whether the transcription factor sequence known for normal osteogenesis generation is followed, or whether abnormal pathways are activated. Preliminary studies show dramatic upregulation of the essential osteogenic transcription factor Runx2 in RPE undergoing transformation towards bone. We will determine whether knocking down Runx2 using an shRNA approach will inhibit RPE osteogenesis. Together, these three aims address the central mechanisms, from exogenous factor through to gene regulation and epigenetic changes, that underlie RPE phenotypic plasticity. Disruption of RPE stability is known to occur in several important retinal diseases such as epiretinal membrane formation, macular degeneration and phthsis bulbii, and understanding this plasticity will provide novel approaches toward developing treatments of these conditions. The findings will also have direct relevance to understanding the factors that maintain the RPE as a stable, pigmented, cobblestone polarized epithelial layer, which is especially timely given that RPE cells derived from human embryonic stem cells are entering clinical trials for retinal disease, and steps must be taken to minimize metaplastic changes to ensure development of safe transplantation conditions.

项目摘要/摘要 在此提案中，我们试图了解人类原发性视网膜色素上皮细胞（RPE）如何区分 进入间充质谱系的细胞。我们已经证明了RPE来自多种来源，包括 人类成年人，胎儿，ESC衍生，ARPE-19和牛，获得间充质表型。而且，主要 从单个细胞产生的脂肪细胞，软骨细胞和骨骼的成年人类RPE扩展。 当放置在各个分化介质中时。这个话题很重要，因为RPE通常是 稳定的上皮可以经历增殖和化生变化，这是有害于视觉的。 RPE Metaplasia 与视网膜疾病相关的间充质相关细胞已经很长 这些变化仍然是一个谜。在AIM 1中，我们将研究这种可塑性如何在 表观基因组。最近的研究已经确定了启动子和增强子的表观遗传学特征，表明 活跃的基因和固执的基因（如果得到适当的提示，可以激活的基因）。我们会问是否 与间充质谱系相关的基因在人RPE细胞中具有平衡的构象，以及是否是否 它们在分化为骨，软骨和脂肪细胞途径后转变为活性形式。在目标2中我们 将通过测试已知作用的因素来确定刺激这种分化的外源性因素 间充质干细胞（MSC）在发育过程中朝着成骨，软骨细胞和脂肪细胞命运， 包括TGF-Beta超家族的成员，并通过使用小分子抑制剂，这将有助于 识别治疗剂以减弱这一过程。与RPE相关的突出的异常细胞命运之一 Metaplasia是骨化，因此了解RPE细胞如何经历特别重要 成骨。在AIM 3中，我们将重点介绍RPE向这种命运过渡时的步骤， 评估是否遵循遵循正常成骨发生的转录因子序列，还是 是否激活异常途径。初步研究表明，基本的急剧上调 RPE中的成骨转录因子Runx2经历向骨骼转变。我们将确定 是否使用SHRNA方法击倒Runx2是否会抑制RPE成骨。一起，这三个 目的解决了从外源性因素到基因调节和表观遗传的中心机制 变化，这是RPE表型可塑性的基础。已知RPE稳定性的破坏发生在几个 重要的视网膜疾病，例如前膜形成，黄斑变性和phthsis bulbii， 了解这种可塑性将为开发这些疗法提供新颖的方法 状况。这些发现还将与理解将RPE保持为一个的因素有直接的相关性 稳定，有色，鹅卵石极化上皮层，尤其是及时的RPE细胞 源自人类胚胎干细胞正在进入视网膜疾病的临床试验，步骤必须为 以最大程度地减少转移变化，以确保发展安全移植条件。