Defining the molecular mechanisms underlying human RPE plasticity

项目概要/摘要 在本提案中，我们试图了解人类原代视网膜色素上皮细胞（RPE）如何分化 进入间充质谱系的细胞。我们已经证明，RPE 的来源多种多样，包括 成人、胎儿、ESC 衍生的、ARPE-19 和牛获得间充质表型。此外，初级 成人 RPE 由单细胞克隆扩增产生脂肪细胞、软骨细胞和骨 当置于各自的分化培养基中时。这个主题很重要，因为 RPE 虽然通常是 稳定的上皮，可能会发生不利于视力的增殖和化生变化。 RPE化生 间充质相关细胞长期以来一直与视网膜疾病相关，但其潜在机制 这些变化仍然是个谜。在目标 1 中，我们将研究这种可塑性是如何在 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特征，表明 活跃基因和平衡基因（如果给予适当的提示则可以被激活的基因）。我们会询问是否 与间充质谱系相关的基因在人 RPE 细胞中处于平衡构象，并且是否 它们在分化成骨细胞、软骨细胞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因素 间充质干细胞（MSC）在向成骨细胞、软骨细胞和脂肪细胞发育过程中， 包括 TGF-β 超家族的成员，并使用小分子抑制剂，这将有助于 确定治疗方法来减弱这一过程。与 RPE 相关的显着异常细胞命运之一 化生就是骨化，因此了解 RPE 细胞如何经历尤为重要 成骨。在目标 3 中，我们将重点关注 RPE 在向这种命运过渡时所经历的步骤， 评估是否遵循正常成骨生成已知的转录因子序列，或 异常通路是否被激活。初步研究表明必需的物质显着上调 RPE 中的成骨转录因子 Runx2 正在向骨转化。我们将确定 使用 shRNA 方法敲除 Runx2 是否会抑制 RPE 成骨。这三个人在一起 目标解决从外源因素到基因调控和表观遗传的核心机制 变化是 RPE 表型可塑性的基础。众所周知，RPE 稳定性的破坏发生在多种情况中。 重要的视网膜疾病，如视网膜前膜形成、黄斑变性和灯泡痨， 了解这种可塑性将为开发这些疾病的治疗方法提供新的方法 条件。研究结果还将直接关系到理解维持 RPE 的因素 稳定、有色素、鹅卵石偏振的上皮层，考虑到 RPE 细胞，这是特别及时的 来自人类胚胎干细胞的细胞正在进入视网膜疾病的临床试验，必须采取措施 采取措施尽量减少化生变化，以确保安全移植条件的发展。

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.