Defining the molecular mechanisms underlying human RPE plasticity

项目摘要/摘要 在这项建议中，我们试图了解人类原代视网膜色素上皮细胞(RPE)是如何分化的 转化为间充质系的细胞。我们已经证明，RPE来自各种来源，包括 成人、胎儿、胚胎干细胞来源、ARPE-19和牛获得间充质表型。此外，初级的 从单个细胞克隆扩增的成人RPE产生脂肪细胞、软骨细胞和骨 当放置在各自的分化介质中时。这个主题很重要，因为RPE虽然通常是 稳定的上皮细胞，可经历不利于视力的增殖和化生变化。RPE化生 转化成间充质相关细胞长期以来一直与视网膜疾病有关，但其背后的机制 这些变化仍然是一个谜。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 活性基因和稳定基因(如果给予适当的提示就可以被激活的基因)。我们会问是否 与间充质系相关的基因在人类RPE细胞中处于稳定构象，以及 它们在分化为骨、软骨和脂肪细胞途径后转变为活跃的形式。在《目标2》中，我们 将通过测试已知的作用因素来确定刺激这种分化的外部因素 间充质干细胞(MSCs)在向成骨、软骨细胞和脂肪细胞方向发展过程中， 包括转化生长因子-β超家族的成员，并使用小分子抑制剂，这将有助于 找出减缓这一过程的治疗方法。与RPE相关的显著异常细胞命运之一 化生是骨化，因此了解RPE细胞是如何经历的尤为重要。 成骨作用。在目标3中，我们将重点介绍RPE在过渡到这一命运时所经历的步骤， 评估是否遵循了已知的正常成骨生成的转录因子序列，或者 异常通路是否被激活。初步研究表明，必要的基因显著上调 成骨转录因子Runx2在RPE向骨转化过程中的作用。我们将决定 使用shRNA方法敲除Runx2是否会抑制RPE成骨。加起来，这三个人 旨在解决从外源因子到基因调控和表观遗传的核心机制 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以下几个方面 重要的视网膜疾病，如视网膜前膜形成、黄斑变性和球痰， 了解这种可塑性将为开发这些疾病的治疗方法提供新的途径 条件。这些发现也将与理解维持RPE作为一种 稳定的、着色的、鹅卵石极化的上皮层，尤其是考虑到RPE细胞 从人类胚胎干细胞中提取的干细胞正在进入视网膜疾病的临床试验，步骤必须是 采取最大限度地减少化生改变，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.