Defining the molecular mechanisms underlying human RPE plasticity

项目概要/摘要 在这个建议中，我们试图了解人类原发性视网膜色素上皮细胞（RPE）的分化 转化为间充质细胞。我们已经证明，RPE来源于多种来源，包括 成人、胎儿、ESC衍生、ARPE-19和牛获得间充质表型。此外，主要 从单个细胞克隆扩增的成年人RPE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虽然通常是一个 稳定的上皮，可经历对视力有害的增殖和化生变化。视网膜色素上皮化生 长期以来，间充质相关细胞与视网膜疾病有关，但其机制 这些变化仍然是个谜。在aim 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 活跃基因和稳定基因（如果给予适当的提示，可以被激活的基因）。我们会问， 与间充质谱系相关的基因在人RPE细胞中处于稳定的构象， 它们在分化成骨细胞、软骨细胞和脂肪细胞途径时转变为活性形式。在目标2中， 将确定刺激这种分化的外源性因素，通过测试已知的作用于 间充质干细胞（MSC）在向成骨、软骨细胞和脂肪细胞命运发育期间， 包括TGF-β超家族成员，以及使用小分子抑制剂，这将有助于 确定治疗方法来减弱这一过程。与RPE相关的显著异常细胞命运之一 化生是骨化，因此了解RPE细胞如何经历 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， 评估是否遵循已知用于正常成骨生成的转录因子序列，或 是否激活了异常通路初步研究表明，在细胞内， 成骨转录因子Runx 2在RPE向骨转化中的作用。我们将确定 使用shRNA方法敲低Runx 2是否会抑制RPE成骨。在一起，这三个 目的是解决中心机制，从外源因素到基因调控和表观遗传 这些变化是RPE表型可塑性的基础。已知RPE稳定性的破坏发生在几种情况下， 重要的视网膜疾病如视网膜前膜形成、黄斑变性和眼球萎缩， 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， 条件这些发现也将直接关系到理解维持RPE作为一种细胞的因素。 稳定的、色素沉着的、鹅卵石极化的上皮层，这对于RPE细胞来说是特别及时的， 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， 采取最小化化生变化，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.