Retinal Pigmented Epithelium Reprogramming and Retina Regeneration

视网膜色素上皮重编程和视网膜再生

• 批准号: 8598851
• 负责人: Katia Del Rio-Tsonis
• 金额: $ 21.3万
• 依托单位: MIAMI UNIVERSITY OXFORD
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-01 至 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8598851
• 关键词: Age related macular degeneration; American; Animal Model; Back; Bioinformatics; Blindness; Cell Death; Cell Differentiation process; Cell Proliferation; Cells; Chick Embryo; Data; Degenerative Disorder; Diabetic Retinopathy; Disease; Electroporation; Embryo; Epithelial; Excision; Eye; Fibroblast Growth Factor; Fibroblast Growth Factor 2; Figs - dietary; Gene Expression Profile; Genes; Glaucoma; Human; Injury; Knowledge; Mammals; Mesenchymal; Messenger RNA; MicroRNAs; Molecular; Monitor; National Eye Institute; Natural regeneration; Neuroglia; Neurons; Outcome; Phase; Pigments; Plasmids; Play; Population; Process; Proliferating; Proliferative Vitreoretinopathy; Publishing; Quantitative Reverse Transcriptase PCR; RNA-Binding Proteins; Regenerative Medicine; Reporting; Retina; Retinal Degeneration; Retinal Detachment; Role; Series; Signal Transduction; Somatic Cell; Staging; Structure of retinal pigment epithelium; Technology; Therapeutic; Visual Fields; Visual impairment; Work; Zebrafish; base; c-myc Genes; cell type; effective therapy; gain of function; genome-wide; injured; innovation; knock-down; loss of function; neuroepithelium; next generation; next generation sequencing; pluripotency; public health relevance; regenerative; repaired; research study; response; retinal neuron; small molecule; tissue regeneration; transcriptome sequencing; transdifferentiation

Project Summary Retinal degeneration leading to vision loss is the ultimate outcome of age related macular degeneration (AMD), diabetic retinopathy and glaucoma. Current therapies offer few options to those suffering from late stages of these diseases. In order to explore possible therapies, it is important to use animal models with regenerative capabilities such as the chick embryo. Embryonic chicks regenerate their retina, following retinectomy, via the process of trans-differentiation if exposed to ectopic fibroblast growth factor 2 (FGF2). This process involves the reprogramming of the retinal pigmented epithelium (RPE) to dedifferentiate, losing its pigment, proliferating and forming a neuroepithelium that eventually differentiates to form all major retina cell types. For this proposal, the emphasis will be on the process of dedifferentiation which is key to understanding how transdifferentiation works. Our preliminary data point to a two-step dedifferentiation process where injury (retinectomy) induces the RPE to become competent to respond to FGF2. We have identified a series of factors that are up-regulated with "injury only" (step 1) including some pluripotency inducing factors (PiFs) and eye field transcriptional factors. In addition, we have found that Lin-28, a PiF and a critical player in Muller glia transdifferentiation in zebrafish, is only up-regulated upon addition of FGF2 (step 2) in the chick eye after retina removal. Based on our preliminary data, we will investigate whether Lin-28 is required and sufficient to induce RPE transdifferentiation in retinectomized chick eyes. We will perform gain-of-function experiments by electroporating a plasmid containing Lin-28 and loss-of-function using morpholinos against Lin28. Our hypothesis is that Lin-28 is sufficient to complete the RPE reprogramming process initiated by injury signals to make new retina. The other focus of this proposal is on dissecting regulatory components including signaling networks and mRNA-miRNA regulatory modules using an unbiased, genome wide approach and taking advantage of state of the art technology such as Next Generation Sequencing to perform mRNA-Seq and miRNA-Seq. We hypothesize that the two-step process implicated in chick RPE dedifferentiation requires a unique set of regulatory molecules at each step. This study will have a significant impact on the field of regenerative medicine since the information obtained can be extrapolated to the process of retina repair in mammals including humans, and specifically on the potential reprogramming of human RPE to generate new neurons. Also the significance of identifying key miRNA molecules that could reprogram RPE is high considering these are small molecules highly desirable for human therapeutics.

项目概要 导致视力丧失的视网膜变性是年龄相关性黄斑变性的最终结果 (AMD)、糖尿病性视网膜病变和青光眼。目前的疗法为患有此病的患者提供的选择很少 这些疾病的晚期阶段。为了探索可能的疗法，重要的是使用动物 具有再生能力的模型，例如鸡胚胎。胚胎雏鸡再生 视网膜，视网膜切除术后，如果暴露于异位成纤维细胞，则通过转分化过程 生长因子 2 (FGF2)。这个过程涉及视网膜色素上皮的重新编程 （RPE）去分化，失去色素，增殖并形成神经上皮，最终 分化形成所有主要的视网膜细胞类型。对于该提案，重点将放在流程上 去分化的过程是理解转分化如何发挥作用的关键。我们的初步数据 指出了一个两步去分化过程，其中损伤（视网膜切除术）导致 RPE 变成 能够对 FGF2 做出反应。我们已经确定了一系列因“伤害”而上调的因素 仅”（步骤 1），包括一些多能性诱导因子 (PiF) 和眼场转录因子。 此外，我们发现Lin-28是PiF，也是Muller胶质细胞转分化的关键参与者。 在斑马鱼中，仅在去除视网膜后在鸡眼中添加 FGF2（步骤 2）后才会上调。 根据我们的初步数据，我们将调查 Lin-28 是否是必需的并且足以诱导 视网膜切除鸡眼中的 RPE 转分化。我们将通过以下方式进行功能获得实验 电穿孔含有 Lin-28 的质粒，并使用吗啡啉针对 Lin28 使其功能丧失。我们的 假设 Lin-28 足以完成由 损伤信号会产生新的视网膜。该提案的另一个重点是剖析监管 组件，包括信号网络和 mRNA-miRNA 调节模块，使用无偏见的、 全基因组方法并利用最先进的技术，例如下一代 测序以执行 mRNA-Seq 和 miRNA-Seq。我们假设两步过程 参与雏鸡 RPE 去分化的每个细胞都需要一组独特的调节分子 步。这项研究将对再生医学领域产生重大影响 获得的信息可以推断到包括人类在内的哺乳动物的视网膜修复过程， 特别是对人类 RPE 进行重新编程以产生新神经元的潜力。还有 考虑到识别可以重编程 RPE 的关键 miRNA 分子的重要性很高 这些是人类治疗非常需要的小分子。