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: 8712501
• 负责人: Katia Del Rio-Tsonis
• 金额: $ 17.4万
• 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 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8712501
• 关键词: Age related macular degeneration; American; Animal Model; Back; Bioinformatics; Blindness; Cell Death; Cell Differentiation process; Cell Proliferation; Cells; Chick Embryo; Data; 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; Age related macular degeneration; American; Animal Model; Back; Bioinformatics; Blindness; Cell Death; Cell Differentiation process; Cell Proliferation; Cells; Chick Embryo; Data; 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

DESCRIPTION (provided by applicant): 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 som 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, isonly 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.

描述（由申请人提供）：导致视力丧失的视网膜变性是与年龄相关的黄斑变性（AMD），糖尿病性视网膜病和青光眼的最终结果。当前的疗法为那些患有这些疾病晚期的人提供了很少的选择。为了探索可能的疗法，重要的是使用具有再生能力的动物模型，例如雏鸡胚胎。胚胎小鸡在视网膜切除术后通过反差异的过程再生其视网膜，如果暴露于异位成纤维细胞生长因子2（FGF2）。该过程涉及视网膜色素上皮（RPE）的重编程以去分化，失去其色素，增殖和形成神经上皮，最终区分以形成所有主要的视网膜细胞类型。对于此提案，重点将放在去分化的过程上，这是了解转变方式的关键。我们的初步数据指出了一个两步的去分化过程，其中损伤（视网膜切除术）引起RPE有能力对FGF2做出反应。我们已经确定了一系列因“仅损伤”（步骤1）上调的因素（包括SOM多能诱导因子（PIF）和眼场转录因子。此外，我们发现Lin-28，斑马鱼中穆勒神经胶质转变的PIF和关键参与者，在视网膜移除后在鸡眼中添加FGF2（步骤2）后，Isonly上调了。根据我们的初步数据，我们将研究是否需要LIN-28且足以诱导视网膜鸡眼触发RPE转分化。我们将通过对含有LIN-28的质粒和使用吗啡对LIN28的质粒进行电孔实验。我们的假设是，LIN-28足以完成由伤害信号启动的RPE重编程过程，以制造新的视网膜。该提案的另一个重点是解剖调节组件，包括信号网络和mRNA-miRNA调节模块，使用公正的基因组范围广泛的方法，并利用先进的技术状态，例如下一代测序以执行mRNA-SEQ和miRNA-SEQ。我们假设与雏鸡RPE去分化有关的两步过程需要在每个步骤中一组独特的调节分子。这项研究将对再生医学领域产生重大影响，因为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，特别是人类RPE的潜在重编程以产生新的神经元。同样，鉴定可能重编程RPE的关键miRNA分子的意义也很高，因为这些分子是人类疗法非常需要的小分子。