Reprogramming adult murine Müller glia via L-Myc expression

通过 L-Myc 表达对成年小鼠 Müller 胶质细胞进行重编程

基本信息

批准号：
10751295
负责人：
Megan Stone
金额：
$ 3.3万
依托单位：
VANDERBILT UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-09-01 至 2026-08-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10751295
关键词：
ATAC-seq Adult Affect Alleles BHLH Protein Binding Biological Process Blindness Bromodeoxyuridine Cell Cycle Cell Reprogramming Cell division Cells Chromatin Color Competence DNA Data Disease Electroporation Euchromatin Family Fibroblasts Fishes Fluorescence Future Gene Expression Gene Expression Regulation Genes Genetic Genetic Transcription Heritability Histone H3 Histones Hour In Vitro Injury Label MYCL1 gene Mammals Measures Modeling Muller&apos s cell Multiomic Data Mus Natural regeneration Neuroglia Neurons Ontology Orthologous Gene Pattern Plasmids Play Population Process Proliferating Proteins Regenerative capacity Retina Role Signal Transduction Somatic Cell Sorting Source Stains System Testing Time Tissue-Specific Gene Expression Tissues Vertebrates Vision Visual Visual impairment Zebrafish bioinformatics network cell growth cell type data integration design fluorophore functional restoration gene regulatory network gene therapy induced pluripotent stem cell insight mouse model overexpression prevent progenitor programs response response to injury restoration retinal neuron retinal progenitor cell retinal regeneration selective expression sight restoration stem cells teleost fish tissue regeneration transcriptome sequencing transcriptomics vector

项目摘要

PROJECT SUMMARY In response to damage from injury or disease, lost neurons in the adult mammalian retina are irreplaceable which can lead to visual impairment including blindness. Some non-mammalian vertebrates, such as teleost fish, can regenerate the retina in response to damage via the Müller glia. In teleost fish, Müller glia will respond to signals from damaged neurons by asymmetrically dividing to produce retinal progenitor cells. These progenitor cells then differentiate into neuronal cell types and functionally restore tissue. Mammalian Müller glia are quiescent and do not display regenerative capacity in response to damage. However, mammalian Müller glia demonstrate some cell state plasticity and can be reprogrammed into induced pluripotent stem cells (iPSC) in vitro. Mammalian Müller glia likely have the capacity for reprogramming but lack the cell intrinsic- or extrinsic-signaling necessary to do so. I have found that overexpression of L-Myc, a robust reprogramming factor used to reprogram many somatic cell types to iPSCs, leads to cell cycle re-entry in adult mammalian Müller glia in ex vivo retinal tissue. Using murine retinal explant tissue as an injury model, I will characterize the effects of L-Myc overexpression on proliferation and dedifferentiation in mammalian Müller glia. I hypothesize that L-Myc overexpression in Müller glia leads to direct and indirect changes in gene expression and chromatin accessibility that reprogram Müller glia toward a progenitor-like state. To test this hypothesis, I will first characterize the effects of L-Myc expression on proliferation. I will do so by defining the interval of proliferation as it relates to L-Myc expression and determining if Müller glia or their descendants are able to divide repeatedly following L-Myc overexpression. Next, I will characterize the effect of L-Myc overexpression in Müller glia on cell identity and gene regulation. Using data integration of RNA-sequencing, ATAC-sequencing, and CUT&Tag of FACS sorted L-Myc overexpressing Müller glia, I will identify gene regulatory networks affected directly or indirectly by L-Myc overexpression and identify changes in cell state. Combining Müller glia, a highly promising population within the mammalian retina for reprogramming, and L-Myc, a potent reprogramming factor to promote proliferation and dedifferentiation, creates an ideal scenario to reveal targets to promote regeneration in the mammalian retina through the analysis of gene regulatory networks activated or inhibited by L-Myc overexpression. Regardless of the cell state changes that occur in response to L-Myc overexpression in Müller glia, this project will reveal what signaling barriers exist that prevent tissue regeneration via Müller glia in the mammalian retina. This study is designed to identify future targets for genetic therapies to promote restoration of tissue and visual function after injury in the mammalian retina, which one day could lead to treatments for currently untreatable visual diseases.

项目概要为了应对损伤或疾病造成的损害，成年哺乳动物视网膜中丢失的神经元不可替代，可能导致一些非哺乳动物脊椎动物失明。例如硬骨鱼，可以通过穆勒神经胶质细胞再生视网膜以应对硬骨鱼的损伤。神经胶质细胞将通过不对称分裂来响应受损神经元的信号，产生视网膜祖细胞然后，这些祖细胞分化成神经细胞类型并恢复组织的功能。哺乳动物的米勒神经胶质细胞处于静止状态，不会表现出对损伤做出反应的再生能力。然而，哺乳动物穆勒神经胶质细胞表现出一定的细胞状态可塑性，可以被重新编程为体外诱导的多能干细胞（iPSC）可能具有哺乳动物米勒神经胶质细胞的能力。重编程，但缺乏这样做所需的细胞内在或外在信号传导。我发现 L-Myc 的过度表达是一种强大的重编程因子，可用于重编程许多体细胞类型转化为 iPSC，导致离体视网膜组织中成年哺乳动物穆勒神经胶质细胞的细胞周期重新进入。使用小鼠视网膜外植体组织作为损伤模型，我将描述 L-Myc 过度表达的影响我领导了哺乳动物 Müller 神经胶质细胞的增殖和去分化的 L-Myc 过度表达。穆勒胶质细胞导致基因表达和染色质可及性的直接和间接变化，从而重新编程为了检验这一假设，我将首先描述 L-Myc 的作用。我将通过定义与 L-Myc 表达相关的增殖间隔来实现这一点。并确定 Müller 胶质细胞或其后代是否能够按照 L-Myc 反复分裂接下来，我将描述 Müller 胶质细胞中 L-Myc 过度表达对细胞身份和特征的影响。基因调控。使用RNA测序、ATAC测序和FACS排序的CUT&Tag的数据集成 L-Myc 过度表达穆勒胶质细胞，我将确定受 L-Myc 直接或间接影响的基因调控网络过度表达并识别细胞状态的变化。结合穆勒神经胶质细胞（哺乳动物视网膜内非常有希望进行重编程的群体）， L-Myc，一种促进增殖和去分化的有效重编程因子，创造了理想的通过基因分析揭示促进哺乳动物视网膜再生的目标的场景无论细胞状态如何变化，L-Myc 过度表达都会激活或抑制调节网络。这是对 Müller 胶质细胞中 L-Myc 过度表达的反应，该项目将揭示存在哪些信号传导障碍这项研究旨在确定通过穆勒神经胶质细胞阻止哺乳动物视网膜的组织再生。基因疗法的未来目标是促进损伤后的组织恢复和视觉功能哺乳动物的视网膜，有一天可能会导致目前无法治疗的视觉疾病的治疗。