Retinal Ganglion Cell Subtype Specification in Human Retinal Organoids

人视网膜类器官中视网膜神经节细胞亚型规范

• 批准号: 10389368
• 负责人: Brian Guy
• 金额: $ 4.68万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-03-01 至 2025-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10389368
• 关键词: Adult; Animal Model; Axon; Biological Assay; Biological Models; Blindness; Brain; Cellular biology; Cessation of life; Classification; Clustered Regularly Interspaced Short Palindromic Repeats; Code; Complement; Data; Development; Dyes; Ectopic Expression; Eye; Fluorescent Dyes; Gene Expression; Generations; Glaucoma; Goals; Human; Immunohistochemistry; Interneurons; Knock-out; Label; Macaca; Modeling; Molecular; Molecular Profiling; Morphology; Mus; Mutagenesis; Neurons; Organoids; Pharmacology; Photoreceptors; Play; Population; Primates; Protocols documentation; Publishing; RNA; Radial; Reporter; Retina; Retinal Diseases; Retinal Ganglion Cells; Role; Science; Specific qualifier value; Stains; Stratification; Testing; Time; Transfection; Viral; Work; base; cell type; combinatorial; experimental study; human tissue; in vivo; insight; lipophilicity; neuronal cell body; regenerative therapy; retinal neuron; single-cell RNA sequencing; stem cells; transcription factor; visual information

Project Summary Retinal ganglion cells (RGCs) are the interneurons that transmit visual information from the eye to the brain. Degeneration or death of RGCs results in a number of blinding conditions, including glaucoma. RGCs can be classified into many subtypes, each with distinct morphologies, functions, and gene expression profiles. While RGC subtypes have been identified in many vertebrate model organisms, RGC subtypes in the human retina have not been well-characterized molecularly. Moreover, the mechanisms controlling the specification of RGC subtypes remain poorly understood, especially in the human retina. In this project, I will study human retinas and organoids to identify human RGC subtypes and determine mechanisms controlling their generation. My work will yield mechanistic insights into human RGC subtype specification and facilitate the use of stem cell-derived organoids in regenerative therapies for retinopathies like glaucoma. One of the major goals of this project is to molecularly classify the RGC subtypes of the adult human retina. Based on published single cell RNA sequencing (scRNA-seq) data in human and macaque retinas, I generated a testable gene expression code to uniquely identify each RGC subtype. I will use this gene expression code to guide combinatorial expression analysis (i.e. immunohistochemistry and RNA FISH) and identify human RGC subtypes. I have already distinguished three human RGC subtypes using my gene expression code. I will also characterize the morphologies of these RGC subtypes. I will use combinatorial IHC and the lipophilic dye DiD to molecularly and morphologically classify human RGC subtypes (Aim 1). A major challenge to studying human RGC biology is the limited access to genetically and pharmacologically manipulatable human tissue. Human retinal organoids provide a powerful model to study developing human tissue in controlled conditions. The Johnston lab advanced the use of human retinal organoids to study the mechanisms controlling photoreceptor fate specification. I showed that organoids are a tractable model system to study RGC biology by developing an RGC axon outgrowth assay, which demonstrated that RGCs rapidly extend axons, recapitulating their in vivo capabilities. Furthermore, I identified two subtypes of RGCs in organoids. To identify the repertoire of RGC subtypes in human organoids, I will assess expression based on the testable gene expression code and complement this approach by conducting scRNA-seq over a time course of organoid development (Aim 2). The transcription factors EOMES/TBR2, TBR1, and TBX5 have been implicated in RGC subtype specification. To investigate mechanisms that specify human RGC subtypes, I will utilize CRISPR mutagenesis to knock out and viral transfection to ectopically express these three transcription factors and examine changes in RGC subtype populations in human retinal organoids (Aim 3).

项目摘要 视网膜神经节细胞（RGC）是将视觉信息从眼睛传递到视网膜的中间神经元。 个脑袋RGC的变性或死亡导致许多致盲病症，包括青光眼。RGC可以 可分为许多亚型，每种亚型具有不同的形态、功能和基因表达谱。而 RGC亚型已在许多脊椎动物模式生物中鉴定，RGC亚型在人类视网膜中 还没有很好的分子特征。此外，控制研资局规格的机制 亚型仍然知之甚少，特别是在人类视网膜中。在这个项目中，我将研究人类视网膜， 类器官鉴定人类RGC亚型并确定控制其产生的机制。我的工作将 产生对人RGC亚型特化的机制见解，并促进干细胞衍生的 类器官在青光眼等视网膜病变的再生治疗中的应用。 该项目的主要目标之一是对成人RGC亚型进行分子分类 视网膜。基于已发表的人类和猕猴视网膜单细胞RNA测序（scRNA-seq）数据， 产生了可测试的基因表达密码以唯一地识别每种RGC亚型。我会利用这个基因 表达编码以指导组合表达分析（即免疫组织化学和RNA FISH）， 鉴定人RGC亚型。我已经用我的基因区分了三种人类RGC亚型 表达式代码。我还将描述这些RGC亚型的形态学特征。我会用免疫组化 和亲脂性染料DiD对人RGC亚型进行分子和形态学分类（目的1）。 研究人类RGC生物学的一个主要挑战是获得基因和 可操作的人体组织。人类视网膜类器官提供了一个强大的模型来研究 在受控条件下发育人体组织。约翰斯顿实验室推进了人类视网膜类器官的使用 研究光感受器命运特化的调控机制。我证明了类器官是一个易于处理的 通过开发RGC轴突生长测定来研究RGC生物学的模型系统，其证明， RGC迅速延伸轴突，重现其体内能力。此外，我还发现了两种亚型， 类器官中的RGC。为了鉴定人类类器官中RGC亚型的库，我将评估RGC亚型的表达。 基于可测试的基因表达代码，并通过在一个 类器官发育的时间进程（目的2）。转录因子EOMES/TBR 2、TBR 1和TBX 5具有 与RGC亚型规范有关。为了研究指定人类RGC亚型的机制，我 将利用CRISPR诱变敲除和病毒转染异位表达这三个 转录因子，并检查人视网膜类器官中RGC亚型群体的变化（Aim 3）。