The Role of Retinal Progenitor microRNAs for Late-stage Progenitor Cell State and Muller Glia Reprogramming

视网膜祖细胞 microRNA 在晚期祖细胞状态和 Muller 胶质细胞重编程中的作用

• 批准号: 10570874
• 负责人: Stefanie G Wohl
• 金额: $ 40.5万
• 依托单位: STATE COLLEGE OF OPTOMETRY
• 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 2027-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10570874
• 关键词: 3' Untranslated Regions; 3-Dimensional; Address; Age related macular degeneration; Architecture; Binding; Biological Assay; Blindness; Cell Cycle; Cell Proliferation; Cell Reprogramming; Cell physiology; Cells; Cyclin D1; Data; Development; Disease; Disease Progression; Electrophysiology (science); Enzymes; Fishes; Gene Expression; Gene Expression Profile; Generations; Genes; Goals; In Vitro; Ions; Lead; Malignant Neoplasms; Measures; Messenger RNA; MicroRNAs; Molecular; Monitor; Morphology; Muller' s cell; Mus; Natural regeneration; Neural Retina; Neuroglia; Neurons; Organism; Pathologic; Photoreceptors; Play; Postdoctoral Fellow; Proliferating; Property; Regenerative Medicine; Regulation; Reporter; Resources; Retina; Retinal Degeneration; Retinal Diseases; Retinitis Pigmentosa; Rod; Role; Source; Supplementation; Testing; Therapeutic Agents; Therapeutic Uses; Tissues; Validation; Visual impairment; Visualization; Work; antagonist; biomarker identification; candidate selection; cell behavior; cell fate specification; cell replacement therapy; cell type; experimental study; in vivo; innovation; overexpression; patch clamp; postnatal; postnatal development; preservation; prevent; programs; repaired; restoration; retinal neuron; retinal progenitor cell; retinal regeneration; retinal rods; retinogenesis; sensor; sight restoration; single-cell RNA sequencing; stem cell therapy; stem cells; therapy development; tool; transcription factor; transplantation therapy

PROJECT SUMMARY Our long-term goal is to prevent blindness, either by interfering with disease progression or by developing cell replacement therapies to restore vision. We believe that microRNAs (miRNAs) are a very powerful and innovative tool to accomplish this, but we first need to identify the set of miRNAs required for retinal cell fate specification and proper cell function in the developing retina. These miRNAs might represent potential therapeutic agents to not only restore imbalances that occur with the onset of retinal disorders, but also to induce specific cell fates for Müller glia (MG) reprogramming. We therefore propose to investigate the role of retinal progenitor cell (RPC)- miRNAs for late-stage RPC state and function and MG reprogramming by Aim 1: identifying the specific miRNAs required in early postnatal development, when rod photoreceptors (PR), bipolar cells (BCs), and MG are generated. Aim 2: testing miRNAs to reprogram MG into functional retinal neurons with focus on BCs and rod PR. We propose to use a mouse line that will not be able to produce miRNAs in their RPCs to better understand the function of miRNAs in retinal development and diseases. We will analyze tissue and cells with regard to morphological and functional alterations and determine which miRNAs are responsible for changes in cellular behavior. If we discover certain cell types that do not form properly without miRNAs, this could mean that specific miRNAs are required for proper development of that cell type. Rescue experiments will show whether disturbed tissue can be restored by miRNA supplementation and would imply potential therapeutic use. These miRNAs might also be new, additional reprogramming factors to regenerate specific cell types from stem cells or MG. My previous work has shown that miRNAs can reprogram MG into neuronal-like cells similar to BCs. However, whether these reprogrammed neurons are functional, and whether other neurons are generated, is still unknown. To address these questions, we will reprogram primary MG from reporter mice to visualize the conversion of MG into RPCs and neuronal-like cells, use patch clamp to measure ion currents, and profile their gene expression. miRNA candidates that successfully converted MG into functional neurons will be tested subsequently in 3D organotypic cultures (intact retinas outside the organism). Explants will be treated with miRNAs and evaluated with regard to cell proliferation and proper differentiation capability. miRNA candidates that can induce MG reprogramming ex vivo will be utilized for in vivo reprogramming approaches. To reveal underlying mechanisms and true miRNAs targets, we will use target prediction and target validation tools to narrow down and test selected candidates via sensors (in vitro proof of miRNA:mRNA prediction) and rescue experiments. This work provides a comprehensive study of miRNAs by combining molecular and cellular analyses with functional testing, in vitro, ex vivo and in vivo and will reveal (1) the set miRNAs required for proper retinal development and cell fate specification of late-born retinal neurons and (2) the set of miRNAs that can reprogram MG into functional neurons.

项目摘要 我们的长期目标是通过干扰疾病进展或发展细胞来防止失明 替代疗法以恢复视力。我们认为microRNA（miRNA）是一个非常有力和创新的 实现此目的的工具，但我们首先需要确定残留细胞脂肪规范所需的miRNA集合 和发育中的视网膜中的适当细胞功能。这些miRNA可能代表潜在的治疗剂 不仅可以恢复残留障碍发作的失衡，而且还引起特定的细胞命运 MüllerGlia（mg）重编程。因此，我们建议研究永久祖细胞（RPC）的作用 - 后期RPC状态和功能和MG重编程的miRNA通过目标1：识别特定miRNA 在产后早期发育中需要的是，当杆感光体（PR），双极细胞（BC）和MG是 生成。 AIM 2：测试miRNA以重新编程MG为功能残留神经元，重点是BC和ROD PR。我们建议使用将无法在其RPC中产生miRNA的鼠标线来更好地理解 miRNA在残留发育和疾病中的功能。我们将分析组织和细胞 形态学和功能改变，并确定哪些miRNA负责细胞的变化 行为。如果我们发现某些没有miRNA的细胞类型，但这可能意味着特定 需要miRNA才能正确开发该细胞类型。救援实验将显示是否受到影响 可以通过补充miRNA来恢复组织，并意味着潜在的治疗用途。这些miRNA 从干细胞或mg中再生特定细胞类型的新的重新编程因素也可能是新的，还可能是新的重编程因素。我的 先前的工作表明，miRNA可以将MG重新编程为类似于BCS的神经元样细胞。然而， 这些重编程的神经元是否功能性，以及是否产生其他神经元，仍然未知。 为了解决这些问题，我们将从记者小鼠中重新编程主要MG，以可视化MG的转换 进入RPC和神经元样细胞，使用斑块夹测量离子电流并介绍其基因表达。 miRNA候选成功转化为功能神经元的miRNA候选者将随后在3D中进行测试 有机培养物（有机体外部的视网膜完整）。外植体将用miRNA处理并进行评估 关于细胞增殖和适当的分化能力。可以诱导MG的miRNA候选者 重编程Ex Vivo将用于体内重编程方法。揭示基本机制 和真正的miRNA目标，我们将使用目标预测和目标验证工具来缩小和测试 通过传感器（miRNA的体外证明：mRNA预测）和救援实验的选定候选者。这项工作 通过将分子和细胞分析与功能测试相结合，提供对miRNA的全面研究， 体外，体内和体内，将揭示（1）适当剩余发育所需的集合miRNA和细胞 后期残留神经元的命运规范和（2）可以将MIRNA重新编程为功能的miRNA集合 神经元。