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: 10366875
• 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/10366875
• 关键词: 3' Untranslated Regions; 3-Dimensional; Address; Age related macular degeneration; Architecture; Binding; Biological Assay; Blindness; Cell Cycle; Cell Proliferation; Cell physiology; Cells; Cyclin D1; Data; Development; Dicer Enzyme; Disease; Disease Progression; Electrophysiology (science); 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; 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; Work; antagonist; base; biomarker identification; cell behavior; cell fate specification; cell replacement therapy; cell type; experimental study; in vivo; innovation; overexpression; patch clamp; postnatal; postnatal development; preservation; prevent; repaired; restoration; retinal neuron; retinal progenitor cell; 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 我们的长期目标是预防失明，无论是通过干预疾病进展，还是通过开发细胞， 替代疗法来恢复视力。我们相信microRNAs（miRNAs）是一种非常强大和创新的 但是我们首先需要鉴定视网膜细胞命运特化所需的一组miRNAs 以及发育中的视网膜中正常的细胞功能。这些miRNAs可能是潜在的治疗药物， 不仅恢复视网膜疾病发作时发生的不平衡，而且还诱导特定的细胞命运， Müller神经胶质（MG）重编程。因此，我们建议研究视网膜祖细胞（RPC）的作用- 通过Aim 1鉴定晚期RPC状态和功能以及MG重编程的miRNAs： 出生后早期发育所需，当视杆细胞（PR），双极细胞（BC）和MG 生成的.目的2：测试miRNAs将MG重编程为功能性视网膜神经元，重点是BCs和杆 PR.我们建议使用一种不能在其RPC中产生miRNAs的小鼠品系，以更好地理解 miRNAs在视网膜发育和疾病中的作用我们将分析组织和细胞， 形态和功能的改变，并确定哪些miRNAs负责细胞的变化， 行为如果我们发现某些细胞类型在没有miRNAs的情况下不能正常形成，这可能意味着特定的 miRNAs是该细胞类型正常发育所必需的。救援实验将显示是否受到干扰 组织可以通过补充miRNA来恢复，并且将暗示潜在的治疗用途。这些mirna 也可能是新的、额外的重编程因子，用于从干细胞或MG再生特定细胞类型。我 先前的工作已经表明，miRNA可以将MG重编程为类似于BC的神经元样细胞。然而，在这方面， 这些重新编程的神经元是否有功能，以及是否产生了其他神经元，仍然是未知的。 为了解决这些问题，我们将重新编程报告小鼠的原代MG，以可视化MG的转化。 进入RPC和神经元样细胞，使用膜片钳测量离子电流，并描绘其基因表达。 成功将MG转化为功能性神经元的miRNA候选物随后将在3D中进行测试。 器官型培养物（在生物体外的完整视网膜）。将用miRNA处理外植体并进行评价 关于细胞增殖和适当的分化能力。可诱导MG的miRNA候选物 离体重编程将用于体内重编程方法。为了揭示潜在的机制 和真正的miRNA靶点，我们将使用靶点预测和靶点验证工具来缩小和测试 通过传感器（miRNA的体外证明：mRNA预测）和拯救实验选择候选物。这项工作 通过将分子和细胞分析与功能测试相结合，提供了对miRNA的全面研究， 在体外、离体和体内，并将揭示（1）正常视网膜发育和细胞增殖所需的miRNAs组， 晚期出生的视网膜神经元的命运特化和（2）可以将MG重编程为功能性的miRNA集。 神经元