A stem cell-based model of the human muscle spindle for studying proprioceptive dysfunction in distal arthrogryposis syndromes

基于干细胞的人体肌梭模型，用于研究远端关节挛缩综合征的本体感觉功能障碍

• 批准号: 10664301
• 负责人: Alec Simon Tulloch Smith
• 金额: $ 42.71万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-19 至 2025-08-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10664301
• 关键词: Afferent Neurons; Arthrogryposis; Benchmarking; Biology; Biopsy Specimen; Cells; Coculture Techniques; Collection; Congenital Disorders; Contracture; Coupled; Data; Data Set; Defect; Development; Distal; Dominant-Negative Mutation; Electrophysiology (science); Embryo; Embryonic Development; Etiology; Exhibits; Feedback; Fiber; Functional disorder; Future; Gene Expression; Gene Expression Profile; Genes; Genetic; Genotype; Growth; Human; Impairment; In Vitro; Investigation; Ion Channel; Joints; Kinetics; Length; Membrane; Missense Mutation; Morphology; Muscle; Muscle Cells; Muscle Contraction; Muscle Development; Muscle Fibers; Muscle Proteins; Muscle Spindles; Muscular Dystrophies; Musculoskeletal Development; Mutation; Myoblasts; Myosin ATPase; Myosin Heavy Chains; Organism; Parkinson Disease; Pathology; Patients; Phenotype; Piezo 2 ion channel; Play; Pluripotent Stem Cells; Population; Proprioception; Proprioceptor; Protein Isoforms; Protocols documentation; Reflex action; Research; Rodent; Role; Sampling; Sensory; Signal Transduction; Skeletal Muscle; Stretching; Structure; Syndrome; Techniques; Testing; Time; Tissues; age-related muscle loss; autosome; cell type; embryo tissue; experience; experimental study; functional loss; genetic analysis; human stem cells; human tissue; in vitro Assay; in vivo; induced pluripotent stem cell; mechanical signal; mechanotransduction; mutant; novel; novel marker; orofacial; postnatal; response; sarcopenia; screening; single-cell RNA sequencing; stem cell model; theories; tool; transcriptome; transcriptomics; voltage sensitive dye

PROJECT SUMMARY Distal arthrogryposis (DA) syndromes are a collection of congenital disorders characterized by joint contractures and orofacial dysmorphisms. The most common genetic cause of DA phenotypes are autosomal dominant missense mutations in the MYH3 gene, encoding the embryonic myosin heavy chain. It has been suggested that expression of mutant MYH3 within contractile muscle fibers is responsible for the developmental defects that characterize DA pathology. However, the contribution of intrafusal fibers to the etiology of DA phenotypes has not yet been investigated. Intrafusal fibers are specialized cells of the muscle spindle; a proprioceptive structure responsible for regulating contractile activity in response to stretch. Since rapid tissue growth during embryogenesis leads to dynamic changes in mechanical cues throughout the organism, it seems logical to assume that defects in spindle function could severely impact the ability for the musculature to respond correctly to these signals. This, coupled with the fact that MYH3 expression persists in intrafusal fibers past embryonic stages of development, highlight the importance of studying the contribution of intrafusal fibers to DA pathology. One of the reasons that intrafusal fibers have not been studied in relation to DA syndromes is the scarcity of spindle structures in normal muscle. Only about 50,000 spindles are present in the entire human musculature, making in extremely unlikely that one will be present within a given biopsy sample. To overcome this issue, we will utilize induced pluripotent stem cells (iPSCs) to produce human intrafusal fibers with both normal and MYH3 mutant genotypes. In Aim 1, optimization of these cells from iPSCs will be performed using primary rodent tissue as a benchmark for spindle morphology. Optimized iPSC-derived spindle cells will then be subjected to controlled stretch to quantify their activation in response to mechanical cues and to characterize any functional differences that arise between mutant and control cells. As it is not yet known whether mechano-sensitive ion channels are present in the intrafusal fiber membrane or in the membrane of associated sensory neurons (or both), these experiments will be conducted in isolation and in co-culture with type 1a sensory neurons. In Aim 2, iPSC-derived intrafusal fibers will be subjected to single cell RNA sequencing to characterize the transcriptome of MYH3 mutant and normal spindle cells and identify whether the expression of mutant MYH3 contributes to an altered phenotype that persists to later stages of development. Again, primary rodent cells will be used to establish a benchmark transcriptomic signature for spindle cell types. Results from iPSC-derived intrafusal cells will be compared to those obtained from mutant and wild type extrafusal (contractile) muscle fibers to determine whether the transcriptomic impact of mutant MYH3 is more pronounced in the spindle than in the surrounding cells of the musculature. Overall, this project will increase our understanding of intrafusal fiber biology, provide a new in vitro assay for probing spindle function, and help determine whether mutant intrafusal fibers contribute to DA etiology.

项目概要 远端关节挛缩 (DA) 综合征是一系列以关节挛缩为特征的先天性疾病 和口面部畸形。 DA 表型最常见的遗传原因是常染色体显性遗传 编码胚胎肌球蛋白重链的 MYH3 基因发生错义突变。有人建议 突变型 MYH3 在收缩肌纤维内的表达是导致发育缺陷的原因 表征 DA 病理学。然而，梭内纤维对 DA 表型病因学的贡献 尚未被调查。梭内纤维是肌梭的特殊细胞；本体感受结构 负责调节收缩活动以响应拉伸。由于在此期间组织快速生长 胚胎发生导致整个生物体机械线索的动态变化，这似乎是合乎逻辑的 假设纺锤体功能缺陷可能严重影响肌肉组织正确反应的能力 对这些信号。再加上 MYH3 表达在胚胎期后的梭内纤维中持续存在这一事实 发育阶段，强调研究梭内纤维对 DA 病理学的贡献的重要性。 尚未研究梭内纤维与 DA 综合征的关系的原因之一是缺乏 正常肌肉中的纺锤体结构。整个人体肌肉组织中仅存在约 50,000 个纺锤体， 使得在给定的活检样本中出现这种情况的可能性极小。为了克服这个问题，我们 将利用诱导多能干细胞 (iPSC) 生产具有正常和 MYH3 的人类梭内纤维 突变基因型。在目标 1 中，将使用原代啮齿动物组织对来自 iPSC 的这些细胞进行优化 作为纺锤体形态的基准。然后，优化的 iPSC 衍生的梭形细胞将受到控制 拉伸以量化其响应机械线索的激活并表征任何功能差异 突变细胞和对照细胞之间出现的。由于尚不清楚机械敏感离子通道是否 存在于梭内纤维膜或相关感觉神经元的膜（或两者）中，这些 实验将单独进行，并与 1a 型感觉神经元共培养。在目标 2 中，iPSC 衍生的 梭内纤维将进行单细胞 RNA 测序，以表征 MYH3 的转录组 突变体和正常纺锤体细胞，并确定突变体 MYH3 的表达是否有助于改变 持续到发育后期的表型。同样，原代啮齿动物细胞将用于建立 梭形细胞类型的基准转录组特征。 iPSC 衍生的梭内细胞的结果将是 与从突变型和野生型梭外（收缩）肌纤维获得的纤维进行比较，以确定是否 突变体 MYH3 的转录组影响在纺锤体中比在纺锤体周围细胞中更明显 肌肉组织。总体而言，该项目将增加我们对梭内纤维生物学的理解，提供新的体外 检测纺锤体功能，并帮助确定突变的梭内纤维是否有助于 DA 病因。