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.

项目摘要 远端关节炎（DA）综合征是以联合染色体为特征的先天性疾病的集合 和口面畸形。 DA表型的最常见遗传原因是常染色体显性 MYH3基因中的错义突变，编码胚胎肌球蛋白重链。有人建议 收缩肌纤维中突变体MYH3的表达是导致发育缺陷的原因 表征DA病理学。但是，恒星内纤维对DA表型病因的贡献具有 尚未进行调查。恒星纤维是肌肉纺锤体的专门细胞。本体感受结构 负责调节收缩活动以响应拉伸。由于组织生长在 胚胎发生会导致整个生物体机械提示的动态变化，这似乎是合乎逻辑的 假设主轴功能的缺陷可能会严重影响肌肉正确反应的能力 这些信号。这与MYH3表达在过去的胚胎纤维中持续存在的事实 发育阶段突出了研究恒星纤维对DA病理学的贡献的重要性。 尚未研究与DA综合症有关的恒星纤维的原因之一是缺乏 正常肌肉中的主轴结构。整个人类肌肉组织中只有大约50,000个纺锤体， 在给定的活检样本中极不可能存在一个。为了克服这个问题，我们 是否会利用诱导的多能干细胞（IPSC）产生正常和MYH3的人体内纤维 突变基因型。在AIM 1中，将使用初级啮齿动物组织对这些细胞进行优化 作为主轴形态的基准。然后，优化的IPSC衍生的纺锤体将受到控制 伸展以量化其对机械提示的激活并表征任何功能差异 这是在突变细胞和对照细胞之间产生的。因为尚不知道机械敏感的离子通道是 存在于恒星纤维膜或相关感觉神经元的膜中（或两者） 实验将与1A型感觉神经元分离和共培养。在AIM 2中，IPSC衍生 恒星纤维将进行单细胞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 持续到后来发展阶段的表型。同样，一级啮齿动物细胞将用于建立 主轴细胞类型的基准转录组签名。来自IPSC衍生的恒星内细胞的结果将是 与从突变体和野生型外交（收缩）肌肉纤维获得的相比 突变体myh3的转录组影响在主轴上比在周围细胞中更明显 肌肉。总体而言，该项目将增加我们对余纤维生物学的理解，提供新的体外 测定探测纺锤体功能的测定，并有助于确定突变体内纤维是否有助于病因。