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

基本信息

  • 批准号:
    10664301
  • 负责人:
  • 金额:
    $ 42.71万
  • 依托单位:
  • 依托单位国家:
    美国
  • 项目类别:
  • 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

项目摘要

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.
项目总结 远端关节紊乱(DA)综合征是以关节痉挛为特征的先天性疾病的集合 和口腔畸形。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基因的错义突变。有人建议, 突变的MYH3在收缩肌纤维中的表达是导致发育缺陷的原因 DA的病理特征。然而,梭内纤维在DA表型的病因中所起的作用 还没有被调查。梭内纤维是肌梭的特化细胞;一种本体感觉结构 负责调节收缩活动以应对拉伸。因为在此期间组织快速生长 胚胎发生导致整个有机体机械信号的动态变化,这似乎是合乎逻辑的 假设纺锤体功能缺陷会严重影响肌肉系统正确反应的能力 这些信号。这一点,再加上MYH3在胚胎后的梭内纤维中持续表达的事实 发展阶段,强调研究梭内纤维对DA病理学的贡献的重要性。 尚未对梭内纤维与DA综合征的关系进行研究的原因之一是缺乏 正常肌肉中的纺锤形结构。整个人类肌肉系统中只有大约50,000个纺锤体, 这使得在给定的活检样本中出现这种病毒的可能性极小。为了解决这个问题,我们 将利用诱导多能干细胞(IPSCs)产生人类融合内纤维,包括正常和MYH3 突变的基因型。在目标1中,将使用原始啮齿动物组织从IPSCs中优化这些细胞 作为纺锤体形态的基准。然后,优化的IPSC衍生的纺锤形细胞将受到控制 拉伸以量化它们对机械提示的激活,并表征任何功能差异 出现在突变细胞和对照细胞之间。因为目前还不知道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中(或两者兼有)。 实验将在隔离和与1a型感觉神经元共同培养的情况下进行。在AIM 2中,IPSC派生 将对融合内纤维进行单细胞RNA测序,以表征MYH3的转录组 突变的和正常的梭形细胞并鉴定突变的MYH3的表达是否有助于 持续到发育后期的表型。同样,原代啮齿动物细胞将被用来建立一个 纺锤形细胞类型的基准转录签名。来自IPSC的融合内细胞的结果将是 与从突变型和野生型梭外(收缩)肌纤维中获得的那些进行比较,以确定 突变的MYH3在纺锤体中的转录影响比在周围细胞中更明显 肌肉学。总体而言,该项目将增加我们对融合内纤维生物学的了解,为体外培养提供新的 检测纺锤体功能,并帮助确定突变的梭内纤维是否与DA的病因有关。

项目成果

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Alec Simon Tulloch Smith其他文献

Alec Simon Tulloch Smith的其他文献

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{{ truncateString('Alec Simon Tulloch Smith', 18)}}的其他基金

Using functional readouts from engineering models of innervated skeletal muscle to assess the efficacy of CRISPR-based c9orf72 ALS gene therapies
使用受神经支配的骨骼肌工程模型的功能读数来评估基于 CRISPR 的 c9orf72 ALS 基因疗法的功效
  • 批准号:
    10653223
  • 财政年份:
    2022
  • 资助金额:
    $ 42.71万
  • 项目类别:
High-throughput nanoMEA-based Proarrhythmia Assay
基于 nanoMEA 的高通量致心律失常检测
  • 批准号:
    9046607
  • 财政年份:
    2016
  • 资助金额:
    $ 42.71万
  • 项目类别:

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国际利益相关者合作进行关节弯曲研究:以客户为中心的护理网络(iSPARC-网络)
  • 批准号:
    468196
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了解和治疗关节弯曲-肾功能障碍-胆汁淤积 (ARC) 综合征中的肾脏疾病
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    2396509
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    2020
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    $ 42.71万
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    Studentship
Adult Arthrogryposis Research Registry: For patients by patients.
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  • 批准号:
    363788
  • 财政年份:
    2016
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    $ 42.71万
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The Stakeholders Partnering for Arthrogryposis Research: Client-Centered Care and Collaboration (SPARC-­Collaboration)
利益相关者合作进行关节弯曲研究:以客户为中心的护理与协作 (SPARC-协作)
  • 批准号:
    340503
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    $ 42.71万
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Analysis of the neural network formation in NZF-2/-3 double knock-out mice that exhibit symptoms of arthrogryposis
表现出关节弯曲症状的 NZF-2/-3 双敲除小鼠的神经网络形成分析
  • 批准号:
    26460259
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Elucidation of pathogenic mechanism of arthrogryposis multiplex congenita by Ctdnep1 gene deficiency
Ctdnep1基因缺陷阐明先天性多发性关节弯曲的发病机制
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导致远端关节弯曲的粗丝和细丝突变
  • 批准号:
    8734889
  • 财政年份:
    2012
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    $ 42.71万
  • 项目类别:
Thick and Thin Filament Mutations that cause Distal Arthrogryposis
导致远端关节弯曲的粗丝和细丝突变
  • 批准号:
    8734213
  • 财政年份:
    2012
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    $ 42.71万
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Thick and Thin Filament Mutations that cause Distal Arthrogryposis
导致远端关节弯曲的粗丝和细丝突变
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    8317383
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