Mass Cytometry Analysis of Signaling Dysfunction in Duchenne Muscular Dystrophy

杜氏肌营养不良症信号传导障碍的质谱流式分析

基本信息

批准号：
9084275
负责人：
Helen M Blau
金额：
$ 32.35万
依托单位：
STANFORD UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-09-30 至 2018-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9084275
关键词：
Accounting Address Age Behavior Biological Assay Biomedical Engineering Biomimetics Biopsy Biotechnology Cell Culture Techniques Cell Lineage Cell Survival Cell physiology Cell surface Cells Cellular biology Cessation of life Clinical Cultured Cells Cytometry DNA Data Defect Development Disease Duchenne muscular dystrophy Dystrophin Exhibits Functional disorder Genes Health Heterogeneity Human Hydrogels Image Inherited Isotopes Knowledge Label Laboratories Length Life Luciferases MAPK14 gene Maps Measures Mediator of activation protein Medicine Methodology Methods Microscopy Modeling Molecular Mus Muscle Muscle Fibers Muscle Weakness Muscle satellite cell Muscular Atrophy Muscular Dystrophies Mutation NOD/SCID mouse Natural regeneration Nature Pathway interactions Patients Phenotype Phosphoproteins Population Sciences Process Protein Array Reagent Recording of previous events Regulation Sampling Science Signal Pathway Signal Transduction Skeletal Muscle Stem cells Surface System Technology Telomere Shortening Testing Time Transition Elements Translating Transplantation Trees Wasting Syndrome antibody conjugate base bioluminescence imaging cell injury disease-causing mutation high throughput analysis human disease in vivo injured innovation meetings mouse model neuromuscular new technology new therapeutic target notch protein novel novel therapeutics regenerative repaired self-renewal senescence stem cell niche stem cell population targeted treatment telomere transcription factor

项目摘要

DESCRIPTION (provided by applicant): Duchenne Muscular Dystrophy (DMD), the most common inherited muscular dystrophy, leads to progressive muscle weakness and death by the third decade of life. A conundrum is that the mouse model that has the same genetic defect, absence of dystrophin, does not mimic the human disease, which has limited the development of efficacious therapies. Recently, we hypothesized that telomere length differences between mice and humans could account for this discrepancy and developed a mouse model that lacks dystrophin and has shortened telomeres (mdx/mTRKO ). This model exhibits all of the major pathological hallmarks of human DMD. In particular, the mdx/mTRKO mice exhibit impaired regeneration and progressive muscle wasting due to functional defects in their muscle stem cells (MuSCs). Here we address a major challenge: MuSCs are known to be functionally heterogeneous, but the nature of their diversity has yet to be characterized which is essential for targeting therapies. We propose to capitalize on two groundbreaking technologies developed in our laboratories to elucidate the defects in the signaling networks that underlie the dysfunctional MuSC subsets in a manner previously not possible. To this end we will use novel (1) state-of-the-art single cell mass cytometry (CyTOF) and (2) artificial bioengineered stem cell niches. The CyTOF is uniquely suited to identify dysfunctional MuSC subsets. Using new parameters identified by CyTOF, we will purify these subsets and in conjunction with our biomimetic hydrogel microwell platform perform single-cell fate mapping. The combination of highly multivariate single cell CyTOF analyses and real time single cell time-lapse imaging will reveal dysfunctional signaling profiles and behaviors within subsets of stem cells. Defects in signaling pathways identified in specific MuSC subsets in the mouse model will be validated in MuSCs isolated from human DMD patient samples. These subsets and pathways can then be targeted, leading to novel therapeutic strategies that will enhance muscle fiber repair in DMD patients.

描述（由申请人提供）：杜氏肌营养不良症 (DMD) 是最常见的遗传性肌营养不良症，会导致进行性肌肉无力，并在 30 岁时导致死亡。一个难题是，具有相同遗传缺陷（缺乏肌营养不良蛋白）的小鼠模型无法模拟人类疾病，这限制了有效疗法的开发。最近，我们假设小鼠和人类之间的端粒长度差异可以解释这种差异，并开发了一种缺乏肌营养不良蛋白且端粒缩短的小鼠模型（mdx/mTRKO）。该模型展示了人类 DMD 的所有主要病理特征。特别是，mdx/mTRKO 小鼠由于肌肉干细胞 (MuSC) 的功能缺陷而表现出再生受损和进行性肌肉萎缩。在这里，我们解决了一个重大挑战：众所周知，MuSC 在功能上是异质的，但其多样性的本质尚未被表征，这对于靶向治疗。我们建议利用我们实验室开发的两项突破性技术来阐明导致功能失调的信号网络缺陷 MuSC 子集以以前不可能的方式出现。为此，我们将使用新颖的（1）最先进的单细胞质谱流式细胞术（CyTOF）和（2）人工生物工程干细胞生态位。 CyTOF 特别适合识别功能失调的 MuSC 子集。使用 CyTOF 识别的新参数，我们将纯化这些子集，并结合我们的仿生水凝胶微孔平台进行单细胞命运图谱。高度多元的单细胞 CyTOF 分析和实时单细胞延时成像的结合将揭示干细胞亚群内功能失调的信号传导谱和行为。小鼠模型中特定 MuSC 亚群中发现的信号通路缺陷将在从人类 DMD 患者样本中分离出的 MuSC 中得到验证。然后可以针对这些子集和途径，从而制定新的治疗策略，增强 DMD 患者的肌纤维修复。