Mass Cytometry Analysis of Signaling Dysfunction in Duchenne Muscular Dystrophy

描述（由申请人提供）：杜氏肌营养不良症（DMD），最常见的遗传性肌营养不良症，导致进行性肌无力和死亡的第三个十年的生活。一个难题是，具有相同遗传缺陷的小鼠模型，缺乏抗肌萎缩蛋白，不能模拟人类疾病，这限制了有效疗法的发展。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，并开发了一种缺乏抗肌萎缩蛋白并缩短端粒的小鼠模型（mdx/mTRKO）。该模型表现出人DMD的所有主要病理学特征。特别地，mdx/mTRKO小鼠由于其肌肉干细胞（MuSC）的功能缺陷而表现出受损的再生和进行性肌肉萎缩。在这里，我们解决了一个主要的挑战：已知MuSC在功能上是异质的，但其多样性的性质尚未被表征，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信号网络中的缺陷，这些缺陷是功能失调的基础。 MuSC子集以以前不可能的方式。为此，我们将使用新的（1）最先进的单细胞质谱仪（CyTOF）和（2）人工生物工程干细胞龛。CyTOF特别适合鉴定功能障碍的MuSC亚群。使用CyTOF确定的新参数，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作图。高度多变量单细胞CyTOF分析和真实的时间单细胞延时成像的组合将揭示干细胞亚群内功能失调的信号传导谱和行为。将在从人DMD患者样品分离的MuSC中验证在小鼠模型中的特定MuSC子集中鉴定的信号传导途径的缺陷。然后可以靶向这些子集和途径，从而产生新的治疗策略，增强DMD患者的肌纤维修复。

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.