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）是最常见的遗传性肌营养不良症，可导致进行性肌肉无力并在生命的第三个十年死亡。一个难题是，具有相同遗传缺陷的小鼠模型，缺乏肌营养不良蛋白，并不能模仿人类疾病，这限制了有效治疗方法的发展。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，并开发了一种缺乏肌营养不良蛋白和端粒缩短（mdx/mTRKO）的小鼠模型。该模型显示了人类DMD的所有主要病理特征。特别是，mdx/mTRKO小鼠由于其肌肉干细胞（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，这是至关重要的

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.