Mass Cytometry Analysis of Signaling Dysfunction in Duchenne Muscular Dystrophy

描述(申请人提供)：Duchenne肌营养不良症(DMD)，最常见的遗传性肌营养不良症，导致进行性肌肉无力和死亡的生命的第三个十年。一个难题是，具有相同遗传缺陷的小鼠模型不能模仿人类的疾病，这限制了有效治疗的开发。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dystrophin并缩短了端粒的小鼠模型(MDX/mTRKO)。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(MuSCs)的功能缺陷而表现出再生障碍和进行性肌肉萎缩。在这里，我们解决一个主要的挑战：已知的MSC在功能上是不同的，但它们的多样性的性质还没有被表征，这对于 有针对性的治疗。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令网络中的缺陷 以以前不可能实现的方式进行MUSC子集。为此，我们将使用新的(1)最先进的单细胞质量细胞术(CyTOF)和(2)人工生物工程干细胞壁龛。CyTOF是唯一适合于识别功能失调的MUSC亚群的方法。使用CyTOF确定的新参数，我们将提纯这些亚群，并与我们的仿生水凝胶微孔平台一起执行单细胞命运图谱。高度多变量的单细胞细胞时间推移分析和实时单细胞时间推移成像相结合，将揭示干细胞亚群中功能失调的信号特征和行为。在小鼠模型中特定的MUSC亚群中发现的信号通路缺陷将在从人类DMD患者样本中分离的MUSCs中得到验证。然后，这些亚群和通路可以被靶向，导致新的治疗策略，将加强DMD患者的肌肉纤维修复。

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.