Understanding and regulating skeletal muscle progenitor and stem cell states through metabolic and epigenetic modulation

项目摘要 骨骼肌是人体内再生能力最强的组织之一。骨骼肌祖细胞 (SMPC)有助于发育性肌肉发生，而骨骼肌干细胞(卫星细胞，SCs) 有助于出生后肌肉的动态平衡和再生。在杜氏肌营养不良症(DMD)中， 流行但致命的X连锁肌肉萎缩病，影响1：5000的活男婴，一种功能丧失 DMD基因的突变导致肌营养不良蛋白缺乏稳定肌肉纤维的功能。这 导致肌肉反复退化和再生而导致持续损伤，并损害 干细胞的再生能力。目前还没有治愈DMD的方法。分化人多能干细胞的实验研究 将hPSCs转化为SCs是开发DMD细胞替代疗法的宝贵资源。但是，当前 HPSC诱导的肌源性分化方案导致不能维持的胚胎/胎儿样SmPC 在文化中的多个阶段(即自我更新)。SMPC也不像出生后的SCs那样有效地植入， 目前还不知道如何将SmPC成熟为SCs，因为尚不清楚它们在分子上的差异。新陈代谢 在干细胞发育过程中，在调节细胞状态和功能方面起着关键作用。我们进行了单细胞实验 RNA测序分析表明，大多数基因在糖酵解、三羧酸循环和 氧化磷酸化在整个人类肌肉发生发育过程中减少。然而，新陈代谢活动如何 支持SMPC或可用于在人类中将SMPC转换为SCs的研究尚未超出 转录水平。通过更密切地评估组织来源的干细胞和hPSC来源的代谢特征 肌源性亚群包括hPSC来源的SMPC(hPSC-SMPC)、特异性代谢酶和 上述途径中的代谢物将作为支持hPSC-SMPC自我更新的候选物质 或在培养中促进hPSC-SMPC向SCs成熟。与新陈代谢密切相关的是表观基因组 在细胞状态之间的转换中起直接作用，特别是通过调节染色质的可及性 顺式调控区域，介导转录因子(TF)结合和控制基因表达。正在评估 同一肌源性群体间TF结合基序染色质可及性的差异识别 支持和规范SMPC和SC状态的TFS。几个特遣部队的候选人已被确定为潜在的 SMPC-to-SC成熟的调节器。候选转移因子的表达将被调节以支持hPSC-SMPC 或者促使他们走向成熟，走向SC的命运。这项工作将首次揭示新陈代谢和 表观遗传学在维持和转换人类肌肉祖细胞和干细胞状态中的作用。这些 这些发现将增强hPSC来源的肌肉细胞的再生潜力，并使 改良的肌源性细胞疗法治疗DMD。这项研究将在加州大学洛杉矶分校进行 在我的努力中，在hPSC和骨骼肌生物学专家阿普里尔·派尔博士的指导下， 成为再生医学领域的研究科学家。

Project Summary Skeletal muscle is one of the most regenerative tissues in the human body. Skeletal muscle progenitor cells (SMPCs) contribute to developmental myogenesis, and skeletal muscle stem cells (satellite cells, SCs) contribute to postnatal muscle homeostasis and regeneration. In Duchenne Muscular Dystrophy (DMD), a prevalent but fatal X-linked muscle wasting disease that affects 1:5000 live male births, a loss-of-function mutation in the DMD gene results in the absence of functional dystrophin protein to stabilize muscle fibers. This leads to continuous damage from repeated muscle degeneration and regeneration and compromises the regenerative ability of SCs. There is currently no cure for DMD. Differentiating human pluripotent stem cells (hPSCs) into SCs is a valuable resource for developing cell replacement therapies for DMD. However, current hPSC directed myogenic differentiation protocols result in embryonic/fetal-like SMPCs that cannot be maintained over multiple passages (i.e. self-renew) in culture. SMPCs also do not engraft as efficiently as postnatal SCs, and it is not known how to mature SMPCs to SCs because it is unclear how they molecularly differ. Metabolism plays a key role in regulating cell state and function in stem cells across development. We performed single cell RNA sequencing analysis that demonstrated that expression of most genes in glycolysis, tricarboxylic cycle, and oxidative phosphorylation decrease across human myogenic development. However, how metabolic activity supports SMPCs or can be used to transition SMPCs to SCs in humans has not been investigated beyond the transcriptional level. By more closely evaluating the metabolic profiles of tissue-derived SCs and hPSC-derived myogenic subpopulations including hPSC-derived SMPCs (hPSC-SMPCs), specific metabolic enzymes and metabolites in the aforementioned pathways will be targeted as candidates to support hPSC-SMPC self-renewal or promote hPSC-SMPC maturation to SCs in culture. Tightly connected to metabolism is the epigenome which has a direct role in transitioning between cell states, particularly through modulating chromatin accessibility at cis-regulatory regions to mediate transcription factor (TF) binding and control gene expression. Evaluating differences in chromatin accessibility of TF binding motifs between the same myogenic populations identifies TFs that support and regulate SMPC and SC states. Several TF candidates have been identified as potential regulators of SMPC-to-SC maturation. Expression of TF candidates will be modulated to support hPSC-SMPCs or promote their maturation toward SC fate. This work will for the first time shed light on the metabolic and epigenetic roles in maintaining and transitioning between human muscle progenitor and stem cell states. These findings will enhance the regenerative potential of hPSC-derived muscle cells and enable the development of improved myogenic cell therapies for DMD. This study will be performed at the University of California, Los Angeles under the mentorship of Dr. April Pyle, an expert in hPSC and skeletal muscle biology, in my endeavor to becoming a research scientist in the field of regenerative medicine.