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

项目摘要 骨骼肌是人体中再生能力最强的组织之一。骨骼肌祖细胞 （SMPCs）有助于发育性肌生成，而骨骼肌干细胞（卫星细胞，SC） 有助于产后肌肉的体内平衡和再生。在杜氏肌营养不良症（DMD）中， 一种流行但致命的X连锁肌肉萎缩性疾病，影响1：5000的活产男婴，功能丧失 DMD基因的突变导致缺乏稳定肌纤维的功能性肌营养不良蛋白。这 导致肌肉反复变性和再生的持续损伤， SC的再生能力。目前还没有治疗DMD的方法。使人多潜能干细胞分化 将人PSC转化为SC是开发DMD细胞替代疗法的宝贵资源。但目前的 hPSC指导的肌源性分化方案导致不能维持的胚胎/胎儿样SMPC 在培养中多次传代（即自我更新）。SMPC也不像出生后的SC那样有效地植入， 并且不知道如何使SMPC成熟为SC，因为不清楚它们在分子上如何不同。代谢 在整个发育过程中调节干细胞的细胞状态和功能中起着关键作用。我们进行了单细胞 RNA测序分析表明，糖酵解，三羧酸循环， 氧化磷酸化在人肌源性发育中减少。然而，代谢活动如何 支持SMPC或可用于将SMPC转化为SC的研究尚未超过 转录水平。通过更密切地评估组织来源的SC和hPSC来源的SC的代谢谱， 肌原性亚群，包括hPSC衍生的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 或在培养中促进hPSC-SMPC成熟为SC。与新陈代谢紧密相连的是表观基因组， 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接近性， 顺式调节区介导转录因子（TF）结合和控制基因表达。评价 相同肌源性群体之间TF结合基序的染色质可及性差异鉴定了 支持和管理SMPC和SC状态的TF。已经确定了几个工作队候选人， SMPC-to-SC成熟的调节剂。将调节TF候选物的表达以支持hPSC-SMPC 或促进其向SC命运的成熟。这项工作将首次阐明代谢和 表观遗传在维持和人类肌肉祖细胞和干细胞状态之间的转换中的作用。这些 这些发现将增强hPSC衍生的肌肉细胞的再生潜力，并使其能够发展。 改善DMD的生肌细胞疗法。本研究将在洛杉矶加州大学进行 在hPSC和骨骼肌生物学专家April Pyle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.