Control of Muscle Stem Cells to Enhance Regeneration

项目摘要 根据最近的美国人口普查，老年人口将增加一倍以上，达到8000万， 到2050年，每五个美国人中就有一个人。衰老的特征是组织功能和再生能力下降 容量肌肉减少症，也称为年龄依赖性骨骼肌质量和力量的丧失，是一个主要的公众- 影响15%的老年人的健康问题，导致行动能力丧失和生活质量下降。年龄- 相关的肌肉损失是由肌肉干细胞（MuSC）功能的丧失引起的， 体内平衡和再生。然而，与年龄相关的MuSC功能障碍的机制 仍然难以捉摸获得对MuSC老化的机械见解的两个主要障碍是（1） 老年MuSC人群，这使得标准批量分析无效，以及（2）缺乏解决问题的工具 这种异质性，强调需要单细胞研究。 我们之前证明了衰老的MuSC是由功能性细胞组成的异质群体， 和功能失调的子集。这一关键的观察结果提出了一种治疗策略， 弹性功能性MuSC的活性。在这里，我们探索这种可能性使用一个特定的细胞表面标记 以及解决MuSC子集所需的一系列创新单细胞技术。我们的初步数据显示- 将CD 47作为细胞表面标志物，其表达水平，而不是存在或不存在， CD 47 lo和功能失调的CD 47 hi MuSC亚群。作为SIRPα的受体，CD 47也是一种受体， 凝血酶敏感蛋白-1（THBS 1）。我们发现，CD 47 hi MuSCs在老化肌肉中积累，并且异常地表达。 按THBS 1。我们假设在衰老过程中，CD 47 hi MuSC的积累损害了细胞的增殖。 CD 47 lo MuSC通过分泌THBS 1，阻碍再生并导致肌肉减少症。这里我们的 具体的目标是（1）确定CD 47信号如何在衰老中出错，（2）阐明转录后 CD 47的调节在衰老过程中改变，导致功能失调的CD 47 hi MuSC的积累，以及（3） 确定老化MuSC小生境中异常THBS 1分泌对再生的影响。我们利用 尖端的单细胞技术，包括多维单细胞质谱仪（CyTOF）和多细胞流式细胞仪， 复合组织成像（CODEX）。这些技术使我们能够追踪同时发生的 在CD 47 lo和CD 47 hi MuSC（CyTOF）中发现了40+不同的细胞和信号表型，并解决了空间 多细胞生态位结构的变化导致衰老中的MuSC功能障碍（CODEX）。我们联合收割机 这一知识与老年小鼠体内再生能力和力量的研究相结合。最后，我们扰动 使用阻断抗体体内CD 47信号传导以克服老年小鼠中的再生缺陷。亲- 对新发现的可以前瞻性分离的老化MuSC亚群的设定分析将提供新的 对衰老的机械见解，并为增强内源性肌肉修复的治疗策略提供信息。

PROJECT SUMMARY According to the most recent U.S. Census, the elderly population will more than double to 80 million, encompassing 1 in 5 Americans by 2050. Aging is characterized by a decline in tissue function and regenerative capacity. Sarcopenia, also known as age-dependent loss of skeletal muscle mass and strength, is a major public- health problem that affects 15% of the elderly, leading to loss of mobility and diminished quality of life. Age- related muscle loss is paralleled by a loss in the function of muscle stem cells (MuSCs), key players in muscle homeostasis and regeneration. However, the mechanisms responsible for age-associated MuSC dysfunction remain elusive. Two major barriers to gaining mechanistic insights into MuSC aging are (1) the heterogeneity of the aged MuSC population, which renders standard bulk analysis ineffective, and (2) the lack of tools to resolve this heterogeneity, underscoring the need for single-cell studies. We previously demonstrated that aged MuSCs are a heterogeneous population comprised of functional and dysfunctional subsets. This key observation suggests a therapeutic strategy to regenerate muscle - boosting the activity of resilient functional MuSCs. Here we explore this possibility using a specific cell surface marker and a series of innovative single-cell technologies required to resolve MuSC subsets. Our preliminary data iden- tify CD47 as a cell surface marker whose expression level, not presence or absence, distinguishes functional CD47lo and dysfunctional CD47hi MuSC subsets. Known widely as a receptor for SIRPα, CD47 is also a receptor for thrombospondin-1 (THBS1). We found that CD47hi MuSCs accumulate in aged muscle and aberrantly ex- press THBS1. We hypothesize that during aging the accumulation of CD47hi MuSCs impairs the proliferation of CD47lo MuSCs through secretion of THBS1, hindering regeneration and contributing to sarcopenia. Here, our specific aims are to (1) determine how CD47 signaling goes awry in aging, (2) elucidate how post-transcriptional regulation of CD47 is altered during aging leading to the accumulation of dysfunctional CD47hi MuSCs, and (3) determine the effects of aberrant THBS1 secretion in the aged MuSC niche on regeneration. We capitalize on cutting-edge single-cell technologies, including multidimensional single-cell mass cytometry (CyTOF) and multi- plexed tissue imaging (CO-Detection by indexing (CODEX)). These technologies allow us to track simultane- ously 40+ distinct cell and signaling phenotypes in CD47lo and CD47hi MuSCs (CyTOF) and resolve how spatial changes in the architecture of the multicellular niche lead to MuSC dysfunction in aging (CODEX). We combine this knowledge with in vivo investigation of regenerative capacity and strength in aged mice. Finally, we perturb CD47 signaling in vivo using blocking antibodies to surmount the regenerative deficits in aged mice. The pro- posed analyses of newly identified aged MuSC subsets that can be prospectively isolated will provide fresh mechanistic insights into aging and inform therapeutic strategies to augment endogenous muscle repair.