Control of Muscle Stem Cells to Enhance Regeneration

项目总结 根据美国最新的人口普查，老年人口将增加一倍以上，达到8000万， 到2050年，覆盖五分之一的美国人。衰老的特征是组织功能下降和再生 容量。骨质疏松症，也被称为年龄相关性骨骼肌质量和力量的丧失，是一种主要的公共- 影响15%老年人的健康问题，导致行动不便和生活质量下降。年龄- 相关的肌肉丧失与肌肉中的关键角色--肌肉干细胞(MuSCs)的功能丧失是平行的 动态平衡和再生。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老化的机制洞察的两个主要障碍是(1) MUSC人口老龄化，这使得标准批量分析无效，以及(2)缺乏工具来解决 这种异质性，强调了单细胞研究的必要性。 我们先前证明了老年间充质干细胞是一个由功能性 和功能失调的子集。这一关键的观察结果提出了一种再生肌肉的治疗策略 有弹性的功能性间充质干细胞的活性。在这里，我们使用一种特定的细胞表面标记来探索这种可能性 以及解析MUSC子集所需的一系列创新的单细胞技术。我们的初步数据表明- TIFY CD47作为细胞表面标志物，其表达水平，而不是有无，区分功能 CD47lo和功能障碍的CD47在MUSC亚群中。作为sirpα的受体，cd47也是一种受体。 用于凝血酶敏感蛋白-1(THBS1)。我们发现CD47hi MUSCs在衰老的肌肉中积聚，并异常地排出。 按THBS1。我们推测，在衰老过程中，CD47hi MUSCs的积累损害了 CD47lo MUSCs通过分泌THBS1，阻碍再生，导致骨质疏松症。在这里，我们的 具体目标是(1)确定CD47信号如何在衰老中出错，(2)阐明转录后如何 CD47的调节在衰老过程中发生改变，导致功能障碍的CD47在MuSCs中积聚，以及(3) 确定老年MUSC壁龛中THBS1的异常分泌对再生的影响。我们利用 尖端单细胞技术，包括多维单细胞质量细胞术(CyTOF)和多细胞 复合组织成像(CODEX)。这些技术使我们能够追踪同步者- CD47lo和CD47hi间充质干细胞(CyTOF)中明显不同的40+细胞和信号表型及其空间分布 多细胞生态位结构的改变导致MUSC衰老功能障碍(CODEX)。我们结合了 这一知识与在体研究衰老小鼠的再生能力和力量有关。最后，我们扰乱了 CD47体内信号传递利用封闭抗体克服衰老小鼠的再生缺陷。支持- 对新发现的可预期分离的陈旧MUSC亚群进行的建议性分析将提供最新的 对衰老的机械性洞察，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.