Quiescence of Muscle Stem Cells During Growth and Repair

描述（由申请人提供）：成体干细胞具有自我更新和分化的能力。虽然它们具有自我更新的特性，但这种潜力不是无限的。这就引出了以下问题：干细胞是如何在整个生命中维持的？答案在于可逆的静止状态。成体干细胞主要处于静止状态，穿插着罕见的进入细胞周期的穿越，不像它们的下游后代在周期中花费更多的时间。利用细胞标记技术监测细胞分裂历史，使分裂频率较低的干细胞具有自我更新的潜力。在肌肉萎缩症中，肌肉干细胞（卫星细胞，SCs）经历连续的增殖，随着时间的推移导致干细胞池的消耗。在衰老的肌肉中，SC池减少，更新和分化受损。因此，了解“增殖受限”干细胞如何可能是优化衰老和疾病期间干细胞功能的关键。在这项拨款提案中，我们将询问1)慢速和快速分裂SCs之间的功能异质性。2)生长因子信号通路抑制剂Spry是如何指定和维持SC缓慢分裂群体的。3)衰老过程中失去SC慢分裂亚群的后果，SC功能和肌肉稳态。基于标签保留特性（LRC），使用转基因H2B-GFP方法在体内鉴定慢分裂SCs；较少分裂的细胞保留更多的标签。卫星单元将分为LRC和非LRC；这两个亚群将根据体内和体外分析测试其功能差异。我们已经生成了初步数据，表明Spry1在LRC中表达丰富，Spry1的遗传破坏导致生长肌肉中LRC的损失和衰老过程中sc数量的加速下降。我们将扩展这些研究，以暂时诱导的方式在sc中特异性地删除和过表达Spry1。我们将破坏Spry1以改变LRC的建立和维持，并研究其对SC功能和肌肉表型的影响。本研究的具体目的是：1)研究卫星细胞在发育和出生后肌发生过程中的缓慢分裂动力学；2)确定Spry1是否需要和充分调节LRC细胞；3)了解SC LRC在整个生命过程中丢失的后果。

DESCRIPTION (provided by applicant): Adult stem cells are capable of self renewal and differentiation. Although they possess self-renewal properties, this potential is not limitless. This begs the following question: how are stem cells maintained throughout life? The answer lies in the reversible state of quiescence. Adult stem cells are predominantly in a quiescent state interspersed with rare traverses into the cell cycle unlike their downstream descendents that spend more time in cycle. Using cell labeling techniques to monitor cell division history, stem cells that divide less frequently are enriched for self-renewal potential. In muscular dystrophies, muscle stem cells (satellite cells, SCs) undergo continuous bouts of proliferation, which leads to a depletion of the stem cell pool over time. In aged muscle the SC pool is diminished and displays impaired renewal and differentiation. Therefore understanding how 'proliferation-restricted' stem cells may hold the key to optimizing stem cell function during aging and disease. In this grant proposal we will interrogate 1) the functional heterogeneity between slow and faster dividing SCs. 2) How the slow dividing SC population is specified and maintained throughout life through Spry, an inhibitor of growth factor signaling. 3) The consequences of losing slow dividing subset of SCs SC function and muscle homeostasis during aging. Slow dividing SCs will be identified in vivo based on label retaining character (LRC) using a transgenic H2B-GFP approach; cells that undergo fewer divisions retain more label. Satellite cells will be characterized for LRC and non-LRC; both subpopulations will be tested for their functional differences based on in vivo and in vitro assays. We have generated preliminary data that demonstrates Spry1 expression is enriched in LRCs and genetic disruption of Spry1 leads to a loss of LRC in growing muscle and an accelerated decline in the number of SCs during aging. We will extend these studies to delete and overexpress Spry1 specifically in SCs in a temporally inducible manner. We will disrupt Spry1 to alter LRC establishment and maintenance and study the effects on SC function and muscle phenotype. The specific aims of this proposal are: 1) To study the slow division dynamics of satellite cells during developmental and postnatal myogenesis, 2) to identify whether Spry1 is required and sufficient to regulate LRC SCs, and 3) to understand the consequences of losing SC LRC throughout life. PUBLIC HEALTH RELEVANCE: Identifying a specialized subset of highly functioning muscle stem cells is essential to ameliorate skeletal muscle aging and diseases. Unfortunately, the identity of such cells and how they are established and maintained in not known. In this project we will identify highly functioning SCs and dissect an essential genetic coordinator that defines their functional superiority. We will employ genetic strategies to impair or enhance this SC subset early in life and study the long term effects in aged muscle. Deciphering endogenous stem cell modifiers that can be modulated to maintain stem cell function and tissue homeostasis throughout life is critical for harnessing their potential to treat muscle related diseases. Furthermore, the biology of satellite cells may serve as a paradigm for stem cells in other tissues.