Quiescence of Muscle Stem Cells During Growth and Repair

描述（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，但这种潜力并不是无限的。这就引出了一个问题：干细胞是如何维持生命的？答案在于静止的可逆状态。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过程，不像它们的下游后代在周期中花费更多的时间。使用细胞标记技术来监测细胞分裂历史，使分裂频率较低的干细胞富集以具有自我更新潜力。在肌营养不良症中，肌肉干细胞（卫星细胞，SC）经历连续的增殖，这导致干细胞库随着时间的推移而耗尽。在老年肌肉中，SC池减少，并显示受损的更新和分化。因此，了解“增殖限制”干细胞如何可能是在衰老和疾病期间优化干细胞功能的关键。 在这个资助计划中，我们将探讨1）慢速和快速分裂的SC之间的功能异质性。2)如何通过Spry（一种生长因子信号传导抑制剂）在整个生命过程中指定和维持缓慢分裂的SC群体。3)衰老过程中SC慢分裂亚群功能和肌肉稳态丧失的后果。 将使用转基因H2 B-GFP方法基于标记保留特征（LRC）在体内鉴定缓慢分裂的SC;经历较少分裂的细胞保留更多标记。将对卫星细胞进行LRC和非LRC表征;将基于体内和体外试验检测两个亚群的功能差异。我们已经生成了初步数据，表明Spry 1表达在LRC中富集，Spry 1的遗传破坏导致生长中肌肉的LRC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，以时间诱导的方式在SC中特异性地删除和过表达Spry 1。我们将破坏Spry 1以改变LRC的建立和维持，并研究对SC功能和肌肉表型的影响。 该提案的具体目的是：1）研究发育和出生后肌发生期间卫星细胞的缓慢分裂动力学，2）确定Spry 1是否需要并足以调节LRC SC，以及3）了解在整个生命过程中失去SC LRC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.