Cell-cycle commitment in muscle regeneration

项目摘要/摘要 哺乳动物组织在受伤后表现出显著的再生活性，这一过程中的缺陷包括 与衰老和退行性疾病有关。骨骼肌再生需要持续的增殖性 被称为卫星细胞(SCs)的肌肉干细胞的反应，目前还不清楚这是如何实现的。 细胞增殖是由细胞外生长因子或有丝分裂原控制的。细胞化的过程 增殖，称为细胞周期，涉及细胞退出非增殖G0状态并启动细胞 在G1中循环。在G1期的某个时间点，细胞有效地逃避了对有丝分裂原的要求，并致力于有丝分裂原- 独立完成细胞周期，但随后的每一轮增殖都需要有丝分裂原信号 在G1期间或正好在G1之前。对细胞周期承诺机制的见解主要来自于体外 成纤维细胞和永生化上皮细胞的研究，但尚不清楚这些系统与 干细胞激活的过程，其中干细胞表现出延长的第一个G0/G1期，随后是多轮 扩散。我们的初步工作表明，一种双稳机制，其中一个过程保持 尽管有丝分裂原被移除，但无限期地参与，是细胞周期承诺在G0/G1期的基础 SCS.这一提议的中心假设是一个双稳态的G0/G1机制是多轮谈判的基础 不依赖丝裂原的干细胞增殖和有效的肌肉再生。建议的工作内容包括 以下是具体目标： 1.通过延长多个G0/G1期来确定SC细胞周期承诺的机制。 2.建立肌肉再生过程中的丝裂原信号动力学。 3.确定Sc G0/G1细胞周期承诺在肌肉再生过程中的作用。 G0/G1期细胞周期承诺的机制将通过基于FACS的SC分离和体外实验来确定 分析，包括延时成像、免疫荧光以及自动细胞跟踪和图像分析。在……里面 将使用肌肉损伤模型和采集肌肉来研究体内有丝分裂原信号动力学 生长因子可获得性和SC有丝分裂原信号的生化和转录分析。功能界别 SC G0/G1细胞周期承诺机制在肌肉再生中的作用将使用SC特异性进行测试 条件基因敲除小鼠和异位导入有丝分裂原的挽救。 斯坦福大学是干细胞和再生科学的领先者，将为我提供技术和 合作网络发展成为独立研究的科学家。这项研究将揭示 肌肉再生过程中有丝分裂原与干细胞增殖的基本关系 引导治疗衰老和退行性疾病中的再生缺陷的治疗策略。

PROJECT SUMMARY/ABSTRACT Mammalian tissues exhibit remarkable regenerative activity following injury, and defects in this process are associated with aging and degenerative diseases. Skeletal muscle regeneration requires a sustained proliferative response from muscle stem cells called satellite cells (SCs), yet it is unknown how this is achieved. Cellular proliferation is controlled by extracellular growth factors, or mitogens. The process of cellular proliferation, referred to as the cell cycle, involves cells exiting a non-proliferative G0 state and initiating the cell cycle in G1. At some point in G1, cells effectively escape the requirement for mitogens and commit to mitogen- independent completion of the cell cycle, but subsequent rounds of proliferation each require mitogen signaling during or just before G1. Insights into cell-cycle commitment mechanisms are primarily derived from in vitro studies of fibroblasts and immortalized epithelial cells, but it is unclear how relevant such systems are to the process of SC activation, where SCs exhibit a prolonged first G0/G1 phase, followed by multiple rounds of proliferation. Our preliminary work indicates that a mechanism of bistability, where a process remains indefinitely engaged despite the removal of mitogens, underlies cell-cycle commitment in the G0/G1 phase of SCs. The central hypothesis of this proposal is that a bistable G0/G1 mechanism underlies multiple rounds of mitogen-independent SC proliferation and effective muscle regeneration. The proposed work addresses the following specific aims: 1. Identify the mechanism of SC cell-cycle commitment through prolonged and multiple G0/G1 phases. 2. Establish mitogen signaling dynamics during muscle regeneration. 3. Determine the role of SC G0/G1 cell-cycle commitment during muscle regeneration. The mechanism of G0/G1 cell-cycle commitment will be identified using FACS-based SC isolation and ex vivo assays, including time-lapse imaging, immunofluorescence, and automated cell tracking and image analysis. In vivo mitogen signaling dynamics will be investigated using a muscle injury model and harvesting muscles for biochemical and transcriptomic analysis of growth factor availability and SC mitogen signaling. The functional role of SC G0/G1 cell-cycle commitment mechanisms in muscle regeneration will be tested using SC-specific conditional knockout mice and rescue by ectopic introduction of mitogens. Stanford is a leader in stem cell and regenerative sciences and will provide me with the technologies and collaborative network to develop into an independent research scientist. This research will uncover the fundamental relationship between mitogens and stem cell proliferation during muscle regeneration and may lead to therapeutic strategies for treating regenerative defects in aging and degenerative diseases.