Dissecting and engineering reversible cell cycle states

解剖和工程可逆细胞周期状态

• 批准号: 9567321
• 负责人: Kristin A Knouse
• 金额: $ 48.75万
• 依托单位: WHITEHEAD INSTITUTE FOR BIOMEDICAL RES
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-07 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9567321
• 关键词: Address; Back; Basic Science; Biological Models; Biology; CRISPR screen; CRISPR/Cas technology; Cell Culture System; Cell Culture Techniques; Cell Cycle; Cell Cycle Regulation; Cell Differentiation process; Cells; Classification; Clinical Medicine; Collaborations; Communities; Core Facility; Cyclin D1; Disease; Engineering; Environment; Equipment; Faculty; Familiarity; Fostering; Foundations; Funding; Genes; Goals; Health; Hepatocyte; Human; Institutes; Knowledge; Lead; Left; Liver; Lymphocyte; Mammalian Cell; Massachusetts; Mentors; Modeling; Molecular; Molecular Profiling; Morbidity - disease rate; Mus; Myocardial Infarction; Nerve Degeneration; Organ; Organism; Pathway interactions; Physiological; Principal Investigator; Process; Proliferating; Regenerative Medicine; Research; Research Personnel; Resources; Role; Signal Transduction; Stroke; System; Technical Expertise; Technology; Tissues; Training; Translating; Translations; Vocational Guidance; age effect; body system; cell type; effective therapy; experience; experimental study; extracellular; faculty mentor; functional genomics; genome-wide; genomic platform; hearing impairment; human disease; improved; loss of function; meetings; novel; skills; stem cell population

7. PROJECT SUMMARY/ABSTRACT Numerous diseases across organ systems as well as the deleterious effects of aging are attributable to the loss of terminally differentiated cells that facilitate organ function. In these cases, there is no active stem cell population capable of generating new functional cells. Moreover, any functional cells that remain cannot replace lost cells as the former have permanently exited the cell cycle as part of their differentiation process. A means of making terminally differentiated cells re-enter the cell cycle would dramatically alleviate and potentially cure these disease states. In principle, cellular differentiation does not necessitate cell cycle exit. Indeed some differentiated cell types, such as hepatocytes and lymphocytes, retain the ability to re-enter the cell cycle. These cell types are considered to be quiescent rather than terminally differentiated. Some of the extracellular signals that drive quiescent cells back into the cell cycle as well as the pathway for cell cycle entry, the Cyclin D-Cdk4,6 pathway, have been defined. However, it is unclear what allows a quiescent cell to receive these signals and reactivate the Cyclin D-Cdk4,6 pathway while a terminally differentiated cell cannot do so. As such, the difference between terminally differentiated cells and quiescent cells is a functional classification lacking a molecular explanation. Although mammalian cell culture models have provided tractable systems for investigating quiescence, they have failed to recapitulate the complexity of cell cycle regulation in tissues. There is therefore a need to study quiescence in an organismal context in order to provide a comprehensive understanding of this state and facilitate its translation to regenerative medicine. This project will establish the mouse liver as a tractable physiologic system to understand the reversibility of the quiescent state ultimately in order to confer this ability to terminally differentiated cells and enable their proliferation in the setting of disease. The approach will consider two non-mutually exclusive means of poising the Cyclin D- Cdk4,6 pathway for reactivation: direct modulation of the pathway itself or involvement of a factor extrinsic to this cascade. Aim 1 will determine whether there are functionally relevant differences in the activity of Cyclin D- Cdk4,6 pathway components in quiescent hepatocytes compared to other cell cycle states in tissues. Aim 2 will establish the first genome-wide, loss-of-function screen in the liver to identify any genes outside of this pathway that are required for the reversibility of quiescence. Finally, aim 3 will define conserved molecular features of quiescence by determining which features identified in aims 1 and 2 are also required for the reversibility of this state in lymphocytes. Together, these experiments will elucidate the core molecular signature of quiescence and thus the distinction between quiescence and terminal differentiation in tissues. Core factors would then be introduced into terminally differentiated cells and evaluated for their ability to confer proliferative capacity. These findings will not only fill a critical gap in our understanding of the mammalian cell cycle but also unlock a novel means of treating myriad diseases currently lacking effective therapy.

7.项目摘要/摘要 许多跨器官系统的疾病以及衰老的有害影响都可归因于 丧失促进器官功能的终末分化细胞。在这些情况下，没有活跃的干细胞 能够产生新的功能细胞的群体。此外，任何剩余的功能细胞都不能 替换丢失的细胞，因为前者已永久退出细胞周期，作为其分化过程的一部分。一个 使终末分化细胞重新进入细胞周期的方法将显著缓解和 有可能治愈这些疾病状态。原则上，细胞分化并不一定要退出细胞周期。 事实上，一些分化的细胞类型，如肝细胞和淋巴细胞，保留了重新进入 细胞周期。这些细胞类型被认为是静止的，而不是终末分化的。其中一些 驱动静止细胞重新进入细胞周期的细胞外信号以及细胞周期的途径 进入，Cyclin D-CDK4，6途径已经被定义。然而，目前还不清楚是什么允许静止的细胞 接收这些信号并重新激活Cyclin D-CDK4，6途径，而终末分化的细胞不能 就这么做吧。因此，终末分化细胞和静止细胞之间的差异是一种功能 缺乏分子解释的分类。尽管哺乳动物细胞培养模型提供了易于处理的 研究静止的系统，他们未能概括细胞周期调控的复杂性 纸巾。因此，有必要在生物环境中研究静止，以便提供一种 全面了解这一状态，并促进将其转化为再生医学。这个项目 将建立小鼠肝脏作为一个易于处理的生理系统，以了解静止的可逆性 最终状态，以便将这种能力授予终末分化的细胞，并使它们在 疾病的背景。该方法将考虑两种不相互排斥的平衡Cyclin D的方法： 再激活的CDK4，6通路：直接调节通路本身或参与外部因子 这个瀑布。目标1将确定Cyclin D活性是否存在功能上的相关差异- 静止期肝细胞中CDK4，6途径组分与组织中其他细胞周期状态的比较。目标2将 在肝脏中建立第一个全基因组、功能丧失的筛查，以确定该基因以外的任何基因 静止期可逆性所需的通路。最后，目标3将定义守恒的分子 通过确定AIMS 1和AIMS 2中确定的哪些特征也是 淋巴细胞中这种状态的可逆性。总之，这些实验将阐明核心分子 静止的信号，从而区分组织中的静止和末端分化。 然后将核心因子引入终末分化的细胞，并评估它们授予 增殖能力。这些发现不仅将填补我们对哺乳动物细胞理解的一个关键空白 但也开启了一种治疗目前缺乏有效治疗方法的无数疾病的新方法。