Sox9 Expression-Thresholds Govern Reserve and Active Intestinal Stem Cell Functions

Sox9 表达阈值控制储备和活跃肠干细胞功能

• 批准号: 9395334
• 负责人: Eric Daniel Bankaitis
• 金额: $ 5.86万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-07-01 至 2019-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9395334
• 关键词: Ablation; Adopted; Biological Assay; Cancer Patient; Cell Culture System; Cell Culture Techniques; Cell Cycle; Cell Death; Cell Differentiation process; Cell Proliferation; Cell physiology; Cells; Cessation of life; Chronic; Data; Disease; Down-Regulation; Doxycycline; Epithelium; Equilibrium; Exhibits; Fluorescence-Activated Cell Sorting; Future; Gastrointestinal tract structure; Generations; Genetic; Genetic Models; Goals; Homeostasis; Image; Image Analysis; In Vitro; Inflammatory Bowel Diseases; Injury; Intestines; Label; Lead; Life Cycle Stages; Maintenance; Malignant Neoplasms; Mediating; Methods; Mitotic; Modeling; Mucositis; Mus; Natural regeneration; Pathway interactions; Phenotype; Physiology; Plasticizers; Population; Process; Proliferating; Property; Radiation; Radiation Tolerance; Radiation exposure; Radiation induced damage; Radioresistance; Regenerative response; Reserve Stem Cell; Resistance; Role; Signal Transduction; Stem cells; Supporting Cell; System; Technology; Testing; Therapeutic; Time; Tissues; Training; Transgenic Mice; Transgenic Organisms; career; cell behavior; chemotherapy; crypt cell; experimental study; high throughput screening; improved; in vivo; innovation; interest; intestinal epithelium; irradiation; knock-down; overexpression; premature; programs; radioresistant; radiosensitive; regenerative; repaired; response; response to injury; self-renewal; small hairpin RNA; stem cell division; stem-like cell; stemness; tissue repair; transcription factor; transcriptome sequencing; transcriptomics

ABSTRACT Self-renewing and multipotent intestinal stem cells (ISCs) support homeostatic and injury-induced regeneration of the intestinal epithelium. The mechanisms that regulate the functions of ISCs under such conditions remain poorly understood and represent a topic of great interest. Recently, increasing evidence supports a model whereby ISCs exist in one of two functional states. These include an actively cycling ‘aISC’ state which drives normal renewal of the differentiated and post- mitotic intestinal lineages during homeostasis, and a quiescent reserve ‘rISC’ state which can be mobilized under injury conditions to re-enter the cell cycle and repair damaged epithelium. The process through which ISCs interconvert between rISC and aISC states is called ‘state-switching’, is proposed to be reversible, and is likely fundamental to how intestinal stem cells balance self-renewal with differentiation and mount adaptive responses to tissue injury. Our goal is to understanding the regulatory mechanisms that regulate ‘state-switching’ in ISC. Preliminary data from the Magness lab and others indicates that genetic loss of Sox9 in intestinal epithelium causes ISC to adopt a highly proliferative aISC-like state that is incapable of regeneration after damaging insults such as radiation. Further, our preliminary data indicate that the rISC state is associated with high levels of Sox9 expression, whereas aISC express low levels of Sox9. The central hypothesis of this proposal is that Sox9 is an intrinsic master regulator of ISC state-switching, and directs aISC-rISC interconversions through high and low Sox9-expression thresholds. To test this hypothesis, I will use in vivo genetic models and in vitro high throughput single-cell culture methods to assess the consequences of experimentally-controlled Sox9 levels on ISC proliferation, intestinal lineage allocation, and resistance to radiation, the latter of which is a defining property of ‘reserve’ stemness. The studies proposed here will test if and how Sox9 expression-thresholds diversify stem cell functions in ISCs.

摘要 自我更新和多潜能的肠道干细胞支持内环境平衡和损伤诱导的再生 肠上皮细胞。在这种情况下调节ISCs功能的机制仍然知之甚少。 并代表了一个令人非常感兴趣的话题。最近，越来越多的证据支持一种模型，即ISCs存在于两个细胞中的一个 功能状态。其中包括积极循环的“AISC”状态，这种状态推动分化和后分化的正常更新。 动态平衡中的有丝分裂肠道血统，以及在受伤情况下可以被动员的静止储备的RISC状态 重新进入细胞周期并修复受损上皮的条件。ISCs之间相互转换的过程 RISC和AISC状态被称为状态切换，被认为是可逆的，并且可能是肠道 干细胞在自我更新和分化之间取得平衡，并对组织损伤做出适应性反应。我们的目标是 了解在ISC中调节“状态切换”的调节机制。来自Magness实验室的初步数据 其他研究表明，肠道上皮细胞中Sox9基因的缺失导致ISC采用高度增殖的AISC样细胞 在遭受诸如辐射等破坏性侮辱后不能再生的状态。此外，我们的初步数据表明， RISC状态与高水平的Sox9表达有关，而AISC表达低水平的Sox9。中环 假设Sox9是ISC状态切换的内在主调节器，并指导AISC-RISC 通过高和低Sox9表达阈值进行相互转换。为了验证这一假设，我将使用体内基因 模型和体外高通量单细胞培养方法评估实验对照的后果 SOX9水平对ISC增殖、肠道谱系分配和辐射抵抗的影响，后者是一个决定性的 “保留”词干的属性。这里提出的研究将测试Sox9表达阈值是否以及如何使干细胞多样化 细胞在ISCs中的功能。