In vivo and in vitro Characteization of BMI1 + Intestinal Stem Cells

BMI1肠干细胞的体内和体外表征

• 批准号: 8534992
• 负责人: CALVIN J KUO
• 金额: $ 2.36万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-22 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8534992
• 关键词: Ablation; Address; Adenoviruses; BMI1 gene; Cell Lineage; Cells; Colon; Columnar Cell; Diphtheria Toxin; Evaluation; Fibrous capsule of kidney; Gene Expression Profile; Generations; Genes; Goals; Homeostasis; In Vitro; Inflammatory Bowel Diseases; Intestines; Knock-in Mouse; Label; Malignant Neoplasms; Maps; Mediating; Metabolic Diseases; Methodology; Methods; Molecular Profiling; Mus; Natural regeneration; Pathway interactions; Population; Positioning Attribute; Publishing; Radiation; Regulation; Signal Transduction; Small Intestines; Source; Stem cells; System; Therapeutic; Transplantation; Validation; base; in vivo; intestinal epithelium; irradiation; loss of function; novel; prominin; public health relevance; regenerative; response; self-renewal; stem cell niche; stem cell population; tool

DESCRIPTION (provided by applicant): Intestinal homeostasis is maintained by the robust activity of intestinal stem cells (ISC) which allow complete regeneration of the intestinal epithelium every 5-7 days. Recent functional studies have established the existence of two types of ISC, a crypt base columnar (CBC) cell expressing LGR5 and prominin-1, and a distinct ISC population expressing Bmi1, located higher in the crypt at approximately the +4 position and restricted to the small intestine. Despite potent stem cell attributes of the Bmi1+ cells in lineage tracing studies, their regulation, relationship to LGR5+ cells and transcriptome have remained poorly defined. The overall goal of this application is the analysis of the Bmi1+ lineage in vivo and in vitro, using recently developed Bmi1-CreER knock-in mice, our robust methodology for small intestinal culture, and R-Spondin1 and Dkk1 adenoviruses allowing gain- and loss-of-function Wnt manipulation in vivo. Accordingly, Aim 1 will investigate the regulation and functional relevance of the Bmi1+ lineage during intestinal regeneration. The number and fate of Bmi1+ cells will be examined during regeneration in response to radiation or R-spondin1, both in vivo and in vivo using the Bmi1-CreER mouse or cultures derived thereof. Importantly, the functional contribution of the Bmi1+ ISC to intestinal regeneration after radiation or R-spondin treatment will be assessed by diphtheria toxin-mediated lineage ablation. Aim 2 will address the important question of relationships between the Bmi1+ and LGR5+ ISC lineages. Fate mapping of the Bmi1 lineage will be performed in vivo and in vitro to formally demonstrate if Bmi1+ cells or their progeny can express LGR5. Culture of isolated Bmi1+ cells from Bmi1-CreER mice will be performed both within and without an ISC niche to explore if Bmi1+ cells can give rise to LGR5+ cells in vitro. In Aim 3, transcriptional profiling of Bmi1+ cells will be performed and compared to the published LGR5+ transcriptome and target validation performed exploiting in vitro intestinal culture. Finally, Aim 4 will explore the ex vivo expansion and transplantation of Bmi1+ ISC. Our ISC niche-dependent intestinal culture system, as well as niche-free systems will be used to expand Bmi1+ cells ex vivo, followed by single cell or population transplantation in vivo. Questions of plasticity will also be addressed with introduction of small intestine Bmi1+ cells into the colon both in vitro and in vivo. PUBLIC HEALTH RELEVANCE: The intestine possesses highly active stem cell populations with therapeutic relevance to diverse conditions including inflammatory bowel diseases, metabolic disorders and cancer. Here, the Bmi1+ intestinal stem cell population will be investigated with regards to regenerative potential both in vivo and in vitro.

描述（由申请人提供）：肠道内稳态是由肠道干细胞（ISC）的强大活性维持的，其允许肠道上皮每 5-7 天完全再生。最近的功能研究已经确定存在两种类型的 ISC，一种是表达 LGR5 和 prominin-1 的隐窝基底柱状 (CBC) 细胞，另一种是表达 Bmi1 的独特 ISC 群体，其位于隐窝中较高的位置，大约 +4 位置并仅限于小肠。尽管在谱系追踪研究中 Bmi1+ 细胞具有有效的干细胞属性，但它们的调控、与 LGR5+ 细胞和转录组的关系仍然不明确。该应用的总体目标是使用最近开发的 Bmi1-CreER 敲入小鼠、我们强大的小肠培养方法以及允许体内获得和丧失功能的 Wnt 操作的 R-Spondin1 和 Dkk1 腺病毒，在体内和体外分析 Bmi1+ 谱系。因此，目标 1 将研究 Bmi1+ 谱系在肠道再生过程中的调节和功能相关性。将在体内和体内使用 Bmi1-CreER 小鼠或其衍生培养物在响应辐射或 R-spondin1 的再生过程中检查 Bmi1+ 细胞的数量和命运。重要的是，Bmi1+ ISC 对放疗或 R-spondin 治疗后肠道再生的功能贡献将通过白喉毒素介导的谱系消融进行评估。目标 2 将解决 Bmi1+ 和 LGR5+ ISC 谱系之间关系的重要问题。 Bmi1谱系的命运图谱将在体内和体外进行，以正式证明Bmi1+细胞或其后代是否可以表达LGR5。来自 Bmi1-CreER 小鼠的分离 Bmi1+ 细胞将在 ISC 生态位内和外进行培养，以探索 Bmi1+ 细胞是否可以在体外产生 LGR5+ 细胞。在目标 3 中，将对 Bmi1+ 细胞进行转录分析，并将其与已发表的 LGR5+ 转录组进行比较，并利用体外肠道培养进行靶点验证。最后，Aim 4将探索Bmi1+ ISC的离体扩增和移植。我们的 ISC 生态位依赖性肠道培养系统以及无生态位系统将用于离体扩增 Bmi1+ 细胞，然后进行体内单细胞或群体移植。通过在体外和体内将小肠 Bmi1+ 细胞引入结肠，可塑性问题也将得到解决。 公共健康相关性：肠道拥有高度活跃的干细胞群，对炎症性肠病、代谢紊乱和癌症等多种疾病具有治疗相关性。在这里，我们将研究 Bmi1+ 肠道干细胞群的体内和体外再生潜力。