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; 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）：肠内稳态通过肠道干细胞（ISC）的稳健活性来维持，该活动允许每5-7天完全再生肠上皮。最近的功能研究已经确定了两种类型的ISC，一个表达LGR5和Prominin-1的隐窝基柱（CBC）细胞，以及一个表达BMI1的独特的ISC群体，位于隐窝中较高的+4位置的地下室中较高的位置，并限制在小纯料上。尽管BMI1+细胞在谱系追踪研究中的有效干细胞属性，但其调节，与LGR5+细胞和转录组的关系仍然很差。该应用的总体目标是分析体内和体外BMI1+谱系的分析，使用最近开发的BMI1-Creer敲入小鼠，我们对小肠培养的强大方法，以及R-Spondin1和DKK1腺病毒，允许在Vivo In Vivo In Vivo中获得的增益和功能障碍WNT。因此，AIM 1将研究肠再生过程中BMI1+谱系的调节和功能相关性。使用BMI1-creer小鼠或培养物培养物，将检查BMI1+细胞的数量和命运，以响应辐射或R-Spondin1的响应，以响应辐射或R-Spondin1。重要的是，将通过白喉毒素介导的谱系消融评估BMI1+ ISC对辐射或R-Spondin治疗后肠再生的功能贡献。 AIM 2将解决BMI1+和LGR5+ ISC谱系之间关系的重要问题。 BMI1谱系的命运映射将在体内和体外进行正式证明BMI1+细胞或其后代是否可以表达LGR5。来自BMI1-筛查小鼠的分离的BMI1+细胞的培养将在内部和不具有ISC生态裂市场内进行，以探索BMI1+细胞是否会在体外引起LGR5+细胞。在AIM 3中，将进行BMI1+细胞的转录分析，并将其与已发表的LGR5+转录组和目标验证进行比较，从而利用体外肠道培养。最后，AIM 4将探索BMI1+ ISC的离体扩展和移植。我们的ISC利基依赖性肠道培养系统以及无小裂系统将用于在体内扩展BMI1+细胞，然后在体内进行单细胞或种群移植。可塑性的问题也将通过在体外和体内引入小肠BMI1+细胞的引入结肠。 公共卫生相关性：肠道具有高度活跃的干细胞群体，与包括炎症性肠病，代谢性疾病和癌症在内的各种疾病具有治疗性相关。在这里，将研究BMI1+肠干细胞种群，以在体内和体外进行再生潜力。