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

描述(申请人提供)：肠道内环境稳定是由肠道干细胞(ISC)的强大活性维持的，它允许每5-7天完全再生肠道上皮。最近的功能研究已经证实存在两种类型的ISC，一种是表达LGR5和重要蛋白-1的隐窝基柱状细胞(CBC)，另一种是表达Bmi1的ISC群体，位于隐窝较高的大约+4位置，仅限于小肠。尽管在谱系追踪研究中Bmi1+细胞具有强大的干细胞属性，但它们的调控、与LGR5+细胞和转录组的关系仍未明确。这项应用的总体目标是在体内和体外分析Bmi1+谱系，使用最近开发的Bmi1-Creer敲入小鼠，我们强大的小肠培养方法，以及允许在体内获得和失去功能的Wnt操作的R-pondin1和Dkk1腺病毒。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。Bmi1+细胞的数量和命运将在体内和活体使用Bmi1-Creer小鼠或其衍生的培养物对辐射或R-pondin1做出反应的再生过程中进行检测。重要的是，Bmi1+ISC在辐射或R-Respondin治疗后对肠道再生的功能贡献将通过白喉毒素介导的谱系消融来评估。目标2将解决Bmi1+和LGR5+ISC谱系之间的关系这一重要问题。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。从Bmi1-Creer小鼠分离的Bmi1+细胞将在ISC生态位内和不在ISC生态位内进行培养，以探索Bmi1+细胞是否能在体外产生LGR5+细胞。在目标3中，将进行Bmi1+细胞的转录图谱分析，并与已发表的LGR5+转录组进行比较，并利用体外肠道培养进行靶标验证。最后，目标4将探索Bmi1+ISC的体外扩增和移植。我们的ISC依赖生态位的肠道培养系统以及无生态位系统将被用于体外扩增Bmi1+细胞，随后将在体内进行单细胞或群体移植。在体外和体内，通过将小肠Bmi1+细胞引入结肠，可塑性问题也将得到解决。 公共卫生相关性：肠道拥有高度活跃的干细胞群，对包括炎症性肠道疾病、代谢紊乱和癌症在内的各种疾病具有治疗相关性。在这里，我们将研究Bmi1+肠道干细胞群体在体内和体外的再生潜力。

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.