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

描述（由申请人提供）：肠内稳态由肠干细胞（ISC）的强大活性维持，其允许肠上皮每5-7天完全再生。最近的功能研究已经确定了两种类型的ISC的存在，一种是表达LGR 5和Bmi-1的隐窝基底柱状（CBC）细胞，另一种是表达Bmi 1的独特ISC群体，其位于隐窝中约+4位置的较高位置，并局限于小肠。尽管在谱系追踪研究中Bmi 1+细胞具有有效的干细胞属性，但它们的调节、与LGR 5+细胞的关系和转录组仍然定义不清。本申请的总体目标是使用最近开发的Bmi 1-CreER基因敲入小鼠、我们用于小肠培养的稳健方法以及允许在体内获得和丧失功能Wnt操纵的R-Spondin 1和Dkk 1腺病毒，在体内和体外分析Bmi 1+谱系。因此，目标1将研究在肠再生过程中Bmi 1+谱系的调节和功能相关性。在体内和使用Bmi 1-CreER小鼠或其衍生的培养物在响应辐射或R-spondin 1的再生过程中检查Bmi 1+细胞的数量和命运。重要的是，将通过白喉毒素介导的谱系消融来评估Bmi 1 + ISC对放射或R-spondin治疗后肠再生的功能贡献。目的2将解决Bmi 1+和LGR 5 + ISC谱系之间关系的重要问题。将在体内和体外进行Bmi 1谱系的命运作图，以正式证明Bmi 1+细胞或其后代是否可以表达LGR 5。将在ISC小生境内和不在ISC小生境下培养从Bmi 1-CreER小鼠分离的Bmi 1+细胞，以探索Bmi 1+细胞是否可以在体外产生LGR 5+细胞。在目标3中，将进行Bmi 1+细胞的转录谱分析，并与已发表的LGR 5+转录组进行比较，并利用体外肠培养进行靶标验证。最后，目的4将探索Bmi 1 + ISC的体外扩增和移植。我们的ISC小生境依赖性肠道培养系统以及无小生境系统将用于体外扩增Bmi 1+细胞，然后进行体内单细胞或群体移植。可塑性的问题也将通过将小肠Bmi 1+细胞在体外和体内引入结肠来解决。

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.