A novel colonic crypt base secretory cell that supports colon stem cells

描述（由申请人提供）：K 08奖的申请描述了一个多学科的5年培训计划，以促进迈克尔·罗森伯格博士作为一名独立的学术医生科学家的职业发展。Rothenberg博士最近完成了胃肠病学的临床研究金，现在寻求建立在他的研究经验，以实现他的长期职业目标，成为一名独立的学术研究者，指导肠道干细胞生物学的研究计划。该K 08奖项将为Rothenberg博士提供必要的支持和保护时间，以完成以下培训目标：（1）成为肠道干细胞和生态位生物学方面的专家，（2）掌握细胞培养、流式细胞术和基因表达分析的先进技术，（3）获得用于研究肠道干细胞的小鼠模型操作方面的专业知识，以及（4）在第四年提交R 01申请。为了实现这些目标，Rothenberg博士组建了一支强大的指导团队，由干细胞、肠道生物学、细胞内信号传导和生物信息学方面的专家组成，他们都成功地指导了年轻科学家。他的主要导师Michael Clarke博士是正常干细胞和癌症干细胞领域的领导者。Rothenberg博士的共同导师Harry Greenberg博士是肠道生理学和病毒学专家。咨询委员会由Irv Weissman博士（干细胞和生态位生物学的先驱）、Rob Tibshirani博士（基因表达分析的领导者）和Ken温伯格博士（细胞内信号和cKit的专家）组成。Rothenberg博士的职业发展计划包括与指导团队的定期会议、教学以及出席当地和国际会议并发表演讲。基于他最近发现的支持肠干细胞的cKit+结肠杯状细胞，Rothenberg博士假设这些细胞和相邻肠干细胞之间的双向信号传导协调结肠上皮的稳态。目的I：表征结肠上皮cKit+细胞急性耗竭的影响。这一目标（1-3年）将从体外研究开始，随后转向体外研究。它将利用单克隆cKit阻断抗体、靶向cKit+细胞的细胞毒素和小分子cKit抑制剂。目标二：通过分析cKit+细胞并在体外和体内试验中检测选定的分泌因子，鉴定结肠干细胞生理学的新型调节因子。该目标（第2-4年）将测试在cKit+杯状细胞中高度富集的两种生长因子的体外作用，并将在cKit+细胞中使用纯化的蛋白质、封闭抗体和慢病毒敲减。其他因素将通过基因表达分析进行鉴定、验证，并在体外和体内进行检测。目的3：表征人结肠上皮中的cKit+隐窝基础群体。这个目标（3-5年）将使用新鲜人结肠的单细胞基因表达谱，也将使用体外类器官培养。初步数据支持所有3个目标的可行性。最终，对结肠干细胞的进一步了解 利基可能使消化系统疾病的新的靶向治疗的发展。

DESCRIPTION (provided by applicant): This application for a K08 award describes a multidisciplinary 5-year training program to facilitate Dr. Michael Rothenberg's career development as an independent academic physician-scientist. Dr. Rothenberg recently completed a clinical fellowship in Gastroenterology and now seeks to build on his research experience to attain his long-term career goal of becoming an independent academic investigator, directing a research program in intestinal stem cell biology. This K08 award will provide Dr. Rothenberg with the support and protected time necessary to accomplish the following training objectives: (1) to become an expert in intestinal stem cell and niche biology, (2) to master advanced techniques of cell culture, flow cytometry, and gene expression analysis, (3) to acquire expertise in the manipulation of mouse models for the study of intestinal stem cells, and (4) to submit an R01 application during year 4. To achieve these goals, Dr. Rothenberg has assembled a strong mentoring team of experts in stem cells, intestinal biology, intracellular signaling, and bioinformatics-all of whom have successfully mentored young scientists. His primary mentor, Dr. Michael Clarke, is a leader in the field of normal and cancer stem cells. Dr. Rothenberg's co-mentor, Dr. Harry Greenberg, is an expert in intestinal physiology and virology. The advisory committee consists of Dr. Irv Weissman (a pioneer in stem cell and niche biology), Dr. Rob Tibshirani (a leader in gene expression analysis), and Dr. Ken Weinberg (an expert in intracellular signaling and cKit). Dr. Rothenberg's career development plan includes regular meetings with the mentoring team, didactics, and attendance and presentation at local and international meetings. Building on his recent discovery of a cKit+ colonic goblet cell that supports intestinal stem cells, Dr. Rothenberg hypothesizes that bidirectional signaling between these cells and adjacent intestinal stem cells coordinates colonic epithelial homeostasis. Aim I: To characterize the effects of acute depletion of cKit+ cells from the colonic epithelium. This aim (years 1-3) will start with in-vitro studies, later moving to in-vvo work. It will make use of monoclonal cKit blocking antibodies, cytotoxins targeted to cKit+ cells, and small molecule cKit inhibitors. Aim 2: To identify novel regulators of colon stem cell physiology by profiling cKit+ cells and testing selected secreted factors in both in-vitro and in-vivo assays. This aim (years 2-4) will test in-vitro effects of two growth factors that are highly enriched in cKit+ goblet cells, and will use purified protein, blocking antibodies, and lentiviral knockdown in cKit+ cells. Additional factors will be identified with gene expression analysis, validated, and tested both in-vitro and in-vivo. Aim 3: To characterize a cKit+ crypt base population in human colon epithelium. This aim (years 3-5) will use single cell gene expression profiling on fresh human colon and will also use in-vitro organoid culture. Preliminary data support the feasibility of all 3 aims. Ultimately, an improved understanding of the colon stem cell niche may enable development of novel targeted treatments for digestive diseases.