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

描述(由申请人提供)：这份K08奖项的申请书描述了一个多学科的5年培训计划，以促进Michael Rothenberg博士作为一名独立的学术内科科学家的职业发展。Rothenberg博士最近完成了胃肠病学的临床研究，现在他正在寻求以自己的研究经验为基础，实现自己的长期职业目标，即成为一名独立的学术研究员，领导一个肠道干细胞生物学研究项目。这一K08奖项将为Rothenberg博士提供必要的支持和保护时间，以实现以下培训目标：(1)成为肠道干细胞和利基生物学的专家，(2)掌握细胞培养、流式细胞仪和基因表达分析的先进技术，(3)获得用于肠道干细胞研究的小鼠模型操作方面的专业知识，以及(4)在第四年提交R01申请。为了实现这些目标，Rothenberg博士组建了一支强大的干细胞、肠道生物学、细胞内信号和生物信息学专家指导团队-所有这些专家都成功地指导了年轻科学家。他的主要导师迈克尔·克拉克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方面的专家。咨询委员会由Irv Weissman博士(干细胞和利基生物学的先驱)、Rob Tibishani博士(基因表达分析的领导者)和Ken Weinberg博士(细胞内信号和cKit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、授课以及出席和介绍当地和国际会议。Rothenberg博士根据他最近发现的支持肠道干细胞的cKit+结肠杯状细胞的发现，假设这些细胞和邻近的肠道干细胞之间的双向信号协调了结肠上皮的动态平衡。目的：研究急性耗竭结肠上皮cKit+细胞的效应。这一目标(1-3年)将从体外研究开始，然后转移到体内工作。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。目的：通过体外和体内检测cKit+细胞和选定的分泌因子，寻找新的结肠干细胞生理调节因子。这个目标(2-4年)将测试两种生长因子的体外效果，这两种生长因子在cKit+杯状细胞中高度浓缩，并将在cKit+细胞中使用纯化蛋白、阻断抗体和慢病毒敲除。其他因素将通过基因表达分析来确定，并在体外和体内进行验证和测试。目的3：鉴定人结肠上皮中cKit+隐窝碱基群的特征。这个目标(3-5年)将在新鲜的人类结肠上使用单细胞基因表达谱，也将使用体外有机物培养。初步数据支持所有3个目标的可行性。最终，对结肠干细胞的了解有所提高 利基可能会使消化疾病的新靶向治疗方法的开发成为可能。

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.