Enteroendocrine cell reprogramming during intestinal injury

研究策略：本提案的主要研究目标是解决 肠内分泌细胞响应损伤信号而改变其活性（分泌）或分化状态。一 更好地了解肠道损伤修复对VA健康优先事项至关重要;炎症的患病率 自1998年以来，VA患者的肠道疾病（IBD）增加了一倍多，至少有一半的患者不会 使用现有疗法体验完全粘膜愈合。肠内分泌细胞产生的激素 (EEC)在IBD患者中改变，可以促进上皮愈合，但对 受伤时EEC分泌是如何调节的对EEC的研究一直很有限，因为它们很罕见， 异源的，并且不适合用现有方法进行长期原代培养。为了弥补这一差距，我们 开发了一种新的实验系统，从原代结肠样本中长期培养EECs 支持所有已知EEC亚型的单层。我们发现EEC的亚型是动态的， 通过体外简单的实验性损伤改变。改变的内皮细胞增加了某些因子的表达， 促进肠道愈合。这项建议的目标是确定细胞和转录基础， EEC亚型如何通过改变活性或分化对肠损伤作出反应。我们的目标是（1） 检验以下假设：内皮细胞的亚型通过损伤诱导的内质网适应性重编程， （2）检验微环境损伤信号重编程EECs以 增加激素分泌，和（3）定义EEC重编程过程中的转录机制 单细胞分辨率下的损伤。这项工作的预期成果是建立新的范式， 内皮细胞在损伤过程中经历适应性重编程，这将为将来独立的 批准提案。此外，我们期望在细胞中发现新的分泌因子或改变的分化状态。 这将为患有肠道或代谢紊乱的退伍军人带来新的潜在治疗方法。 候选人/环境：Brian Muegge博士是内分泌科的高级研究员， 圣路易斯的华盛顿大学和圣路易斯退伍军人医疗中心。他完成了医学博士学位，博士培训 在华盛顿大学，他在杰弗里·戈登博士的实验室进行研究生学习。Muegge博士 在加州大学旧金山分校完成了内科住院医师实习他正在他的实验室里进行他的奖学金研究。 在华盛顿的主要导师Thaddeus Stappenbeck博士和共同导师卡洛斯Bernal-Mizrachi博士 大学Muegge博士在肠道干细胞培养和计算分析方面积累了丰富的经验， 转录数据。他开发了一种新颖的文化系统，使他能够模拟EEC的发展， 使用原代细胞的损伤。他现在寻求扩大他在新领域的专业知识，包括血统追踪，细胞， 细胞信号传导和单细胞RNA测序，然后开始他的独立研究生涯。 职业发展：该奖项将确保Muegge博士能够建立职业生涯， VA医疗保健系统的独立医生科学家。他制定了一个正式的5年培训计划 这将扩大他在肠道发育、体外模型和单细胞转录组学方面的科学技能， 以及在科学出版，资助写作和实验室管理方面的学术技能。Muegge医生的 培训将通过实验研究、教学法、研讨会和全国会议来完成。他 已经组建了一个科学咨询委员会，拥有必要的经验和专门知识， 支持他实现职业目标。在CDA-2完成后，Muegge博士将 具备尖端技术（单细胞转录组学、体外干细胞培养系统）的技能 在一个独特的领域工作，使他有别于其他人在该领域（肠内分泌功能，在肠道 伤害）。他将是唯一准备解决的基本问题，需要知识的肠道 生物学、内分泌学和计算生物学。

RESEARCH STRATEGY: The primary research objective of this proposal is to address how subtypes of enteroendocrine cells change their activity (secretion) or state of differentiation in response to injury signals. A better understanding of intestinal injury repair is critical to VA health priorities; the prevalence of inflammatory bowel disease (IBD) has more than doubled among VA patients since 1998 and at least half of patients won’t experience full mucosal healing with existing therapies. Hormones produced in intestinal enteroendocrine cells (EEC) are altered in patients with IBD and can promote epithelial healing, but there is little understanding of how EEC secretion is regulated during injury. The study of EECs has been limited because they are rare, heterogenous, and not amenable to long-term primary culture with existing methods. To address this gap, we have developed a new experimental system to culture EECs from primary colon samples in long-term monolayers that supports all known EEC subtypes. We have discovered that subtypes of EEC are dynamically altered with a simple experimental injury in vitro. The altered EECs increase the expression of factors that promote intestinal healing. The goal of this proposal is to determine the cellular and transcriptional basis of how subtypes of EEC respond to intestinal injury through altered activity or differentiation. Our Aims are to (1) test the hypothesis that subtypes of EECs are adaptively reprogrammed by injury induced endoplasmic reticulum (ER) stress, (2) test the hypothesis that microenvironmental injury signals reprogram EECs to increase hormone secretion, and (3) define the transcriptional mechanisms of EEC reprogramming during injury at single-cell resolution. The expected outcome of this work is to establish the new paradigm that EECs undergo adaptive reprogramming during injury which will serve as the foundation for future independent grant proposals. Additionally, we expect to discover novel secreted factors or altered states of differentiation in EEC that would lead to new potential treatments for veterans suffering from intestinal or metabolic disorders. CANDIDATE/ENVIRONMENT: Dr. Brian Muegge is a senior fellow in the Division of Endocrinology at Washington University in St. Louis and the St. Louis VA Medical Center. He completed his M.D., Ph.D. training at Washington University where he performed graduate studies in Dr. Jeffrey Gordon’s laboratory. Dr. Muegge completed his residency in internal medicine at UCSF. He is conducting his fellowship research in the lab of his primary mentor Dr. Thaddeus Stappenbeck and co-mentor Dr. Carlos Bernal-Mizrachi at Washington University. Dr. Muegge has gained experience in intestinal stem cell culture and computational analysis of transcriptional data. He has developed a novel culture system that allows him to model EEC development and injury using primary cells. He now seeks to expand his expertise in new areas including lineage tracing, cell- cell signaling, and single cell RNA sequencing prior to starting his independent research career. CAREER DEVELOPMENT: This award will ensure that Dr. Muegge is able to establish a career as an independent physician scientist in the VA health care system. He has developed a formal 5-year training plan that will expand his scientific skills in intestinal development, in vitro models, and single cell transcriptomics, as well as academic skills in scientific publishing, grant writing, and laboratory management. Dr. Muegge’s training will be accomplished with experimental research, didactics, seminars, and national conferences. He has assembled a scientific advisory committee with the requisite experience and expertise to monitor and support his progress towards achieving his career goals. At the completion of the CDA-2, Dr. Muegge will be equipped with skills in cutting edge technology (single cell transcriptomics, in vitro stem cell culture systems) working in a unique area that sets him apart from others in the field (intestinal endocrine function during bowel injury). He will be uniquely prepared to address fundamental questions that require knowledge of intestinal biology, endocrinology, and computational biology.