Enteroendocrine cell reprogramming during intestinal injury

研究策略：本提案的主要研究目标是解决亚型 肠内分泌细胞对损伤信号的反应改变其活动(分泌)或分化状态。一个 更好地了解肠道损伤修复对于退伍军人管理局的健康优先事项至关重要；炎症性疾病的流行 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 用现有的治疗方法体验完全的粘膜愈合。肠道内分泌细胞产生的激素 (EEC)在IBD患者中被改变，并能促进上皮愈合，但对其了解甚少 EEC分泌在损伤过程中是如何被调节的。对EECS的研究一直很有限，因为它们很罕见， 异质性的，不适应现有方法的长期原代培养。为了解决这一差距，我们 我开发了一种新的实验系统，可以长期从原始结肠样本中培养EECs 支持所有已知EEC子类型的单层。我们发现，EEC的亚型是动态的 在体外用简单的实验性损伤进行了改变。改变的EECS增加了因子的表达， 促进肠道愈合。这项提议的目标是确定细胞和转录基础 肠上皮细胞亚型如何通过改变活动或分化对肠道损伤作出反应。我们的目标是(1) 检验损伤诱导的内质网对EECs亚型进行适应性重编程的假设 网状细胞(ER)应激，(2)检验微环境损伤信号重新编程EECs以 增加激素分泌，以及(3)确定EEC重新编程的转录机制 单细胞分辨率损伤。这项工作的预期结果是建立新的范式， EECs在受伤期间进行适应性重新编程，这将为未来的独立奠定基础 批准提案。此外，我们希望发现新的分泌因子或分化状态的改变 这将为患有肠道或代谢障碍的退伍军人带来新的潜在治疗方法。 候选人/环境：Brian Muegge博士是哈佛大学内分泌科的高级研究员 位于圣路易斯的华盛顿大学和圣路易斯退伍军人医学中心。他完成了医学博士和博士学位的培训 在华盛顿大学，他在杰弗里·戈登博士的实验室进行研究生研究。米格博士 完成了他在加州大学旧金山分校的内科实习。他正在他的实验室里进行他的研究员研究 华盛顿的主要导师塔迪厄斯·斯塔彭贝克博士和共同导师卡洛斯·贝尔纳尔-米兹拉奇博士 大学。Muegge博士在肠道干细胞培养和计算分析方面获得了经验 转录数据。他开发了一种新的文化系统，使他能够模拟欧洲经济共同体的发展和 使用原代细胞进行损伤。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- 在开始他的独立研究生涯之前，他从事细胞信号转导和单细胞RNA测序。 职业发展：这个奖项将确保Muegge博士能够建立一个职业生涯 退伍军人医疗保健系统中的独立内科科学家。他已经制定了一个正式的五年培训计划 这将扩大他在肠道发育、体外模型和单细胞转录方面的科学技能，如 以及科学出版、基金撰写和实验室管理方面的学术技能。米格医生的 培训将通过实验研究、教学、研讨会和国家会议来完成。他 已经组建了一个拥有必要经验和专业知识的科学咨询委员会，以监督和 支持他在实现职业目标方面取得的进步。在CDA-2完成后，Muegge博士将 掌握尖端技术(单细胞转录学、体外干细胞培养系统) 在一个独特的领域工作，使他有别于该领域的其他人(肠道内分泌功能 受伤)。他将独一无二地准备好解决需要肠道知识的基本问题 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.