Enteric Nervous System Development and Function in Human Intestinal Organoids

人肠类器官中肠神经系统的发育和功能

基本信息

批准号：
10662539
负责人：
Allison Leigh Speer
金额：
$ 15.34万
依托单位：
UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-07-11 至 2027-05-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10662539
关键词：
3-Dimensional Affect Agonist Award Calcium Caring Cessation of life Child Childhood Collaborations Complex Constipation Creativeness Data Dehydration Development Development Plans Developmental Biology Developmental Process Disease Ensure Enteral Enteric Nervous System Environment Epithelial Cells Epithelium Failure Funding Genetic Goals Human Image In Vitro Incidence Institution Intestines Knock-out Knockout Mice Knowledge Left Live Birth Maintenance Malnutrition Measures Medical center Mentors Mesenchyme Mission Modeling Morbidity - disease rate Morphology Muscle Near-infrared optical imaging Nervous System Physiology Neural Crest Cell Neuroglia Neurons Nutritional Operative Surgical Procedures Organoids Parenteral Nutrition Peristalsis Permeability Principal Investigator Procedures Process Production Publishing Research Research Design Research Methodology Research Personnel Resistance Role Scientist Serotonin Stains Surgeon Synapses Syndrome Testing Texas Therapeutic Therapeutic Intervention Tight Junctions Time Tissue Engineering Training Translational Research Transmission Electron Microscopy Transplantation United States National Institutes of Health Western Blotting Work antagonist career career development cell motility cost experimental study gain of function gastrointestinal gastrointestinal transplantation human embryonic stem cell imaging modality improved in vivo in vivo imaging innovation intestinal barrier intestinal epithelium loss of function mortality multidisciplinary nervous system development neurogenesis novel nutrient absorption pathogen pharmacologic preservation programs receptor serotonin receptor skills success teduglutide therapeutically effective

项目摘要

This application outlines a proposal to advance the research career of a promising young investigator within a mentored setting. Successful completion will enable the candidate to establish a career as an independent NIH-funded surgeon-scientist, conducting translational research focused on identifying the processes required to generate functional tissue-engineered intestine as a cure for pediatric intestinal failure. Background: Intestinal failure is a survivable but morbid syndrome that afflicts tens of thousands of children. Contemporary management is inadequate and costly. Tissue-engineered intestine is a potential solution; however, complex intestinal functions have yet to be confirmed in existing models. Thus, understanding the processes required to generate a functional intestine are a critical unmet need and a high priority for the NIH. The enteric nervous system (ENS) is crucial for intestinal functions including barrier maintenance and peristalsis. Serotonin receptors (e.g. 5-HT4R), expressed by intestinal epithelial cells and enteric neurons, promote neurogenesis and regulate intestinal barrier function and motility. Based on preliminary data and published work, we hypothesize that human intestinal organoid (HIO) function depends on the ENS, and 5-HT4R is essential for proper ENS development and function within HIOs. Research Design and Methods: Three aims will test this hypothesis in HIOs in vitro and transplanted HIOs (tHIOs) in vivo± ENS. We will employ gain and loss of function experiments using 5-HT4R genetic knockouts and pharmacological 5-HT4R agonists/antagonists. Aim 1 will evaluate the role of 5-HT4R during ENS development in HIOs utilizing 3-D immunofluorescent (IF) advanced imaging as well as RT-qPCR to locate and quantify the ENS. Aim 2 will assess if 5-HT4R is required for barrier function in tHIOs by measuring tight junction production and morphology with IF staining, Western Blot, Transmission Electron Microscopy, and RTqPCR as well as determining transepithelial resistance and permeability in an Ussing Chamber ex vivo. Aim 3 will ascertain if 5-HT4R is indispensable for peristalsis in tHIOs using non-invasive, dynamic imaging in vivo and measuring contractility ex vivo. This project is novel because no prior study has examined the role of the ENS in barrier function in HIOs nor applied these new imaging methods. This project is novel and innovative in proposing a mechanism by which ENS development and function in HIOs are regulated by 5-HT4R. Career Development Plan & Goals: Over this 5 year award, the principal investigator will have 75% protected time to expand her background in intestinal tissue-engineering to gain new skills in ENS development and neurogastroenterology. Completion will propel the field forward and launch her independent research career. Research Environment: The proposed research will be carried out at UTHealth within the Texas Medical Center, the world's largest medical center which seeks to nurture cross-institutional collaboration, creativity, and innovation. This environment has an established success of nurturing the careers of junior investigators.

该申请概述了一项促进有前途的年轻人的研究生涯的提案在指导下成功完成调查将使候选人能够建立自己的职业生涯。作为美国国立卫生研究院 (NIH) 资助的独立外科医生科学家，开展转化研究，重点是确定产生功能性组织工程肠道以治疗小儿肠道衰竭所需的过程。背景：肠衰竭是一种可存活但病态的综合征，困扰着数以万计的人当代的管理不充分且成本高昂。然而，复杂的肠道功能尚未在现有模型中得到证实。了解产生功能性肠道所需的过程是一个未满足的关键需求，也是一个高度重视的问题 NIH 优先考虑肠神经系统 (ENS) 对于肠道功能（包括屏障）至关重要。维持和蠕动。 5-羟色胺受体（例如 5-HT4R），由肠上皮细胞表达和肠神经元，促进神经发生并调节肠屏障功能和运动。根据初步数据和已发表的工作，我们发现人类肠道类器官（HIO）功能取决于在 ENS 上，5-HT4R 对于 HIO 中 ENS 的正常开发和功能至关重要。研究设计和方法：三个目标将在体外 HIO 和移植 HIO 中检验这一假设 (tHIO) 体内± ENS 我们将使用 5-HT4R 基因敲除进行功能获得和丧失实验。目标 1 将评估 ENS 期间 5-HT4R 的作用。 HIO 的开发利用 3-D 免疫荧光 (IF) 先进成像以及 RT-qPCR 来定位和量化 ENS 的目标 2 将通过测量紧密度来评估 tHIO 中的屏障功能是否需要 5-HT4R。通过 IF 染色、蛋白质印迹、透射电子显微镜和 RTqPCR 观察连接的产生和形态以及在离体 Ussing Chamber 中测定跨上皮阻力和渗透性。将使用体内非侵入性动态成像确定 5-HT4R 对于 tHIO 的蠕动是否必不可少该项目是新颖的，因为之前没有研究检验过其作用。 ENS 在 HIO 的屏障功能中也应用了这些新的成像方法，该项目是新颖且创新的。提出了一种由 5-HT4R 调节 HIO 中 ENS 发育和功能的机制。职业发展计划和目标：在这 5 年奖励中，首席研究员将获得 75% 的保护是时候扩展她在肠道组织工程方面的背景，以获得 ENS 开发和神经胃肠病学的完成将推动该领域的发展并开启她的独立研究生涯。研究环境：拟议的研究将在德克萨斯医学中心内的 UTHealth 进行中心是世界上最大的医疗中心，致力于培养跨机构合作、创造力、这种环境在培养初级研究人员的职业生涯方面取得了既定的成功。