Enteric Nervous System Development and Function in Human Intestinal Organoids

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.