Establishment of Anoxia/cytokine-induced Injury and Regeneration in Colonoids

Abstract

Intestinal epithelial cells (IECs) play key roles in nutrient absorption and fluid secretion, providing a barrier to protect cells. IECs, derived from intestinal stem cells (ISCs), are crucial in maintaining intestinal homeostasis and regeneration, which is crucial in gastrointestinal diseases such as inflammatory bowel disease (IBD). To study epithelial injury/regeneration, primary cultured colonoids were utilized as a model system to mimic injury. They retain the composition and differentiation of the tissue, making it a suitable model for studying homeostasis and regeneration of ISCs in vitro. Anoxia and proinflammatory cytokines frequently coexist and exacerbate one another during intestinal injury. However, anoxic conditions have been mainly ignored in previous research. Thus, we developed a regeneration model using cytokines/anoxia injury to better recapitulate intestinal inflammation. Colonoids were treated with cytokines TNF-α and IFN-γ (5 ng/μL) in anoxic conditions for 24 hours to provoke an injury. After 24 hours of injury, the colonoids were exposed to normal oxygen and media conditions for regeneration. Regeneration was observed for 24, 48, and 72 hours and documented using photomicrographs. Morphological analysis detailed that injury was most severe 24 hours post-injury with the combined induction of cytokines and anoxia resulting in decreased colonoid area and number of buds compared to control groups. RT-PCR analysis detailed mRNA expression of stem cell markers ( Lgr5, Bmi1, and Hopx), Wnt ( Axin2, Myc2, and Ascl2), and YAP target genes ( Sca1 and Ctgf) were increased at peak injury and returned to pre-injury levels at 72 hours of recovery. Regeneration of the colonoids is consistent with in vivo observations, thus confirming the validity of the model. This versatile model can be used to study responses to injury and regeneration of intestinal epithelium and the function of ISCs with applications in intestinal disease therapeutics. University of Maryland College of Agriculture and Natural Resources and the Maryland Agricultural Experiment Station Undergraduate Research Grant Grant #4307572 from the Maryland Stem Cell Research Fund. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.