Introduction: The intestinal epithelium is a self-renewing organ that regenerates every 3-5 days. Intestinal Stem Cells (ISCs) are central to the continuous renewal of the intestinal epithelium throughout an individual’s lifetime. Declined ability of ISCs to regenerate rapidly renewing tissues drives development of cancers and inflammatory bowel disease, especially in the aging population. It is unknown whether epithelial renewal and turnover rates in the intestine are region specific. Hypotheses: (1) Sorted and single Intestinal Stem Cells (ISCs) from the distal small intestine exhibit declining stemness, compared to those from the proximal small intestine, (2) Declining stemness in distal ISCs is due to decreased mitochondrial oxygen consumption rate (OCR). Methods: Single ISCs were isolated from Lgr5-EGFP-IRES-CreERT2 reporter mice (that fluorescently labels ISCs) by sorting for enhanced green fluorescent protein (EGFP) fluorescence using Fluorescence Activated Cell Sorting (FACS). Hallmarks of stemness were evaluated using the intestinal organoid (enteroid) model, generated by harvesting sorted, single ISCs with high intensity of GFP (GFP high ) from proximal and distal small intestine. Mitochondrial OCR, total ATP, and contribution of mitochondrial oxidation (mitoATP) and glycolytic pathway (glycoATP) to total ATP were measured in single cell generated enteroids using Agilent Seahorse XF Analyzer. Results: Enteroids generated from distal GFP high ISCs showed significantly reduced enteroid forming efficiency (-37%), bud numbers (-53%) and bud area (-61%), compared to those generated from proximal small intestines. Expression of stem cell markers, Lgr5 and Olfm4 remained unchanged. Distal ISCs demonstrated lower mitochondrial OCR (-56%) and mitoATP production (-66%) with significant increase in glycoATP production (45%), compared to proximal ISCs. Conclusion: ISCs harvested from the distal small intestine exhibit reduced stemness ex vivo in culture. Diminished stemness is associated with lower mitochondrial OCR, mitoATP along with concomitant surge in glycoATP production, indicating shift from mitochondrial aerobic oxidation to glycolysis. The role of region-specific differences in the observed shift in cellular metabolic preference is only beginning to be studied and contributes to the mechanistic basis for inflammatory and degenerative diseases prevalent in the distal small intestine. If our findings are translatable in humans, they set the groundwork for design of therapeutic strategies for amelioration of inflammatory and degenerative diseases of the distal gut. Significance: 1. FIRST study to show zonal differences in stemness along the SI.2. Differences in mitochondrial bioenergetics unravels the role of mitochondria in regulation of stemness in the gut. 3. Sets the groundwork and mechanistic basis for increased incidences of inflammatory diseases and cancer in the distal SI, compared to the proximal SI. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.
Sundaresan et al. (Fri,) studied this question.