Abstract
The intestinal epithelium maintains tissue homeostasis through a dynamic balance of stem cell proliferation and differentiation. This process is spatially regulated along the crypt-villus axis, with intestinal stem cells in the crypt regions proliferating and progenitor cells differentiating as they migrate toward the villus tips. Because the lumen of the gut contains very low levels of oxygen (i.e., hypoxia), an oxygen gradient is established within the crypt-villus axis, placing the crypt regions under normoxic conditions while the villus tips reside under hypoxic conditions. Hence, intestinal epithelial cells encounter distinct oxygen microenvironments throughout their life span as they migrate along the crypt-villus structures during their proliferation and differentiation process. To investigate how oxygen availability influences intestinal stem cell proliferation and differentiation, we cultured patient-derived human ileum organoids (i.e., enteroids) under normoxic (20% oxygen) or hypoxic (1% oxygen) conditions. Under hypoxia, enteroid growth was reduced, and expression of several stem cell markers, such as OLFM4 and LGR5, was decreased. Bulk and single-cell RNA sequencing revealed that hypoxia suppressed Wnt signaling pathways and reduced stem cell activity. Importantly, pharmacological stabilization of HIF-1α under normoxic conditions recapitulated the hypoxia-induced loss of stemness, demonstrating that HIF-1α is a key mediator of oxygen-dependent stem cell regulation in enteroids. These findings establish that physiological hypoxia in the intestinal epithelium directly regulates stem cell fate through HIF-1α stabilization, providing mechanistic insight into how oxygen availability along the crypt-villus structures controls intestinal homeostasis.