HIF-1alpha activity affects stem cell fate in patient-derived ileum organoids

The intestinal epithelium maintains tissue homeostasis through a dynamic balance of stem cell proliferation and differentiation along the crypt-villus axis. This process is spatially regulated, where intestinal stem cells in crypts proliferate and progenitor cells differentiate as they migrate toward the villus tips. An oxygen gradient is present along the crypt-villi structure setting the villi into a low-oxygen environment (physiological hypoxia) and the crypts into normoxia. Hence, during this differentiation and migration process, intestinal epithelial cells encounter distinct oxygen microenvironments throughout their life span. Inflammatory bowel diseases and other chronic inflammatory conditions disrupt this homeostatic balance and alter local oxygen dynamics, leading to dysregulated tissue repair and regeneration. To investigate how oxygen availability influences intestinal stem cell fate, we cultured patient-derived human ileum organoids under normoxic (20% oxygen) or hypoxic (1% oxygen) conditions. Under hypoxia, organoid growth was reduced and expression of the stem cell marker OLFM4 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-1a under normoxic conditions recapitulated the hypoxia-induced loss of stemness, demonstrating that HIF-1a is a key mediator of oxygen-dependent stem cell regulation. These findings establish that physiological hypoxia in the intestinal epithelium directly regulates stem cell fate through HIF-1a stabilization, providing mechanistic insight into how oxygen availability controls intestinal homeostasis. Overall design: Single-cell RNA sequencing on human ileum-derived organoids generated from three different donors were incubated for 24 h or 48 h normoxia (20% O2) vs. hypoxia (1% O2). For each condition, organoids were seeded 3D-sheroids in Matrigel domes submerged in low Wnt media.