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

ABSTRACT: 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-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. 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 controls intestinal homeostasis.

ORGANISM(S): Homo sapiens

PROVIDER: GSE302085 | GEO | 2026/01/14

REPOSITORIES: GEO

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Project description:To investigate the detailed molecular mechanisms for the regulatory role of HIF-2α in experimental colitis, microarray gene expression analysis was performed on colon RNA isolated from 6- to 8-week-old Hif-2αF/F, Hif-2αlΔIE mice treated with 3%DSS for 3 days. Background & Aims: Hypoxic inflammation is characterized by decreased oxygen tension in inflammatory foci, and is a notable feature in inflammatory bowel disease (IBD). Hypoxic response is mediated by transcription factors hypoxia-inducible factor (HIF)-1α and HIF-2α, both of which are highly induced in IBD. HIF-1α is a protective factor that limits intestinal barrier dysfunction during inflammation. However, the role of HIF-2α has not been assessed in hypoxic inflammation and IBD. Methods: A hypoxia reporter mouse model was used to test the presence of hypoxia and HIF-2α in dextran sulfate sodium (DSS) and Citrobacter rodentium (C.rod)-induced colitis. The role of HIF-2α in these mouse models of colitis was further assessed in mice with an intestinal epithelium-specific gain- and loss-of-function of HIF-2α. Results: Induction of hypoxia and HIF-2α was confirmed in both murine experimental colitis models and human IBD samples. Disruption of HIF-2α attenuated colonic inflammation whereas stabilization of HIF-2α potentiated inflammation in mouse models of colitis. Interestingly, intestine specific overexpression of HIF-2α but not HIF-1α leads to spontaneous colitis and premature death in mice. Further mechanistic analysis showed that HIF-2α is a driver for pro-inflammatory response and is critical regulator of intestinal epithelial-derived tumor necrosis factor (TNF)-α. Blocking TNF-α completely ameliorated HIF-2α potentiated intestinal inflammation. Conclusions: These data demonstrate that HIF-2α is a critical transcription factor essential in intestinal epithelium elicited inflammatory response. Global gene expression profiling in colon RNAs isolated from 7-week-old Hif-2αF/F (n=6, Shah 007) and Hif-2αΔIE (n=5, Shah 008).

Project description:Genes encoding transcription factors function as hubs in gene regulatory networks because they encode DNA-binding proteins, which bind to promoters that carry their binding sites. In the present work we have studied gene regulatory networks defined by genes with transcripts belonging to different mRNA abundance classes in the small intestinal epithelial cell. The focus is the rewiring that occurs in transcription factor hubs in these networks during the differentiation of the small intestinal epithelial cell while it migrates along the crypt-villus axis and during its development from a fetal endodermal cell to a mature adult villus epithelial cell. We have generated transcriptome data for mouse small intestinal villus, crypt and fetal intestinal epithelial cells. In addition we have generated metabolome data from crypt and villus cells. Our results show that the intestinal crypt transcription factor hubs that are rewired during differentiation are involved in the cell cycle process (E2F, NF-Y) and stem cell maintenance (c-Myc). In contrast the villi are dominated by a HNF-4 villus hub, which is rewired during differentiation by the addition of network genes with relevance for lipoprotein synthesis and lipid absorption. Moreover, we have identified a villus NF-kB hub, which was revealed by comparison of the villus and endoderm transcriptomes. The rewiring of the NF-kB villus hub during intestinal development reflects transcriptional activity established by host and microflora interactions. To aid in the mining of our results we have developed a web portal (http://gastro.imbg.ku.dk/mousecv/) allowing easy linkage between the transcriptomic data, biological processes and functions. Experiment Overall Design: Four different sample categories were analyzed. Experiment Overall Design: 1) Small intestinal crypts isolated form 12-weeks old C57BL/6 mice. These samples are in triplicates. Experiment Overall Design: 2) Small intestinal villi isolated form 12-weeks old C57BL/6 mice. These samples are in triplicates. Experiment Overall Design: 3) Embryonic day 12 mesenchyme. These samples are in quadruplicate. each sample is derived from a pool of mesenchymes (10-40) Experiment Overall Design: 4) Embryonic day 12 endoderm. These samples are in quadruplicate. each sample is derived from a pool of endoderms (10-40)

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

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