Hypoxia Affects Stem Cell Fate in Patient-Derived Ileum Enteroids in a HIF-1α-Dependent Manner

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.

Keywords: enteroids; epithelium; hypoxia; ileum; intestinal stem cells; organoid formation.

Figure 1

Growth of human ileum-derived enteroids is reduced in hypoxia. (a) Ileum-derived enteroids were lysed after 24 h incubation under normoxia (N, red) or hypoxia (H, blue), and stabilization of HIF-1α was assessed by Western blot analysis. A representative image from enteroid donor 1 is shown, and β-actin was used as a loading control. (b) The expressions of the HIF-target genes CA9, VEGF, and GLUT1 were quantified using qRT-PCR after 48 h incubation of enteroids derived from donor 1 in normoxia (red) or hypoxia (blue). Figures show the mean ± SEM (n = 3 independent experiments), and an unpaired t-test with Welch’s correction was applied. p < 0.05 = *, <0.001 = ***. (c) Schematic depicting the experimental setup of enteroid seeding followed by incubation in normoxia (20% oxygen, red) or hypoxia (1% oxygen, blue) two days post-seeding. (d–f) Enteroids derived from donor 1 were cultured in normoxia or hypoxia according to (c), and enteroid growth was monitored over time. (d) Brightfield images were acquired each day using a ZEISS Celldiscoverer 7 microscope using a 5× 0.5× magnification. Representative images are shown, and yellow arrows point towards enteroids (not all enteroids were marked). Scale bar = 200 μm. (e,f) The number of enteroids per well (e) and enteroid size (f) were determined. Enteroids from ≥2 wells per independent experiment were counted and measured (n = 3 independent experiments). (e,f) The graphs depict the mean ± 95% confidence interval. A 2-way ANOVA with multiple comparisons was applied. p ≥ 0.05 = ns (not significant), <0.05 = *, <0.0001 = ****.

Figure 2

Bulk RNA sequencing of human ileum-derived enteroids suggests a loss of stemness in hypoxia. (a) Schematic depicting the experimental setup for bulk RNA sequencing. Enteroids from donor 1 were incubated in normoxia (red) or hypoxia (blue) for 6, 12, 24, or 48 h in quadruplicates in one experiment. (b) Dimensionality reduction was assessed by multi-dimensional scaling (MDS). Duration of incubation in normoxia (red) or hypoxia (blue) is illustrated by color intensity. (c) Pathway RespOnsive GENes for activity inference (PROGENy) analysis was performed to infer pathway activity. Hypoxia, VEGF, and Wnt pathway activities were plotted for each different time point. An unpaired signed-rank Wilcoxon test was applied. p ≥ 0.05 = ns (not significant), <0.05 = *. (d) Differential expression analysis of 16 stem cell-associated genes between enteroids incubated under normoxia and hypoxia is represented as a heat map. The color scale indicates relative expression levels, with magenta representing upregulated and green representing downregulated gene expression in hypoxia compared to normoxia. (e) Correlation analysis between hypoxia and stemness using stem cell signatures from a previously published single-cell RNA sequencing dataset [38]. A Spearman rank correlation test was performed. (f) Cell type deconvolution was performed via CIBERSORTX using our previously published single-cell RNA sequencing dataset of the same ileum enteroids (derived from donor 1) [41] to infer the stem cell fractions of the different samples. An unpaired signed-rank Wilcoxon test was performed. p ≥ 0.05 = ns (not significant), <0.05 = *.

Figure 3

Single-cell RNA sequencing confirms decreased stem cell number in hypoxia. (a) Schematic depicting the experimental setup for single-cell RNA sequencing (scRNAseq). Ileum-derived enteroids from three different donors were incubated in normoxia (red) or hypoxia (blue) for 24 h and 48 h. (b) Uniform manifold approximation and projection (UMAP) was plotted to create a sample overview with donor-specific differences. The different colors represent the three different donors. (c) UMAP plot of donors 1, 2, and 3 comparing normoxia (red) and hypoxia (blue). (d) UMAP plot depicting the different cell types present in the analyzed enteroids (cycling TA, dark green; enterocyte 1, beige; enteroendocrine cells, grey; goblet cells, red; immature enterocyte 1, pink; immature enterocyte 2, brown; stem cells, light green; TA cells, blue). (e) Gene expression signatures of the different cell types present in ileum-derived enteroids are shown as a dot plot of the top marker genes. Dot sizes represent the percentage of cells expressing the gene, and the color represents the average relative expression across the cell type. (f) Cytotrace was used to determine the stemness score of each enteroid at 24 h and 48 h under normoxia (red) or hypoxia (blue). An unpaired signed-rank Wilcoxon test was applied. p < 0.01 = **, <0.001 = ***, <0.0001 = ****. (g) Fractions of stem cells present in the enteroids derived from donor 1, 2, or 3 incubated in normoxia or hypoxia for 48 h. A ratio-paired t-test was performed. p < 0.05 = *.

Figure 4

Hypoxia leads to a loss of stem cells. Human ileum-derived enteroids from donors 1, 2, and 3 were incubated in normoxia (red) or hypoxia (blue) for 48 h. (a–c) Enteroids were cultured in high-Wnt media without media changes, and transcript levels of OLFM4, LGR5, and AXIN2 were analyzed using qRT-PCR. (d) Human ileum-derived enteroids from three different donors were cultured in high-Wnt media in normoxia or hypoxia for 48 h without media changes and then fixed and stained for flow cytometry analysis. Expression of the proliferation marker Ki-67 was assessed, and the fraction of cells positive for Ki-67 is depicted. (e) Enteroids were cultured in high-Wnt media with daily media changes to supplement Wnt, and transcript levels of OLFM4 were assessed by qRT-PCR. (f) Enteroids were cultured in low-Wnt media to induce differentiation, and OLFM4 gene expression was determined using qRT-PCR. (a–f) The graphs show the mean ± SEM (n ≥ 3 independent experiments), and a 2-way ANOVA with multiple comparisons was applied. p < 0.05 = *, p < 0.01 = **, <0.001 = ***, <0.0001 = ****.

Figure 5

Enteroid formation efficiency of human ileum-derived enteroids is reduced after incubation in hypoxia. (a) Schematic depicting the experimental setup to assess enteroid formation efficiencies. Enteroids from three different donors were seeded into normoxia (red) and then incubated in normoxia or hypoxia (blue) for 12, 24, 48, or 72 h before splitting and re-incubation in normoxia. (b) Brightfield images were acquired two days post-splitting using a ZEISS Celldiscoverer 7 microscope using a 5× 1× magnification. Magnified areas of representative fields of view from enteroid donor 1 are shown, and yellow arrows indicate enteroids (not all enteroids were marked). Scale bar = 200 μm. (c–e) The number of enteroids from donor 1 (c), donor 2 (d), and donor 3 (e) was quantified from ≥7 fields of view per independent experiment (c,d) or from ≥3 fields of view per independent experiment (e) and are depicted as mean ± 95% confidence interval (n = 3 independent experiments). A 2-way ANOVA with multiple comparisons was applied. p ≥ 0.05 = ns (not significant), <0.01 = **, <0.001 = ***, <0.0001 = ****.

Figure 6

HIF-1α stabilization by roxadustat in normoxia reduces stemness and proliferation in human ileum-derived enteroids. Enteroids were treated with 100 μM roxadustat (Rox, purple) to stabilize HIF-1α protein expression in normoxia and compared to solvent (DMSO)-treated enteroids incubated in normoxia (N, red) or hypoxia (H, blue). (a) To confirm HIF-1α stabilization, enteroids were lysed 24 h post-treatment, and HIF-1α protein expression was assessed via Western blotting, and β-actin was used as a loading control. A representative Western blot image of enteroids derived from donor 1 is shown. (b,c) Gene expression of the HIF-1α target gene CA9 (b) and the stem cell-associated gene OLFM4 (c) from all three donors was assessed using qRT-PCR 48 h post-treatment. The graphs depict the mean ± SEM (n = 3 independent experiments), and a 1-way ANOVA with multiple comparisons was applied. p < 0.05 = *, <0.01 = **, <0.001 = ***, <0.0001 = ****. (d) Enteroids from all three donors were seeded into untreated high-Wnt media. After two days, media were exchanged and enteroids were incubated with 100 μM roxadustat (purple) or a solvent control (DMSO, red) in normoxia. Brightfield images were acquired at the indicated time points post-media change using a ZEISS Celldiscoverer 7 microscope using a 5× 1× magnification. Enteroid growth was quantified by counting the number of enteroids per field of view. Means ± 95% confidence intervals are depicted from 8 fields of view. A 2-way ANOVA with multiple comparisons was applied. p ≥ 0.05 = ns (not significant), <0.05 = *, <0.01 = **. (e,f) Enteroids from donor 1 were seeded into normoxia (red) into untreated high-Wnt media. After two days, media were exchanged, and enteroids were incubated with 100 μM roxadustat (purple) or a solvent control (DMSO, red) for the indicated incubation spans. On day 5, enteroids were split into untreated high-Wnt medium, and imaging was performed on day 7. Brightfield images were acquired two days post-splitting using a ZEISS Celldiscoverer 7 microscope using a 5× 0.5× magnification. (e) Schematic depicting the experimental setup to assess enteroid formation efficiencies. (f) Enteroid formation efficiency was determined by quantifying the number of enteroids. Means ± 95% confidence intervals are depicted from ≥7 fields of view per independent experiment (n = 3 independent experiments). A 2-way ANOVA with multiple comparisons was applied. p < 0.01 = **, <0.0001 = ****.