Xenotransplanted human organoids identify transepithelial zinc transport as a key mediator of intestinal adaptation

Abstract

Short bowel syndrome (SBS) leads to severe morbidity and mortality. Intestinal adaptation is crucial in improving outcomes. To understand the human gene pathways associated with adaptation, we perform single-cell transcriptomic analysis of human small intestinal organoids explanted from mice with experimental SBS. We show that transmembrane ion pathways, specifically the transepithelial zinc transport pathway genes SLC39A4 and SLC39A5, are upregulated in SBS. This discovery is corroborated by an external dataset, bulk RT-qPCR, and Western blots. Oral zinc supplementation is shown to improve survival and weight gain of SBS mice and increase the proliferation of intestinal crypt cells in vitro. Finally, we identify the upregulation of SLC39A5 and associated transcription factor KLF5 in biopsied intestinal tissue specimens from patients with SBS. Thus, we identify zinc supplementation as a potential therapy for SBS and describe a xenotransplantation model that provides a platform for discovery in other intestinal diseases.

Fig. 1. Development of a human–mouse chimera model system for the identification of pathways that mediate intestinal adaptation in experimental SBS.

a Schematic overview of the experimental workflow for the human small intestinal organoid xenotransplantation SBS model. Six-week-old Rag1 KO C57BL/6 mice underwent experimental SBS (n = 5) or sham surgery (n = 5). Human intestinal organoids, differentiated from iPSCs, were implanted into the mesentery using surgical glue. Xenotransplants were harvested after 25 days for single-cell RNA sequencing analysis. b Bright field microscopy of human small intestinal organoids differentiated from iPSC (Day 42). Magnification ×20. Scale bar 100 μm. c RT-qPCR analysis of intestinal-specific markers in human organoids pre-xenotransplantation, showing increased expression relative to housekeeping gene RPLP0. Data from three independent organoid cultures are presented as mean ± standard error of the mean (SEM). d Immunofluorescence staining of pre-xenotransplantation organoids for epithelial and intestinal markers. Scale bar 100 µm. e Images of mouse intestine pre-resection, during xenotransplantation, and at harvest on Day 25. f Schematic of intestinal resection and sampling areas in the SBS model. 75% of the small bowel was resected, with a jejuno-ileal anastomosis. Sham controls underwent transection and anastomosis without resection. g Survival curves for SBS (n = 23) and sham (n = 40) mice with human xenotransplants. SBS mice had ~70% survival at 10 days vs ~100% for sham (log-rank test, p = 0.0015). h Growth curves showing significant weight loss in SBS mice (n = 6) compared to steady weight gain in sham controls (n = 7). Weight differences were significant at all indicated time points (t-test, *p < 0.05). Data are mean ± SEM. i H&E staining of jejunum and ileum from Rag1 KO mice post-xenotransplant. SBS jejunal villi were significantly lengthened; ileal villi were modestly increased. Scale bar 100 µm. j Immunofluorescence images of BrdU staining in intestinal tissue post-xenotransplant. BrdU incorporation was higher in SBS ileum compared to sham. Scale bar 100 µm. k Quantification of BrdU-positive cells in intestinal crypts showed significantly higher incorporation in SBS compared to sham controls. Source data are provided as a separate file.

Fig. 2. Distinct transcriptional signatures are revealed by single-cell RNA sequencing analysis of human intestinal organoid explants harvested from mice with SBS.

a UMAP projection of single-cell data from human small intestine organoid implants in SBS (n = 5) and sham mice (n = 5). Each dot represents a cell, with coordinates determined by transcriptome expression via principal components analysis and subsequent UMAP dimensional reduction. Clusters are color-coded and labeled by cell type. b UMAP projection colored by SBS cells (red) and sham cells (blue). The Enterocyte 1 and stem-like fibroblast clusters are predominantly derived from SBS samples. c Barplot showing the proportions of each cell type identified in scRNA-seq analysis of human intestinal organoid explants from mice subjected to either sham or SBS surgery. Each bar represents a cell type, and the x-axis indicates the experimental group. The height of each bar corresponds to the percentage of cells of that type in the total cell population for that sample. d Heatmap illustrating DEGs in human intestinal organoids derived from sham and SBS mice. The top genes within each cluster and the corresponding marker genes identified by scRNA-seq analysis are displayed. Rows represent individual genes, and columns represent cell clusters or individual marker genes. Color intensity reflects gene expression levels, with red indicating high expression and blue indicating low expression. Horizontal bars above the heatmap denote marker genes for each cell cluster. e Network plot generated from gene set enrichment analysis (GSEA) showing upregulated pathways between enterocyte 1 (95% SBS) and enterocyte 2 (59% SBS) as well as stem-like fibroblast (96% SBS) and mature fibroblast (52% SBS). Node size correlates with the number of genes examined within those gene sets. Edges represent connections between nodes, with thickness proportional to the number of shared genes. The top six genes differentially expressed with statistical significance between the compared cell types and listed in the core enrichment of the GSEA analysis are displayed alongside each pathway. f Network plot of downregulated pathways in SBS, generated from GSEA showing downregulated pathways between Enterocyte 1 (95% SBS) and Enterocyte 2 (59% SBS) as well as stem-like fibroblast (96% SBS) and mature fibroblast (52% SBS). Two-sided Wilcoxon rank sum test; filtering for genes with a Bonferroni-corrected p-value of less than 0.05. Source data are provided.

Fig. 3. Zinc transport genes SLC39A4 and SLC39A5 are upregulated in SBS.

UMAP visualization of human small intestinal xenotransplants from five SBS mice and five sham controls. Higher expression of SLC39A4 (a) and SLC39A5 (b) is observed within the enterocyte 1 cluster derived from SBS. Violin plots depict the elevated expression of SLC39A4 (c) and SLC39A5 (d) in human SBS xenotransplants compared to sham. Wilcoxon rank sum test, ****p < 0.0001. RT-qPCR of isolated intestinal epithelial cells of SBS mice (n = 5) shows the increased relative expression of Slc39a4 in the jejunum (e) and ileum (f) and of Slc39a5 expression in SBS jejunum (g) and ileum (h) compared to sham control (n = 5). Mann–Whitney U-test. *p ≤ 0.05; **p ≤ 0.01. Data represent mean ± SEM. i UMAP projection of processed, filtered, and clustered SBS epithelial cell data from the gene expression omnibus GSE130113 generated in Seurat. Feature plots illustrate the expression of j SLC39A4, and k SLC39A5 genes in mouse SBS native epithelial cells in GSE130113. Representative Western blot analysis of Zip4 (l) and Zip5 (m) protein expression in sham and SBS mice. Quantification analysis of Zip4 (n) Zip5 (o) protein band density from blots comparing ileum tissue from sham and SBS mice fed a control diet. Source data are provided as a Source Data file.

Fig. 4. Zinc supplementation promotes intestinal stem proliferation in juvenile mouse jejunal enteroids and human small intestinal organoids in vitro.

Representative light microscopy images of juvenile WT C57BL/6 mice jejunal enteroids following 24 h of treatment with control a 4 µM TPEN and b 40 µM zinc acetate. c Magnification 10×; scale bar 100 mm. d Representative immunofluorescence images of BRDU staining in jejunal enteroids indicating proliferation after an 8-h pre-treatment with control, d 4 µM TPEN, and e 40 µM zinc acetate. f Magnification 20×; scale bar 100 mm. Representative immunofluorescence images showing Ki67 expression in jejunal enteroids treated with control (g), 4 µM TPEN (h), and 40 µM zinc acetate (i). Magnification 20×; scale bar 100 mm. Representative immunofluorescence images visualizing PCNA expression in jejunal enteroids following treatment with control (j), 4 µM TPEN (k), and 40 µM zinc acetate (l). Magnification 20×; scale bar 100 µm. m Quantification of jejunal enteroid sizes (n = 12 per experimental group) and fluorescence for BrdU, Ki67, and PCNA following 24 h of treatment with control, TPEN, or zinc acetate (n = 3 or 4 per group). Data represent compiled measurements derived from two independent experiments. Enteroid size: t-test. ****p ≤ 0.0001. Quantification of fluorescence: Mann–Whitney U-test, *p ≤ 0.05. Error bars indicate ±SEM. Representative immunofluorescence images of human small intestinal organoids treated with control (n), 4 µM TPEN (o), and 40 µM zinc acetate (p) visualizing BrdU to assess proliferation after 8-h pre-treatment. Magnification 25×; scale bar 50 mm. Representative immunofluorescence images displaying Ki67 expression in human small intestinal organoids treated with control (q), 4 µM TPEN treatment (r), and 40 µM zinc acetate treatment (s). Magnification 25×; scale bar 50 μm. Representative immunofluorescence images showing PCNA expression in human small intestinal organoids treated with control (t), 4 µM TPEN, and (u) 40 μM zinc acetate (v). Magnification 25×; scale bar 50 μm. w Fluorescence quantification of BrdU, Ki67, and PCNA staining following 24 h of treatment with control, TPEN, or zinc acetate. Data are compiled from measurements of three intact stained organoids derived from two independent experiments. Error bars indicate ± SEM. Mann–Whitney U-test, *p ≤ 0.05. Source data are provided as a Source Data file.

Fig. 5. Zinc supplementation in murine SBS enhances intestinal proliferation and adaptation in vivo.

a Growth curves for SBS mice treated with control (Ctrl SBS) (n = 10), zinc supplementation ((+) Zn SBS) (n-6), and zinc-depleted diet ((−) Zn SBS) (n = 6) over 7 days. Significant weight change is observed in (+) Zn SBS compared to Ctrl SBS from Day 5 onward. Data points are mean ± SEM. t-test, *p < 0.05 at indicated time points. b Survival curves comparing the three groups within the 7-day SBS model. c Plasma zinc levels measured by colorimetric assay (Abcam). Zinc-supplemented mice (n = 6) achieved plasma zinc levels within or above the normal range (12–25 µmol/l, yellow-shaded area). SBS mice on control (n = 4) and zinc-depleted diets (n = 4) did not reach this range. Data are mean ± SEM. t-test, p = 0.0336. H&E staining of the jejunum (d) and ileum (e) tissue following the 7-day SBS model. Mice were fed either a control or a zinc-supplemented diet. Magnification: 20x; scale bar: 100 µm. RT-qPCR analysis of ileal tissue from −Zn SBS mice (n = 4) vs +Zn SBS mice (n = 3), showing ~ 2-fold increase in Ki67 expression (f, p < 0.05) and a trend towards increased PCNA expression (g) in the zinc-supplemented group (p = 0.0540). Data are mean ± SEM. Representative immunofluorescence images of intestinal tissue from SBS mice following 10 days of post-operative treatment with zinc depleted or zinc-supplemented diet. h BRDU staining 24 h after gavage; Ki67 expression (i), PCNA expression (j), and SI expression (k) under the same conditions. Magnification 10×; scale bar 100 µm. Source data are provided as a Source Data file.

Fig. 6. Intestinal tissue from SBS patients shows increased expression and protein translocation of ZIP4 and ZIP5 to putative sites.

a Table describes a total of 26 patients that were included in this study. b, c RT-qPCR of tissue derived from human intestinal biopsies. Gene expression levels were normalized by SI expression to estimate expression levels in enterocytes. SLC39A4 (b) is expressed at similar levels in control (n = 14) compared to SBS (n = 12). Approximately 1.5-fold higher SLC39A5 (c) expression in SBS (n = 9) compared to control (n = 12). Data points represent means, and bars represent ±SEM. Mann–Whitney U-test, p = 0.0346. Representative immunostaining of ZIP4 in control (d) and SBS (e) human intestinal tissue biopsies; staining of ZIP5 in control (f) and SBS (g) biopsy tissue demonstrate changes in the localization of zinc transport proteins in response to SBS. Tissue from over 25 biopsies from participants across both the SBS group and the control group were analyzed. Magnification 20×, upper panel scale bar 50 μm; lower panel scale bar 200 μm. Source data are provided as a Source Data file.

Fig. 7. Gene regulatory network analysis uncovers KLF5 as a key regulator of ZIP4 expression in SBS.

a Workflow for regulatory analysis of SLC39A4 and SLC39A5. TF was identified using pySCENIC and analyzed for best fit. b Heatmap showing the correlation between TF and gene expression in human intestinal organoid explants from scRNA-seq data. Columns represent genes, and rows represent TFs, with color intensity indicating correlation (red for positive, blue for negative). KLF5, HNF4A, HNF4G, and ESRRA were identified as potential regulators due to their strong correlation with SLC39A4 and SLC39A5 expression. c Violin plots of TF expression in sham and SBS human intestinal explant cells. All four TFs (KLF5, HNF4A, HNF4G, and ESRRA) show significantly higher expression in SBS compared to sham. Wilcoxon rank sum test, ****p < 0.0001. d AUC scores indicate increased regulon activity for each TF in SBS vs sham, suggesting regulon enrichment in cells expressing these TFs. Box plots display AUC score distributions, with the median indicated by a central line, bounds of the box corresponding to the lower (Q1) and upper (Q3) quartiles, and whiskers extending to 1.5 times the interquartile range. Wilcoxon rank sum test, ****p < 0.0001. Data are from 3090 cells (1729 from five SBS mice, 1361 from five sham mice). e Violin plots showing TF expression across cell clusters from human small intestinal explant scRNA-seq analysis, highlighting KLF5 specificity within the regulon to enterocyte cells. f qRT-PCR reveals increased Klf5 expression in ileal enterocyte cells from SBS mice (n = 5) vs sham controls (n = 5), p = 0.004. g Increased Klf5 expression in ileum tissue from SBS patients (n = 13) compared to controls (n = 12) by qRT-PCR, p = 0.0173. Data are mean ± SEM. Mann–Whitney U-test, *p ≤ 0.05; **p ≤ 0.01. Source data is provided as a Source Data file.