Engineering transplantable jejunal mucosal grafts using patient-derived organoids from children with intestinal failure

Abstract

Intestinal failure, following extensive anatomical or functional loss of small intestine, has debilitating long-term consequences for children¹. The priority of patient care is to increase the length of functional intestine, particularly the jejunum, to promote nutritional independence². Here we construct autologous jejunal mucosal grafts using biomaterials from pediatric patients and show that patient-derived organoids can be expanded efficiently in vitro. In parallel, we generate decellularized human intestinal matrix with intact nanotopography, which forms biological scaffolds. Proteomic and Raman spectroscopy analyses reveal highly analogous biochemical profiles of human small intestine and colon scaffolds, indicating that they can be used interchangeably as platforms for intestinal engineering. Indeed, seeding of jejunal organoids onto either type of scaffold reliably reconstructs grafts that exhibit several aspects of physiological jejunal function and that survive to form luminal structures after transplantation into the kidney capsule or subcutaneous pockets of mice for up to 2 weeks. Our findings provide proof-of-concept data for engineering patient-specific jejunal grafts for children with intestinal failure, ultimately aiding in the restoration of nutritional autonomy.

Figure 1. Generation and characterization of primary intestinal organoids derived from targeted pediatric patient group

a, Schematic overview demonstrating the expansion timeline after harvesting intestinal crypts endoscopically from pediatric patients. Each patient biopsy sample yields ~3-5 organoids by week 4, and over 10 million cells by week 8 after successive passaging as indicated by a representative phase contrast image of organoids in culture at week 8. b, Phase contrast images of human intestinal organoids [patient 6] established from isolation at the indicated time points. Original magnifications: X20 (days 0); X10 (days 3, 8); X5 (days 15, 18, 28). c, Representation phase contrast images of first passage expansion cultures of duodenal [patient 9] (left), jejunal [patient 2] (middle) and ileal [patient 3] organoids (right). Scale bars represent 200μm. d, Quantitative RT-qPCR analysis of human duodenal [patient 10], jejunal [patient 2] and ileal [patient 14] organoids for functional duodenal markers (CYBRD1; SLC40A1), jejunal markers (SI; LCT) and ileal markers (SLC10A2; OSTB). e, Quantitative RT-qPCR analysis of human jejunal organoids [patient 2, 7, 8] at passages 5, 15 and 25 for jejunal specific markers SI and LCT. f, Quantitative RT-qPCR analysis of human jejunal organoids [patients 2, 7, 8] cultured in basal media culture conditions as indicated in the method, treated with the GSK3β inhibitor CHIR99021 (CHIR), or the Notch inhibitor DAPT. g, Representative stainings for EdU and DAPI of human jejunal organoids [patient 2] in basal culture conditions and expansion conditions (+CHIR). Scale bars represent 30μm. h,i, Representative immunostaining of human jejunal organoids [patient 2] cultured in expansion conditions (+CHIR) (h) or differentiation conditions (+DAPT) (i) using the indicated antibodies to mark proliferating cells (Ki67), stem cells (LGR5 and SOX9), Paneth cells (LYZ), goblet cells (UEA-1), epithelial cells (E-cad), enterocytes (alkaline phosphatase) and enteroendocrine cells (CHGA). Scale bars represent 100μm. All images are representative of 3 experiments (b, c, h, i) or 2 experiments (g). Quantitative data shown represents mean ± s.e.m. of n = 3 experimental replicates (d) or biologically distinct replicates (e, f). Differences were analyzed by one-way ANOVA with Dunnett’s multiple comparisons test in (d, f) or with Tukey’s multiple comparisons (e). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 were considered significant; n.s., not significant. Detailed p values are stated in the Source Data.

Figure 2. Decellularization and biomolecular characterization of human small intestinal and colon scaffolds

a,b, Representative images of SI [patient 1] (a) and colon [patient 18] (b) samples before (native) and after decellularization. Top, macroscopic images; middle, H&E histological images; bottom, immunofluorescent staining using the indicated antibodies. Scale bars: top, 1cm; middle, 200μm; bottom, 100μm. c, Representative scanning electron micrographs of decellularized SI [patient 1] and colon [patient 18] scaffolds highlighting the microarchitecture of the mucosa (Mu) submucosa (S) and muscularis (M). Yellow arrowheads indicate intestinal crypts. The red arrow head indicates villi structure present on the SI scaffold. Scale bars: top, 100μm; bottom, 10μm. d, Raman spectral analysis of comparable histological regions (mucosa (Mu), submucosa (S) and muscularis (M)) of the native tissue (blue lines) and decellularized scaffolds (red lines) of SI [patient 1] (left) and colon [patient 18] (right) samples. Peaks at 726 and 780 cm^-1 were assigned to ring breathing vibrations of nucleic acids whilst peaks at 1078 and 1303 cm^-1 were assigned to δ(CH₂) and v(C-C)/v(C-O) modes of lipids. e, False colored heat maps representing direct classical least squares component analysis of Raman maps using previously acquired reference spectra of purified biomolecules. Distinct spatial distribution of Phenylalanine (PHE), Collagen (COL) and Glycosaminoglycans (GAGs) in SI [patient 1] and colon [patient 18] scaffolds is shown. Scale bars represent 50μm. Images representative of 2 experiments. f, Score plot from principal component analysis differentiates the distinct Raman biochemical spectral profiles of each distinct histological layer of the SI [patient 1] (blue) and colon [patient 18] scaffolds (red). Images are representative of at least 3 (a-c) or 2 (e) independent decellularization experiments. g, Venn diagrams showing total and extracellular proteins detected in the SI [patients 4, 11, 12, 13] and colon [patients 2, 15, 16, 17] scaffolds by mass spectrometry. Proteomics data represents samples from 4 biologically independent patient samples in each group.

Figure 3. Bioengineering functional human jejunal mucosal grafts in vitro

a, Schematic outline of the scaffold seeding strategies using a bioreactor circuit. The timeline shows seeding of each cellular component onto the scaffolds and the time periods in static and dynamic cultures (top). The bioreactor circuit design and all individual components are indicated (bottom). b, Representative H&E staining of a jejunal graft harvested at day 11, showing a monolayer of epithelial cells with invaginating crypt compartments marked by black arrowheads [colon scaffold - patient 2] (left, overview; right, close up). c,d, Representative histological and immunostaining images of jejunal grafts reconstructed using human SI scaffolds [patient 13] (c) or human colon scaffolds [patient 16] (d) at day 18. New matrix deposition is shown by newly synthesized collagen (white asterisks). Arrowheads indicate AB-PAS-positive goblet cells. Scale bars represent 50μm. e, Representative electron micrographs of a jejunal construct [SI scaffold - patient 5] showing microvilli (MV) (top left and right); basement membrane with basal lamina (BL) and reticular lamina (RL) at the scaffold (Sc) border (bottom left); Goblet cell (G) with mucous vesicles indicated by the orange arrow head (bottom middle) and Paneth cell (P) with secretory vesicles indicated by the green arrow head (bottom right). Scale bars represent 5μm. f, Representative immunofluorescent images of a jejunal graft seeded on human SI scaffolds [patient 5] using the indicated antibodies. Scale bars represent 50μm. g, Timeline indicating the experimental sampling (marked by each colored symbol) of jejunal grafts for functional analyses along the course of the in vitro culture period. h, Immunofluorescent staining showing β-AMCA peptide (red) uptake on a jejunal graft (piglet SI scaffold). Phalloidin staining (green) indicates epithelial cell boundaries. Scale bars represent 30μm. i-m, Functional analysis using jejunal grafts seeded on human colon scaffolds (green lines), human SI scaffolds (red lines), piglet scaffold (blue lines) or unseeded blank scaffolds (black lines). i, Dipeptidyl protease IV activity, as measured by nitroaniline release from the grafts [colon scaffolds - patients 19 & 20]. j, Disaccharidase enzyme activity, as measured by rising glucose concentrations following a sucrose challenge (solid lines) or PBS control (dashed lines) to the grafts [SI scaffolds - patients 11 & 12; colon scaffold - patient 2; piglet scaffold]. k, Barrier function, as measured by FITC-Dextran leakage percentage through jejunal grafts (piglet scaffold) from “luminal side” to “serosal side”. Blank scaffolds show an average of 61% baseline leakage as indicated by dashed line. l, Citrulline concentrations measurement in supernatant collected from the indicated graft cultures [SI scaffolds - patients 11 & 12; colon scaffold - patient 2; piglet scaffold]. All organoids used in this figure originate from patient 2. For all functional assays, data represents mean ± s.e.m. of 3 independently cultured jejunal grafts. Images are representative of 4 (b), 3 (c-f) and 2 (h) graft culture experiments.

Figure 4. Characterization of the engineered jejunal graft following in vivo transplantation

a-g, Kidney capsule transplantation model, 1-week. a, Macroscopic image of the kidney harvested after implantation of a jejunal graft in the kidney capsule. b,c, Histology of a transplanted jejunal graft as analyzed by H&E and human nucleoli staining. d, 3D volume rendered model of the jejunal graft structure after transplantation under the kidney capsule. e-g, Representative immunofluorescent images of transplanted jejunal grafts using the indicated antibodies. h-o, Subcutaneous transplantation model, 1 week (h-i,k-o) or 2 weeks (j). h, Top left, schematic representation of the luciferase-GFP reporter plasmid used to label jejunal organoids (bottom left). Scale bar showing bioluminescent signal intensity (middle). Seeded grafts using labelled organoids were detected using live in vivo bioluminescent imaging of mice in the subcutaneous transplantation model (right). i, Representative histology and immunostaining of transplanted jejunal grafts. AB-PAS, Alcian Blue - Periodic Acid Schiff. j, Quantification of the formation of lumens within jejunal grafts following subcutaneous implantation in Teduglutide-treated versus control mice, measured as the percentage of sections with a lumen per graft. Data represents mean ± s.e.m of n = 4 independently transplanted jejunal grafts at 2 weeks post-transplantation; Unpaired t-test, *P 0.0271. k, Representative histology of a transplanted human jejunal graft indicating the mucosa, submucosa and muscularis structure of the graft. Arrowhead indicates the epithelial layer of polarized columnar jejunal cells. l, Immunostaining analyses of cell proliferation on the scaffold, as indicated by PCNA (top) and EdU (bottom) staining. Arrowhead indicates the jejunal epithelium. m, Electron microscopy analysis identifying mucous granules of goblet cells in the native intestine (top left) and a jejunal construct (bottom left and right). n,o, Representative immunostaining of a jejunal graft formed using epithelial markers (pancytokeratin and E-cad), stromal marker (vimentin) and jejunal brush border enzyme (SI). Grafts in a-i were formed using piglet SI scaffold. Grafts in j were formed using human colon scaffolds from patient 2. Grafts in k-o were formed using human SI scaffolds from patient 1. All organoids and fibroblasts used in this figure originate from patient 2. Images shown are representative of 3 transplanted grafts (a-c, e-g), 6 transplanted grafts (h,i) and 2 transplanted grafts (k-o). All scale bars represent 50μm unless specified otherwise. See Source Data.