A xenogeneic-free system generating functional human gut organoids from pluripotent stem cells

Abstract

Functional intestines are composed of cell types from all 3 primary germ layers and are generated through a highly orchestrated and serial developmental process. Directed differentiation of human pluripotent stem cells (hPSCs) has been shown to yield gut-specific cell types; however, these structures do not reproduce critical functional interactions between cell types of different germ layers. Here, we developed a simple protocol for the generation of mature functional intestinal organoids from hPSCs under xenogeneic-free conditions. The stem cell-derived gut organoids produced here were found to contain distinct types of intestinal cells, including enterocytes, goblet cells, Paneth cells, and enteroendocrine cells, that were derived from all 3 germ layers; moreover, they demonstrated intestinal functions, including peptide absorption, and showed innervated bowel movements in response to stimulation with histamine and anticholinergic drugs. Importantly, the gut organoids obtained using this xenogeneic-free system could be stably maintained in culture for prolonged periods and were successfully engrafted in vivo. Our xenogeneic-free approach for generating gut organoids from hPSCs provides a platform for studying human intestinal diseases and for pharmacological testing.

Figure 1. Generation of peristaltic gut organoids from human pluripotent stem cells on a patterning substrate.

(A) Illustration of gut organoid generation from human pluripotent stem cells cultured on a single device. (B) A micropatterned substrate for cell growth was produced by coating a small mesh net, 1,500 μm in diameter (left panel), with a bioactive polymer (scale bar: 15 mm). The right panel shows that human embryonic stem cells attached and grew only within each zone (scale bar: 500 μm). (C) Time course of organoid growth in culture. The organoid at day 63 is surrounded by intestinal columnar epithelium layers adjacent to mesenchymal tissues; these epithelial layers are delineated by the dotted white line (high-magnification view of the area within the red box). Scale bar: 200 μm (top row and right bottom); 50 μm (left bottom). (D) Six organoids that exhibited gut-like motor activity were maintained in culture at day 129 and did not coalesce. Scale bar: 5 mm.

Figure 2. Characterization of developing gut organoids.

(A) Analysis of expression of cell biomarker genes in differentiated human embryonic stem cells (hESCs) (SEES1 cells). Values were normalized against GAPDH. The data are reported as mean ± SEM. Statistically significant differences were identified between embryonic stem (ES) vs. day 7, ES vs. day 14, and ES vs. day 21 using Student’s t test (**P < 0.01) (n = 3–6). (B) H&E staining of organoids with peristaltic movement at day 100. Scale bar: 2 mm (left); 200 μm (right). (C) Alcian Blue staining of differentiated organoids (day 60) indicating goblet-like cells (white arrowheads). Scale bar: 100 μm. (D) Relative expression of cell biomarker genes in differentiated day 50 organoids and human adult small intestine. Statistical analysis was performed using a t test or a Mann-Whitney rank-sum test (*P < 0.05, **P < 0.01). The data are reported as means (%) ± SEM and were obtained from 3 independent experiments (n = 3–4).

Figure 3. Characterization of gut organoids and detection of LGR5-EGFP–positive cells during gut organogenesis.

(A) Day 50–60 organoids from human embryonic stem cells (hESCs) (SEES1 cells) immunostained with markers for intestinal differentiation: villin, leucine-rich repeat containing G protein–coupled receptor 5 (LGR5), CDX2, E-cadherin (ECAD), chromogranin A (CGA), mucin-2 (MUC2), defensin α-6, Paneth cell–specific (DEFA6), α-smooth muscle actin (SMA), and protein gene product 9.5 (PGP9.5). Cell nuclei were counterstained with DAPI. PGP9.5-positive enteric neuronal cells within the α-SMA–positive myenteric area are indicated by yellow arrowheads. Scale bar: 50 μm (VILLIN, LGR5, SMA); 100 μm (ECAD and MUC2). (B) An enterocyte with a characteristic brush border (left), and Paneth cells with secretory granules (black arrowhead) and goblet cells containing mucin granules (yellow arrowhead) (right). Scale bar: 10 μm (left); 5 μm (right). (C) Organoids that developed from SEES1 cells expressed EGFP under the LGR5 promoter (green), indicating that they were LGR5-positive gut organoids. Gut tube-like architecture in a day 34 organoid (red square; high-magnification view, white square) (top row). A small number of EGFP-positive cells were detected in the day 34 organoid (white square) by fluorescence microscopy (bottom left). The number of EGFP-positive cells increased at day 41 (bottom right). Scale bar: 300 μm (top left), 100 μm (top right and bottom row).

Figure 4. Contractility of gut organoids.

(A) Immunohistochemical staining for α-smooth muscle actin (SMA) in a day 60 gut organoid. Scale bar: 200 μm. (B) Immunostaining for the intestinal cells of Cajal. CKIT and S-100 double-positive cells within the myenteric and submucosal plexuses (white arrowheads). Scale bar: 50 μm. (C) The distribution of the neurotransmitter serotonin, an enteroendocrine cell marker, in a representative hESC-derived day 60 gut organoid. Serotonin-positive cells were observed in the lining epithelium and displayed a triangular shape (yellow arrowheads). Scale bar: 100 μm. (D) Contractile activity of the motile gut organoid in response to pharmacological agents (Supplemental Video 2). The aspect ratios are based on the ratio of the longest diameter to the shortest diameter of the organoid, which were calculated for each frame. The original video was recorded at 30 frames per second. The playback speed of the video was 20 times actual speed. Waves of constant contractions were observed before treatment (frames 1–160), histamine treatment increased the frequency of gut organoid contractile activity in frequency, and atropine treatment decreased contraction amplitude and frequency. (E) Nonmotile organoids (n = 6) did not show contractions in response to histamine (Supplemental Video 3). The original video was recorded at 30 frames per second. (F) Human intestine tissue and motile and nonmotile organoids were immunostained for histamine H1 receptor. Cell nuclei were counterstained with DAPI. Nonmotile organoids showed positive staining in the epithelial and mesenchymal areas. Scale bar: 20 μm.

Figure 5. Absorptive functions of gut organoids.

(A) The expression of the intestinal oligopeptide transporter (PEPT1) and major ATP-binding cassette (ABC) transporters ABCB1 and ABCG2 in hESC-derived day 50 gut organoids was analyzed by qRT-PCR and compared to that in the healthy adult small intestine. Statistical analysis was performed using a t test or a Mann-Whitney rank-sum test (**P < 0.01). Data are shown as means (%) ± SEM; n = 3. (B) Gut organoids were treated with fluorophore-conjugated dipeptide β-Ala-Lys-AMCA with or without angiotensin-converting enzyme inhibitor captopril (top row and bottom left). The gut organoids clearly showed dipeptide uptake (top right) inhibited by captopril (top left). Gut organoids are visible in the the bright-field image (top left), the fluorescence image (top right), and the combined bright-field and fluorescence image (bottom left) (n = 3 for each group). Schematic diagram of the peptide absorption assay (bottom right). Scale bar: 200 μm. (C) To quantify the uptake of β-Ala-Lys-AMCA, the AMCA-related signals in gut organoids cultured with or without 10 μM, 100 μM, and 1 mM of captopril were observed using a fluorescence microscope BZ-X710 (Keyence) equipped a top-stage incubator (5% CO₂ at 37°C) (images on the right), and the fluorescence signal intensity was quantified by using Hybrid Cell Count BZ-H3C (Keyence). The results did not show dose dependency. All sample images were recorded under standard conditions. Each concentration assay was performed with 3 biologically independent replicates.

Figure 6. CFTR transport activity of gut organoids.

(A) Day 115 gut organoids from hESCs (SEES1 cells) immunostained with markers for the cystic fibrosis conductance regulator (CFTR). CFTR was present in the epithelial layers of the gut organoids as well as in the control human intestine. CFTR (green), actin (red), and DAPI (blue). Scale bar: 100 μm. (B) Quantification of forskolin-induced gut organoid swelling. Gut organoids (green) derived from SEES1 cells were monitored by time-lapse fluorescence laser confocal microscopy (Keyence). The organoid surface area (red) was quantified using Hybrid Cell Count BZ-H3C (Keyence). The normalized total organoid surface area was calculated and averaged from 3 individual wells per treatment (n = 3 for each group). A forskolin-induced organoid (19 minutes) was overlaid with a pretreated organoid (0 minutes; 569 μm).