Development of Human Gut Organoids With Resident Tissue Macrophages as a Model of Intestinal Immune Responses.

Intestinal macrophages are largely responsible for the innate immune response and also for intestinal homeostasis. We have developed novel xenogeneic-free human intestinal organoids (XF-HIOs) that are uniquely structured with an apical-out mucosal epithelium and complex mesenchymal tissue, including smooth muscle and intestinal nerve cells., To further develop XF-HIOs containing tissue macrophages, we first prepared human-induced pluripotent stem cell (hiPSC)-derived monocyte-like cells (pMCs). These were directly injected into the cystic cavity of an XF-HIO, followed by differentiation into macrophage-like cells (pGMACs) within XF-HIOs in the presence of macrophage colony-stimulating factor (M-CSF) (Figure 1A). We prepared macrophages/monocytes derived from an enhanced green fluorescent protein (EGFP)–hiPSC line, which constitutively expressed EGFP (Supplementary Figure 1A). The pGMACs were observed to evenly disperse inside the XF-HIOs, and image analysis showed pGMACs with short-elongated projections (Figure 1B). Immunofluorescence staining also revealed that ionized calcium-binding adapter molecule 1 was detectable in the monocyte (MC)-XF-HIOs; however, CD14 was not (Figure 1C). This staining pattern is similar to that observed in human intestinal macrophages., We also identified that ionized calcium-binding adapter molecule 1 and C-X3-C motif chemokine receptor 1 were co-localized in MC-XF-HIOs (Supplementary Figure 1B and C).

Figure 1

Establishment of hiPSC-derived gut organoid residing macrophages. (A) Human iPSC-derived monocyte-like cells (pMCs) were transplanted into human intestinal organoids (XF-HIOs) and then treated with macrophage colony-stimulating factor to differentiate into monocyte-XF-HIOs (MC-XF-HIOs). Scale bars: 500 μm. (B) Macrophages from enhanced green fluorescent protein labeled human-induced pluripotent stem cells (EGFP–hiPSCs) were dispersed in the organoids (1, 2), and image analysis revealed short-elongated projections (red arrowheads) of gut macrophages (pGMACs) in MC-XF-HIOs. (3). Scale bars: black, 500 μm; white, 100 μm; yellow, 20 μm. (C) Immunostaining for macrophage-specific marker IBA1 merged with EGFP-hiPSCs (white arrowheads) but not CD14 (yellow arrowheads). Scale bars: white, 30 μm; yellow, 200 μm. (D) Representative views of pGMACs by transmission electron microscopy showed a characteristic large nucleus, phagocytic vacuoles, and short pseudopodia (white arrowheads). Scale bar: 5 μm.

Supplementary Figure 1

Characterization of hiPSC-derived macrophage-integrated gut organoids. (A) Enhanced green fluorescent protein (EGFP)–human-induced pluripotent stem cells (hiPSCs), which constitutively expressed EGFP under a cytomegalovirus promoter, were cultured under feeder-free conditions in StemFlex medium (1) and differentiated into monocyte-like cells (pMCs) (2). Scale bars: black, 300 μm; white, 100 μm (2) (B) Immunofluorescence staining for ionized calcium-binding adapter molecule 1 (IBA1) in human intestine. Scale bar, 100 μm. (C) Immunofluorescence staining for IBA1 and CX3CR1 in MC-XF-HIOs derived from a non-EGFP hiPSC line (Edom-iPSCs). Scale bar: 50 μm. (D) Immunostaining for caudal type homeobox 2 (CDX2), villin, zonula occludens-1 (ZO-1), E-cadherin (ECAD), glycoprotein 2 (GP2), mucin 2 (MUC2), defensin alpha 6 (DEFA6), protein gene product 9.5 (PGP9.5), and smooth muscle actin (SMA). PGP9.5-positive enteric neuronal cells were surrounded by SMA-positive mesenteric tissue in MC-XF-HIOs. Cell nuclei were counterstained with 4′, 6-diamidino-2-phenylindole, dihydrochloride (DAPI). Scale bars, 100 μm. Anti-CDX2 (1:1000, ab76541; Abcam), anti-villin (1:50, sc-7672; Santa Cruz Biotechnology, Dallas, TX), anti-GP2 (1:1000, HPA016668; Sigma-Aldrich), anti-MUC2 (1:50, sc-7314; Santa Cruz Biotechnology), anti-PGP9.5 (1:10, ab8189; Abcam), anti-SMA (1:400, A2547; Sigma-Aldrich), anti-ECAD (1:50, 610181; BD Pharmingen, San Diego, CA), anti–ZO-1 (1:100, 40-2200; Invitrogen), and anti-DEFA6 (1:500, HPA019462; Sigma-Aldrich) were used as primary antibodies.

Furthermore, the presence of pGMACs under the epithelium of each organoid as indicated by zonula occludens-1 staining was also observed in a three-dimensional image (Supplementary Videos 1A and B). Transmission electron microscopy of a section of MC-XF-HIO showed a pGMAC displayed phagocytic vacuoles, a large nucleus, and several short pseudopodia (Figure 1D). The MC-XF-HIOs have an intestinal tissue structure composed of apical-out epithelial and mesenchymal cells with neuronal cells (Supplementary Figure 1D) and also showed peristaltic-like movements (Supplementary Video 1C), as previously demonstrated in XF-HIOs. By sectionalizing their supernatant and fluid contents, this enabled us to mimic human intestinal physiological conditions in vitro. To assess the abilities of MC-XF-HIOs to produce and secrete soluble cytokines and chemokines, we investigated the fluid content (FC) of the organoids using a bead-based Multiplex cytokine assay (Figure 2A). Consistent with the intestinal epithelial barrier (Supplementary Figure 1D), several differences were apparent in the amounts of soluble cytokines in the FC of MC-XF-HIOs (Supplementary Figure 2A). Quantitative reverse transcription polymerase chain reaction (PCR) analysis based on single-cell sorting of pGMACs in MC-XF-HIOs revealed the distinct expression of macrophage polarization markers such as TNF, NOS2, HLA-DB1, IL-6, KLF4, and VEGFA (Supplementary Figure 2B). MC-XF-HIOs expressed pleiotropic types of cytokines. In addition, lipopolysaccharide (LPS) was used as a potential inflammatory stimulus. However, the expression of inflammatory cytokines, except for interleukin 4, did not exhibit a statistically significant change after exposure to LPS (Figure 2B). We showed that LPS induced a strong response in pMCs (Supplementary Figure 2C). Two possible reasons exist for the very low or no responses to LPS observed in MC-XF-HIOs. Macrophages in MC-XF-HIOs are CD14 negative cells (Figure 1C). Resident intestinal macrophages characterized as lacking CD14 did not show enhanced cytokine production by LPS. We observed that toll-like receptor 4 protein was weakly expressed on the apical surface of MC-XF-HIOs (Supplementary Figure 2D). This observation is consistent with a recent report by Price et al, who observed a weaker expression of toll-like receptor 4 in the small intestine in comparison with that in the stomach or colon and very low responses to LPS in human intestinal organoids compared with colon organoids.

Figure 2

Macrophage-related characterization of MC-XF-HIOs. (A) An illustration that indicates the characteristic structure of a MC-XF-HIO compartmentalizing fluid content (FC). (B) Secretions released in the FC fluid of a single XF-HIO or MC-XF-HIO were assayed for selected interleukin (IL) cytokines, and these were quantified. LPS stimulation of organoids for 24 hours; FC samples were then collected. Data represent the mean ± standard error of 3–6 independent gut organoids generated in at least 3 individual experiments in the presence or absence of LPS. Statistical significance was identified using Student t test (∗P < .05, ∗∗P < .01, NS, not significant. (C) EGFP-expressing pGMACs in MC-XF-HIOs demonstrated red fluorescence (white arrowheads) inside the cells after exposure to pHrodo red Escherichia coli bioparticles. Scale bars: white, 300 μm; gray, 100 μm. (D) A diagram of hiPSC-derived MC-XF-HIOs.

Supplementary Figure 2

Cytokine and chemokine profiles of MC-XF-HIOs. (A) Total of 29 cytokines and chemokines in the fluid content (FC) and supernatant fluid (SF) of the organoids were assayed using a bead-based Multiplex cytokine assay. Secretions released in the SF medium and FC fluid of a single xenogeneic-free human intestinal organoid (XF-HIO) or macrophage–xenogeneic-free human intestinal organoid (MC-XF-HIO) were assayed for selected cytokines and chemokines, and these were quantified. Lipopolysaccharide (LPS) stimulation of organoids for 24 hours; SF and FC samples were then collected. Data represent the mean ± standard error of the mean of 3–6 independent gut organoids generated in at least 4 individual experiments in the presence or absence of LPS. (B) Representative fluorescence-activated cell sorting (FACS) images of MC-XF-HIOs disassembled into single cells. Expression of key macrophage polarization markers determined by quantitative reverse transcription PCR: M1 macrophage–associated genes (TNF, NOS2, HLA-DB1); M2 macrophage–associated genes (IL6, KLF4, VEGFA). Relative expression was calculated using the ΔΔCT method, with GAPDH as an endogenous control and normalization to human blood monocytes. Samples as human-induced pluripotent stem cell (hiPSC)–derived monocyte-like cells (pMCs) and differentiated macrophages (pMACs) were generated in independent experiments. Originally injected monocytes (pMCs) in XF-HIOs or co-cultured human pluripotent stem cell–derived gut macrophages (pGMACs) were isolated from 5 MC-XF-HIOs in 3 individual experiments using FACS. The data represent the mean ± standard error, and statistical significance was identified using Student t test (∗P < .05) (n = 3). (C) Monocytes derived from hiPSCs were stimulated with the indicated concentrations of LPS for 24 hours and analyzed for interleukin 6 by quantitative reverse transcription PCR. Each assay was performed with 3 biologically independent replicates. Values of interleukin 6 were normalized against the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The data represent the mean ± standard error, and statistical significance was identified using Student t test (∗P < .05, ∗∗P < .01; n = 3) versus 0 ng/mL LPS as a control. (D) Immunofluorescence staining for toll-like receptor 4 (TLR4) in an MC-XF-HIO. Scale bar: 50 μm.

Next, we assessed the phagocytosis of pGMACs in response to foreign antigens on the epithelium of MC-XF-HIOs using pH-dependent dye labeled Escherichia coli bioparticles. The bioparticles only fluoresced when localized in the acidic environment of the phagolysosome. A magnified image showed red signals detectable within pGMACs and suggested pGMACs existing in the organoid captured bioparticles in acidified phagolysosomes (Figure 2C).

Here we present the development of hiPSC-derived intestinal organoids inhabited by tissue macrophages that model intestinal immune responses in vitro. One of the important features of the MC-XF-HIO system is that both organoids and macrophages are derived from an identical hiPSC line. We further applied this technique to a novel Crohn’s disease model as a potential platform for studying human intestinal inflammatory disorders (Supplementary Figure 3). The MC-XF-HIO culture system we describe here provides a species-specific in vitro model for temporally and spatially investigating interactions between the gastrointestinal tract and intestinal macrophages (Figure 2D). This represents a powerful addition to the repertoire of methods available to research gut homeostasis and the immune system.

Supplementary Figure 3

XF-HIOs and MC-XF-HIOs derived from Crohn’s disease–specific iPSC lines. Crohn’s disease–specific induced pluripotent stem cell (iPS) lines (CD-iPSCs) were derived from patients with Crohn’s disease (CD). HPS1508 and HPS2816 cell lines were derived from 2 separate patients with an ileal form of CD. HPS2054 was derived from a patient with an ileocolic form of CD. These 3 cell lines were confirmed to differentiate into xenogeneic-free human intestinal organoids (XF-HIOs) and PSC-derived monocyte-like cells (pMCs). Macrophage–xenogeneic-free human intestinal organoids (MC-XF-HIOs) were generated from the CD-iPSC lines and each pMC. Hematoxylin-eosin staining of CD-iPSC derived MC-XF-HIOs (CD-MC-XF-HIOs). The CD-MC-XF-HIOs are structured outward and oriented toward the epithelial layers. Scale bar corresponds to 200 μm. The XF-HIOs are structured outward and oriented toward the epithelial layers. White and black scale bars correspond to 500 μm and 200 μm, respectively.