In vitro expansion of human gastric epithelial stem cells and their responses to bacterial infection

Abstract

Background & aims: We previously established long-term, 3-dimensional culture of organoids from mouse tissues (intestine, stomach, pancreas, and liver) and human intestine and pancreas. Here we describe conditions required for long-term 3-dimensional culture of human gastric stem cells. The technology can be applied to study the epithelial response to infection with Helicobacter pylori.

Methods: We generated organoids from surgical samples of human gastric corpus. Culture conditions were developed based on those for the mouse gastric and human intestinal systems. We used microinjection to infect the organoids with H pylori. Epithelial responses were measured using microarray and quantitative polymerase chain reaction analyses.

Results: Human gastric cells were expanded indefinitely in 3-dimensional cultures. We cultured cells from healthy gastric tissues, single-sorted stem cells, or tumor tissues. Organoids maintained many characteristics of their respective tissues based on their histology, expression of markers, and euploidy. Organoids from healthy tissue expressed markers of 4 lineages of the stomach and self-organized into gland and pit domains. They could be directed to specifically express either lineages of the gastric gland, or the gastric pit, by addition of nicotinamide and withdrawal of WNT. Although gastric pit lineages had only marginal reactions to bacterial infection, gastric gland lineages mounted a strong inflammatory response.

Conclusions: We developed a system to culture human gastric organoids. This system can be used to study H pylori infection and other gastric pathologies.

Keywords: Gastric Epithelium; Primary Cells; Stomach Cancer; Tissue Engineering.

Figure 1. Human gastric cultures expand in vitro

(A) Scheme and image of gland isolation. (B) Nicotinamide (Nic) increases formation of organoids, while p38 inhibitor and TGFβ inhibitor do not. Bars represent average of triplicates of one culture with standard deviation. (C) Effect of several growth factors and inhibitors on the human gastric culture. TGFβi increases lifespan of organoids up to 30 weeks. (D) Removal of any of the factors EGF, noggin, R-spondin1, Wnt, FGF10, gastrin and TGFβi limits the growth of the culture. Removal of nicotinamide enables long term growth (> 1 year). In B and D, each bar represents a newly established culture. (E) Example of an organoid growing from a human gland. The insert shows a budding structure. (F) Karyogram after 3 months in culture. Scale bars 100 µm.

Figure 2. Human gastric organoids differentiate into four gastric lineages and self-organize into gland and pit domains

(A) mRNA expression of gastric marker genes. LGR5 was used as marker for stem cells, MUC5AC for gastric pit mucous cells, PGC for chief cells, SST for enteroendocrine cells, MUC6 for gland mucous cells. Gastric trefoil factors 1 (TFF1) and TFF2 are also expressed. Housekeeping gene GAPDH was used as loading control. MUC2, CDX1 and CDX2 are markers of intestinal tissue and were used to control for tissue specificity. (B) Images of stained paraffin sections of gastric mucosa and organoids. Periodic acid-Schiff (PAS) staining also marks pit mucous cells. (C) Confocal images of wholemount organoids. (D) Proliferating cells are labeled by 5-ethynyl-2’-deoxyuridine (EdU) incorporation. Scale bar 100 µm. Culture condition: ENRWFG_Ti.

Figure 3. Directed differentiation of organoids into pit or gland cell lineages

(A) Examples of the phenotypical change upon Wnt withdrawal. Cultures were grown in ENRWFGNiTi for 10 days and subsequently either kept with Wnt (left panel) or without Wnt (right panel) for 4 days. Cultures under Wnt withdrawal loose budding structures and become large cysts. (B) Differential gene expression in the two growth conditions measured by genome-wide microarray of cultures from 3 donors. Removal of Wnt reduces expression of stem cell markers (LGR5, TROY), and increases expression of MUC5AC. (C) Quantitative PCR of known Wnt-response genes and stomach specific genes. mRNA was normalized to GAPDH housekeeping gene. Bars represent normalized average of triplicates with standard deviation. (D) PCR of gastric and intestinal markers. Intestinal markers MUC2, CDX1 and CDX2 are not expressed. (E) Images of stained paraffin sections show differential expression of MUC5AC and MUC6. PGC is expressed in both types of organoids. (F) Scheme of gastric gland and the two types of organoids. In all experiments organoids were grown as described in A. Scale bar 100 µm.

Figure 4. Single stem cells from human gastric mucosa can form gastric organoids

(A) Scheme of isolation and FACS plot. FACS was used to generate single cells based on FSC. (B) Example of an organoid growing from a single cell. (C) Example of two single cell clones that were cultured for 1 year (here the first 3 passages = 6 weeks). (D) mRNA expression of gastric marker genes. Intestinal markers MUC2, CDX1 and CDX2 are not expressed and confirm tissue specificity. (E) Images of stained paraffin sections of organoids. MUC5AC expression increases after Wnt removal. Culture condition: ENRWFGNiTi (“Wnt”) or 4 days of Wnt withdrawal (“no Wnt”). Scale bar 100 µm.

Figure 5. Normal and tumor organoids

(A) Brightfield image of tumor organoids (left) and normal organoids (right) established from the same patient. (B) Metaphase spread the tumor organoids shown in A. Counts of 7 spreads showed 70–160 chromosomes. (C) Images of stained paraffin sections of the original tumor tissue and organoids shown in A. (D) Organoids from tumor are insensitive to nutlin-3. Organoids were grown for 1 week with indicated concentrations of nutlin-3. Cell viability was assessed using luciferase assay. Bars represent average of triplicates with standard deviation. Scale bar 100 µm

Figure 6. Primary response of gastric organoids to H. pylori infection

(A) Scheme and brightfield image of microinjection of H. pylori into an organoid (left). Confocal image of infected organoid (Lumen infected, LI) and non-infected organoid (NI) (right). Bacteria were visualized with using GFP-expression, organoids were counterstained with E-Cadherin (red) and DAPI (blue). (B) Electron microscopy demonstrates close interaction of bacteria with epithelial cells. Arrows indicate the binding site (C) Differential gene expression upon infection of human gastric organoids. Genome-wide microarray was performed on 3 independent experiments using organoids derived from 3 patients. (D) Confocal images of infected (LI) and non-infected organoids (NI). Staining for NF-κB subunit p65 (red) indicates nuclear p65 in the infected organoid. (E) qPCR for IL-8 mRNA after injection of H. pylori or medium (mock). Bars represent average of duplicates with standard deviation. (F) Organoids were differentiated into gland-type organoids (with Wnt) or pit-type organoids (4 days Wnt withdrawal) and subsequently microinjected with H. pylori. After indicated time, mRNA was extracted and expression of IL-8 was measured with quantitative real time PCR. Bars represent average of duplicates with standard deviation. LI = Lumen infected organoid, NI = Noninfected organoid. Scale bar 200 nm (C) or 100 µm (B, E). * = Matrigel.