Interaction of Salmonella enterica Serovar Typhimurium with Intestinal Organoids Derived from Human Induced Pluripotent Stem Cells

A1ATD-1 iHOs display a morphology characteristic of the human intestine. (A) Time course showing that directed differentiation of hIPSCs with defined concentrations of combinations of activin A, FGF, BMP-4, LY294002, and CHIR99021 results in the formation of a definitive endoderm at day 4. After 8 days, patterning of this definitive endoderm with defined concentrations of CHIR99021 and retinoic acid results in the formation of hindgut. Placement of this hindgut into a 3D intestinal culture system consisting of a supporting matrix of Matrigel and medium supplemented with prointestinal proliferation factors R-Spondin 1, Noggin, EGF, CHIR99021, and prostaglandin E2 results in the formation of small spheroids. Sustained maintenance and passaging of these spheroids result in the formation of established iHO cultures. Images were taken at ×100 magnification. (B) Transmission electron micrograph of established A1ATD-1 iHO wall demonstrates polarization of epithelial cells with a distinct luminal side (L) and microvilli (MV). (C) Transmission electron micrograph showing the presence of structures closely resembling tight junctions between cells (ZO, zonula occludens; ZA, zonula adherens; MA, maculae adherens). (D) Immunohistochemistry with marker-specific antibodies for mucin 2 (goblet cells), lysozyme (Paneth cells), and chromogranin A (enteroendocrine cells) validates the presence of intestinal secretory cell lineages in the iHO ultrastructure.

Microinjection of A1ATD-1 iHOs with S. Typhimurium SL1344 results in upregulation of genes associated with infection and inflammation. After at least 6 weeks in culture, A1ATD-1 iHOs were microinjected with a mixture of phenol red dye and S. Typhimurium SL1344 with the Eppendorf TransferMan NK2-FemtoJet express system. iHOs were incubated at 37°C and 5% CO₂ for 3 h, phenol red-marked organoids were isolated, and RNA was prepared. (A) PCA of RNA-Seq expression data for three biological replicates per condition illustrates distinct differences in gene expression patterns between iHOs stimulated with SL1344 and unstimulated iHOs. (B) Heat map of the RNA-Seq expression data calculated with DESeq2 for the 30 most significantly upregulated genes after the addition of SL1344 to A1ATD-1 iHOs. (C) Enriched biological processes upregulated after stimulation of A1ATD-1 iHOs with S. Typhimurium SL1344 (for the genes associated with each pathway and P values, see Data Set S1 in the supplemental material). (D) RT-qPCR showing that transcripts for the cytokines IL-8, CXCL2, IL23A, TNF-α, and IL1B are significantly upregulated in iHOs injected with SL1344 in comparison to those in iHOs injected with PBS (P = 1.56 × 10⁻⁹, P = 2.35 × 10⁻⁹, P = 4.21 × 10⁻⁸, P = 1.28 × 10⁻⁴, and P = 6.42 × 10⁻⁴, respectively). RT-qPCR data were analyzed via the comparative C_T method with GAPDH as an endogenous control. (E) After stimulation of iHOs with S. Typhimurium SL1344, induction of TNF-α, IL-6, and IL-8 production was shown to be significantly upregulated with Luminex cytokine assays of supernatants collected from stimulated and unstimulated iHOs (P = 3.34 × 10⁻⁴, P = 6.33 × 10⁻⁷, P = 3.89 × 10⁻⁴, respectively). Assays were performed with supernatants from three biological replicates.

Microinjection of organoids derived from hIPSCs provides a model of S. Typhimurium SL1344 infection. Overnight cultures of S. Typhimurium SL1344 were diluted 1:1 with phenol red and injected into the lumen (L) of A1ATD-1 iHOs with the Eppendorf TransferMan NK2-FemtoJet express system. (A) iHOs retained the inoculum for 3 h subsequent to injection. Arrowheads mark the interface between the bacterial inoculum and human cells. (B and C) Transmission electron micrographs 3 h after injection showing SL1344 populating the lumen of the iHO and residing inside the epithelial cells in SCVs after invasion (arrow) (B; enlarged in panel C).