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Host Response and Inflammation

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

Jessica L. Forbester, David Goulding, Ludovic Vallier, Nicholas Hannan, Christine Hale, Derek Pickard, Subhankar Mukhopadhyay, Gordon Dougan
B. A. McCormick, Editor
Jessica L. Forbester
aWellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, United Kingdom
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David Goulding
aWellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, United Kingdom
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Ludovic Vallier
aWellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, United Kingdom
bWellcome Trust-Medical Research Council Stem Cell Institute, Anne McLaren Laboratory, Department of Surgery, West Forvie Site, University of Cambridge, Cambridge, United Kingdom
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Nicholas Hannan
bWellcome Trust-Medical Research Council Stem Cell Institute, Anne McLaren Laboratory, Department of Surgery, West Forvie Site, University of Cambridge, Cambridge, United Kingdom
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Christine Hale
aWellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, United Kingdom
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Derek Pickard
aWellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, United Kingdom
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Subhankar Mukhopadhyay
aWellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, United Kingdom
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Gordon Dougan
aWellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, United Kingdom
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B. A. McCormick
Roles: Editor
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DOI: 10.1128/IAI.00161-15
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    FIG 1

    A1ATD-1 hIPSC-derived intestinal organoids show increased expression of genes for markers of adult intestinal tissue. (A) At differentiation day 30, the expression of adult intestinal markers CHGA (enteroendocrine cells), LYZ (Paneth cells), MUC2 (goblet cells), and VIL1 (enterocytes) was significantly greater in A1ATD-1 iHOs than in A1ATD-1 hIPSCs (P = 0.0023, P = 8.41 × 10−5, P = 0.002, and P = 4.4 × 10−5, respectively). (B) Primers were validated by measuring the expression of the same markers in RNA isolated from human ileum. (C) Log fold change in the expression of pluripotency markers. The expression of NANOG and POUF51 was significantly lower in A1ATD-1 iHOs on both days 25 (25d) (P = 4.64 × 10−5 and P = 1.99 × 10−3) and 55 (55d) (P = 3.55 × 10−5 and P = 3.63 × 10−4) than in their A1ATD-1 hIPSC progenitors. Each RT-qPCR was performed with a TaqMan gene expression assay specific for each gene and analyzed via the comparative CT method with GAPDH as an endogenous control. *, P < 0.05; **, P < 0.001; ***, P < 0.0001.

  • FIG 2
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    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.

  • FIG 3
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    FIG 3

    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 CT 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.

  • FIG 4
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    FIG 4

    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).

  • FIG 5
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    FIG 5

    S. Typhimurium SL1344 invA mutant is deficient in the invasion of epithelial cells of iHOs. A1ATD-1 iHOs were microinjected with wild-type (WT) or invA mutant SL1344 and incubated at 37°C and 5% CO2 for 90 min. (A) Log numbers of CFU per milliliter recovered from cells of iHOs after microinjection and modified gentamicin protection assay, showing that WT SL1344 is significantly (∼30×) more invasive than the invA mutant (P = 0.0092). (B) Fluorescence staining of GMA JB-4- and benzoyl peroxide-fixed sections for CSA-1 (green) and LAMP-1 (red) and an overlay of the two images showing the colocalization (yellow) of bacteria and host protein after invasion by WT SL1344. invA mutant SL1344 was distinguishable from WT SL1344 by its lack of induction of LAMP-1 expression.

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    • Supplemental file 1 -

      Dataset S1. Top 100 genes significantly upregulated after microinjection of Salmonella Typhimurium SL1344 into iHOs, accompanied by gene symbols, gene names, significance P values, and raw counts in stimulated and unstimulated iHOs. Dataset S2. Top 50 biological processes identified using innate DB enriched in A1ATD-1 iHOs after microinjection of A1ATD-1 iHOs with S. Typhimurium SL1344. Dataset S3. Top 100 genes significantly upregulated after stimulation by Salmonella Typhimurium SL1344 in iHOs, accompanied by gene symbols, gene names, significance P values, and raw counts in stimulated and unstimulated iHOs. Dataset S4. Top 50 biological processes identified using innate DB enriched in A1ATD-1 iHOs after stimulation of A1ATD-1 iHOs with S. Typhimurium SL1344.

      XLSX, 41K

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Interaction of Salmonella enterica Serovar Typhimurium with Intestinal Organoids Derived from Human Induced Pluripotent Stem Cells
Jessica L. Forbester, David Goulding, Ludovic Vallier, Nicholas Hannan, Christine Hale, Derek Pickard, Subhankar Mukhopadhyay, Gordon Dougan
Infection and Immunity Jun 2015, 83 (7) 2926-2934; DOI: 10.1128/IAI.00161-15

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Interaction of Salmonella enterica Serovar Typhimurium with Intestinal Organoids Derived from Human Induced Pluripotent Stem Cells
Jessica L. Forbester, David Goulding, Ludovic Vallier, Nicholas Hannan, Christine Hale, Derek Pickard, Subhankar Mukhopadhyay, Gordon Dougan
Infection and Immunity Jun 2015, 83 (7) 2926-2934; DOI: 10.1128/IAI.00161-15
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