Innate immune signaling in trophoblast and decidua organoids defines differential antiviral defenses at the maternal-fetal interface

  1. Liheng Yang
  2. Eleanor C Semmes
  3. Cristian Ovies
  4. Christina Megli
  5. Sallie Permar
  6. Jennifer B Gilner
  7. Carolyn B Coyne  Is a corresponding author
  1. Department of Molecular Genetics and Microbiology, Duke University School of Medicine, United States
  2. Duke Human Vaccine Institute, Duke University, United States
  3. Division of Maternal-Fetal Medicine, Division of Reproductive Infectious Disease, Department of Obstetrics, Gynecology and Reproductive Sciences, University of Pittsburgh Medical Center (UPMC), United States
  4. Magee Womens Research Institute, United States
  5. Department of Pediatrics, Weill Cornell Medical Center, Duke University Medical Center, United States
  6. Division of Maternal-Fetal Medicine, Department of Obstetrics and Gynecology, Duke University Medical Center, United States
8 figures, 1 table and 1 additional file

Figures

Figure 1 with 2 supplements
Long-term three-dimensional organoids cultures can be established from human mid- and late-gestation placental tissue.

(A) Schematic representation of the derivation of trophoblast organoids (TOs) and decidual organoids (DOs) from mid-to-late gestation placental tissue. (B) Representative bright-field images of TOs …

Figure 1—figure supplement 1
Characterization of trophoblast (TO) and decidua organoids (DO).

(A) Immunohistochemistry in human full-term placental villi tissue used to generate TOs. Shown are no primary controls (left) and representative images of tissue immunostained for cytokeratin-19 …

Figure 1—video 1
Three-dimensional image reconstruction of SDC-1 immunostaining in trophoblast organoids (TOs).

Shown is a three-dimensional image reconstruction of an xz-stack of TOs immunostained for SDC-1 (in green) and counterstained for actin (in red). DAPI-stained nuclei are shown in blue. A single xz …

Figure 2 with 1 supplement
Whole genome transcriptional profiling of trophoblast (TO) and decidua organoids (DO).

(A) Volcano plot demonstrating differentially expressed transcripts in TOs or DOs as assessed by DeSeq2 analysis. Gray circles represent genes whose expression was not significantly changed and red …

Figure 2—figure supplement 1
RNASeq of TOs and DOs.

(A, B) Principal component analysis based on transcriptional profiles from DOs (orange) or TOs (blue). (C) Transcripts on the first and second principal components (PC1, PC2) with the greatest …

Figure 3 with 1 supplement
Basal cytokine and chemokine secretion profiles from matched trophoblast (TO) and decidua organoids (DO).

(A, B) Heatmap of 12 conditioned medium (CM) preparations generated from four established TO (A) and DO (B) lines analyzed by Luminex-based multianalyte profiling for the indicated cytokines, …

Figure 3—figure supplement 1
IL-6 and IFN-λ2 release in TOs over time.

Concentrations of IL-6 (blue) and IFN-λ2 (red) in CM generated from a single TO line throughout the first 25 days of culture.

Trophoblast organoids secrete high levels of antiviral IFN-λ2.

(A, B) Comparison of the levels of the type I IFNs IFN-α2 and IFN-β and type III IFNs IFN-λ1 and IFN-λ2 in conditioned medium (CM) isolated from TOs (A) or DOs (B) as determined by Luminex assays. (C

Figure 5 with 1 supplement
Differential cytokine and chemokine release in trophoblast organoids (TOs) and decidua organoids (DOs) treated with poly I:C.

(A, B) Volcano plots demonstrating differentially expressed transcripts in TOs (A) or DOs (B) treated with poly I:C as assessed by DeSeq2 analysis. Gray circles represent genes whose expression was …

Figure 5—figure supplement 1
Factors induced by poly I:C treatment of TOs and DOs.

(A) Venn diagram denoting unique and shared differentially expressed transcripts in TOs (blue circle) or DOs (orange circle) treated with poly I:C. (B) Heatmap (based on log2 RPKM values) of …

Differential susceptibility of TOs and DOs to HCMV infection.

(A, B) Representative confocal micrographs of DOs (A) or TOs (B) infected with human CMV (HCMV) strains EGFP-tagged AD169r and mCherry-tagged TB40E for ~48 hr. Mock infected shown in top row of (A) …

Transcriptional profiling of HCMV infected trophoblast organoids (TOs) and decidua organoids (DOs).

(A, B) Volcano plots demonstrating differentially expressed transcripts in TOs (A) or DOs (B) infected with HCMV strains AD169r or TB40E (as denoted at top) by DeSeq2 analysis. Gray circles …

Figure 8 with 2 supplements
Differential innate immune responses to HCMV infection of TOs and DOs.

(A, B) Heatmaps demonstrating the induction of factors at left (shown as fold change from mock-treated controls) from TOs (A) and DOs (B) infected with HCMV (AD169r, top or TB40E, bottom strains). …

Figure 8—figure supplement 1
Co-cultures of TOs and DOs.

(A) Black and white images of single channel confocal micrographs used to generate the merged images shown in Figure 8I. (B) Representative over the counter (OTC) pregnancy tests for hCG in two …

Figure 8—figure supplement 2
Differential innate responses in TOs and DOs infeced with HCMV.

(A, B) Heatmaps demonstrating the induction of factors at left (shown as fold change from mock-treated controls from TOs (A) and DOs (B) infected with AD169r (left) or TB40E (right) strains of HCMV …

Tables

Appendix 1—key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
OtherMatrigelCorning356231Growth-factor-reduced
for organoid growth.
OtherTrypLE ExpressGibco12605–028For organoid
dissociation during
passaging.
OtherStem Pro AccutaseGibcoA11105-01For organoid
dissociation during
passaging.
OtherCryoStor CS10 stem cell freezing mediumSTEMCell Technologies07930For organoid
cryopreservation.
OtherTrypsin-250Alfa AesarAlfa AesarFor stem cell
isolation from tissue.
OtherCollagenase VSigma-AldrichC9263-100MGFor stem cell
isolation from tissue.
Otherrecombinant human EGFGibcoPHG0314For organoid
growth media.
Otherrecombinant human R-spondin 1R & D systems4645-RS-100For organoid
growth media.
Otherrecombinant human FGF2Peprotech100–18 CFor organoid
growth media.
Otherrecombinant human HGFPeprotech100–39For organoid
growth media.
OtherITS-X supplementGibco51500–056For organoid
growth media.
OtherKO serum replacementThermoFisher10828010For organoid
growth media.
OtherNRG1Cell Signaling5218SCFor organoid
growth media.
OtherA83-01Tocris2939For organoid
growth media.
OtherY27632Sigma or STEMCell TechnologiesY0503 or 72304For organoid
growth media.
OthernicotinamideSigmaN0636For organoid
growth media.
OtherFBSCytiva HyCloneSH30070.03For organoid
growth media.
Heat inactivated
at 50 oC for 30 min.
OtherAdvanced DMEM/F12 mediumLife Technologies12634–010For organoid
growth media.
Other24-well tissue culture platesCorning3526For organoid plating.
Cell line (human)HEK-Blue IFN-λ reporter cellsInvivoGenhkb-ifnl
OtherHCMV AD169r BADrUL131-Y4T. ShenkPMID: 16051825
OtherHCMV TB40/EN. MoormanPMID: 21307184
Other8-well chamber slideMilliporePEZGS0816For organoid plating.
OtherCell recovery solutionCorning354253
OtherVectashieldVector LaboratoriesH-1200–10Mounting medium for
immunofluorescence.
AntibodyMouse monoclonal Cytokeratin-19Abcamab9221(Immunofluorescence, 1:500)
AntibodyRabbit monoclonal Cytokeratin-19Abcamab52625(Immunofluorescence, 1:500)
AntibodyRabbit polyclonal E-cadherinInvitrogenPA5-85088(Immunofluorescence, 1:500)
AntibodyRabbit monoclonal EpCAMAbcamab223582(Immunofluorescence, 1:500)
AntibodyMouse monoclonal Mucin 5ACAbcamab3649(Immunofluorescence, 1:500)
AntibodyRabbit polyclonal SIGLEC6Abcamab262851(Immunofluorescence,
1:500)
AntibodyMouse monoclonal HLA-GAbcamab52454(Immunofluorescence,
1:500)
AntibodyRabbit monoclonal HLA-GAbcamab283260(Immunofluorescence,
1:500)
AntibodyMouse monoclonal Ki67BD Biosciences550609(Immunofluorescence,
1:500;
Immunohistochemistry,
1:200)
AntibodyRabbit monoclonal SDC-1Abcamab128936(Immunofluorescence,
1:500)
OtherAlexa Fluor 594–conjugated phalloidinInvitrogenA12381For actin staining.
(Immunofluorescence
1:1000)
OtherAlexa Fluor 633–conjugated phalloidinInvitrogenA22284For actin staining.
(Immunofluorescence
1:1000)
OtherAlexa Fluor Plus 488 Goat anti-Mouse IgG secondary antibodyInvitrogenA32723Secondary antibody
for immunofluorescence.
(Immunofluorescence 1:1000)
OtherAlexa Fluor 594 Goat anti-Mouse IgG secondary antibodyInvitrogenA11032Secondary antibody
for immunofluorescence.
(Immunofluorescence 1:1000)
OtherAlexa Fluor 488 Goat anti-Rabbit IgG secondary antibodyInvitrogenA11034Secondary antibody
for immunofluorescence.
(Immunofluorescence 1:1000)
OtherAlexa Fluor 594 Goat anti-Rabbit IgG secondary antibodyInvitrogenA11037Secondary antibody
for immunofluorescence.
(Immunofluorescence 1:1000)
Software, algorithmFijiN/Ahttps://imagej.net/software/fiji/For image analysis,
including three-
dimensional
reconstructions.
Software, algorithmCellSensOlympusN/A
Software, algorithmImarisOxford InstrumentsVersion 9.2.; for
image analysis,
including three-
dimensional
reconstructions.
Software, algorithmCLC GenomicsQiagenVersion 20
Software, algorithmGraphPad PrismDotmaticsVersion 9
OtherVectastain Elite ABC HRP KitVector LaboratoriesPK-6100For immunohistochemistry.
OtherBiotinylated Goat Anti-RabbitVector LaboratoriesBA-1000For immunohistochemistry.
(Immunohistochemistry,
1:250)
OtherBiotinylated Goat Anti-MouseVector LaboratoriesBA-9200For immunohistochemistry.
(Immunohistochemistry,
1:250)
OtherDAB substrateVector LaboratoriesSK-4100For immunohistochemistry.
OtherVectamount Permanent Mounting MediumVector LaboratoriesH-5000For immunohistochemistry.
Commercial assay, kitGenElute total mammalian RNA miniprep kitSigmaRTN70-1KTWith on-column
DNAse treatment
Commercial assay, kitiScript cDNA synthesis kitBiorad1708891N/A
Commercial assay, kitiQ SYBR Green SupermixBiorad1708882N/A
Software, algorithmDeSeq2M.Love et alPMID: 25516281https://bioconductor.org/packages/release/bioc/html/DESeq2.html
Software, algorithmpheatmapRaivo Koldehttps://cran.r-project.org/web/packages/pheatmap/pheatmap.pdf
Software, algorithmpcaExplorerMarini and BinderPMID: 31195976https://bioconductor.org/packages/release/bioc/html/pcaExplorer.html
Commercial assay, kitBio-Plex Pro Human Inflammation Panel 1Biorad171AL001M
Commercial assay, kitBio-Plex Human Chemokine Panel, 40-plexBiorad171AK99MR2
Commercial assay, kitBio-Plex Pro Human Cytokine Screening Panel, 48-Plex kitBiorad12007283
Commercial assay, kithCG Human ProcartaPlex Simplex KitThermo FisherEPX010-12388-901
Commercial assay, kitCA125/Mucin-16 Human ProcartaPlex Simplex KitThermo FisherEPX010-12437-901
Chemical compound, drugPoly(I:C) HMWInvivogentlrl-pic
Chemical compound, drugRuxolitinibInvivogentlrl-rux
Biological samplePrimary human trophoblasts from full term placentasPMID: 27066743SRP067137Bulk RNASeq from primary human trophoblasts.
Biological samplePrimary human trophoblasts from full term placentasPMID: 28784796SRP109039Bulk RNASeq from primary human trophoblasts.
Biological sampleFirst trimester placental tissueUniversity of CambridgePRJEB38810Bulk RNASeq from
first trimester
placental tissue.
Biological sampleFull-term placental tissuePMID: 33969332PRJNA721668Bulk RNASeq from
ffull-term placental tissue.

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