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. 2022 Aug 17;11:e79794. doi: 10.7554/eLife.79794

Figure 6. Differential susceptibility of TOs and DOs to HCMV infection.

Figure 6.

(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) and (B). Infected cells are positive for EGFP (AD169r, middle row) or mCherry (TB40E, bottom row). Organoids were stained for actin (red in mock- and AD169r-infected or green in TB40E infected). DAPI-stained nuclei are shown in blue. (C, D) Quantification of HCMV infection in DOs or TOs infected with AD169r (C) or TB40E (D). Shown is the percent HCMV positive cells as assessed by EGFP (AD169r, (C)) or mCherry (TB40E, (D)) over total number of nuclei from confocal micrographs. A minimum of 30 independent organoids taken from biological replicate experiments were quantified by automated image analysis. (E) Comparison of RPKM values for HCMV RNA4.9 and UL63 in TOs (blue) or DOs (orange) as assessed by bulk RNASeq. Data are shown as mean ± standard deviation as a fold of DO values. Significance was determined using a t-test. ***p<0.001, **p<0.01. Each symbol represents an independent sample. (F) Representative confocal micrographs of undifferentiated (top) of EVT differentiated (bottom) TOs infected with EGFP-tagged AD169r and immunostained for HLA-G (in red). DAPI-stained nuclei are shown in blue and actin is in purple. Middle panels are black and whote imaged of HCMV infection and HLA-G immunostaining. (G) Quantification of HCMV (AD169r strain) infection in TOs cultured under standard conditions (Undiff., left) or under conditions that promote EVT differentiation (Diff., right). Shown are the percentage of total cells that were positive for HCMV, as assessed by the presence of GFP, over total nuclei as assessed by DAPI staining. Each symbol represents an independent field from replicates, with at least 2000 total nuclei quantified under both conditions. Significance was determined using a t-test, ns, not significant. In (A, B), F scale bar, 50 µm. DO, decidua organoid; EVT, extravillous trophoblast; HCMV, human cytomegalovirus; TO, trophoblast organoid.