The identity of iPSCs and derived lineages was maintained in the presence of rubella virus (RV). (A) Overview of the methods applied to initiate differentiation in mock- and RV-infected iPSCs. (A) [i] Undirected differentiation in the respective induction medium started at the rim region of iPSC colonies (indicated by white arrows) and extended to their center over time of incubation. (A) [ii] Additionally, directed differentiation into the primary germ layers ectoderm, mesoderm, and endoderm was induced with the STEMdiff differentiation kit. (B) Immunofluorescence analysis with anti-E1 antibody (shown in red) was performed to monitor distribution of RV-positive cells within iPSC colonies. Nuclei are shown in blue. Ph, phase contrast (C) To assess RV replication in iPSCs and derived lineages, (i) virus progeny, and (ii) the amount of genomic viral RNAs was determined by standard plaque assay (n = 11 for passaged iPSCs, otherwise n = 3) and TaqMan-based reverse transcription-quantitative PCR (passaged iPSCs and ectodermal cells n = 3, mesodermal cells n = 7, endodermal cells n = 4), respectively. (D) Expression heatmap of selected marker genes of pluripotency and lineage identity (based on microarray whole transcriptome data) in mock- and RV-infected iPSCs and iPSC-derived lineages. Shading indicates overlap in the expression of some of the marker genes between iPSCs and iPSC-derived lineage cells, respectively. (E) The expression of indicated target genes in mock- and Wb-12-infected iPSCs and derived cells after initiation of undirected and directed differentiation was determined by real-time quantitative PCR (RT-qPCR) and analyzed by qbase+ software. For normalization, chromosome 1 open reading frame 43 (C1orf43) and hypoxanthine guanine phosphoribosyl transferase 1 (HPRT1) were used. Relative gene expression was calculated as normalized relative quantity (NRQ) and given as means ± SD (n = 3 to 5). As a reference, lineage-specific expression levels were assigned based on literature data and transcriptomics data by Stemcell Technologies for the Trilineage differentiation kit. (F) Analysis of stemness-related expression signatures was based on the GO gene set telomere maintenance. An embryonal stem cell signature and a gene set collecting OCT4 targets were both taken from [].

RV supports expression of markers for definite endoderm. (A) Similarity tree of the gene expression portraits of the cell systems studied. Both, mock- and RV-infected mesodermal and ectodermal cells share relatively high mutual similarity of their transcriptomes. In contrast, mock- and RV-infected endodermal cells form a separate branch that is closer to the mock- and RV-infected iPSCs. (B) Spot summary map (I) provides an overview of activated cellular programs and their functional context, which is depicted in more detail in (II). (C) Barplot representation of the expression profiles of gene sets related to different functions. Their genes were enriched in the spots that were identified in the SOM portraits. They are thus indicated accordingly (see also the overview map in part B of the Figure and ). (D) Selected marker genes for definite endoderm are illustrated in the heatmap of the transcriptome of mock- and RV-infected iPSCs and iPSC-derived lineages. (E) The expression of marker genes for definite endoderm was determined by RT-qPCR in mock- and Wb-12-infected iPSCs and iPSC-derived lineages by qbase+ software. For normalization, C1orf43 and HPRT1 were used. Relative gene expression was calculated as normalized relative quantity (NRQ) and given as means ± SD (n = 3 to 5). See also .

RV infection activates an IFN response in iPSCs and derived lineages. (A) Expression portraits of the embryonal germ layers before and after RV infection and the respective difference portraits reveal characteristic spot patterns, where spots C, D, and E are specific for endoderm, ectoderm, and mesoderm cells, respectively. Spot B appears after RV infection mainly in endodermal cells. (B) IFN response genes with signaling and stimulated functions in the IFN response pathways accumulate in spots B and C and were upregulated predominantly in iPS and endodermal cells after RV infection as also indicated by the IFN and viral response gene signature profiles. (C) Expression heatmap of selected marker genes involved in IFN-sensing and -signaling in mock- and RV-infected iPSCs and iPSC-derived lineages. Interferon-stimulated genes (ISGs) that were identified by the SOM analysis shown in (A) are included. (D) The mRNA expression level of the IFN-signaling components IRF9 and STAT1 and indicated ISGs was verified by RT-qPCR analysis. Data are given as means ± SD (n = 3 and n = 5 for RV-infected mesoderm, IRF9 and STAT1). (E) The IFN profile for RV-infected iPSCs and derived lineages was determined by the LEGENDplex IFN panel for undiluted supernatants collected after five (iPSCs and mesodermal cells) and seven days (endodermal and ectodermal) of cultivation. (F) The mRNA expression level of type III IFNs was verified by qPCR analysis and given as means ± SD (n = 3). (G) Mean expression of a gene set (gene set Z-score, GSZ) that is controlled by KSRP, which appears to keep inflammatory gene expression within defined limits []. (H) Similarity tree of the gene expression portraits of mock- and RV-infected iPSCs in comparison to iPSCs after cultivation in the presence of exogenous type III IFNs. (D,F) For normalization of qRT-PCR data in the 2^−∆∆Ct method, the HPRT1 gene was used. See also .

RV infection is accompanied by an altered expression of components of the SWI/SNF and NURF chromatin remodeling complexes. (A) Mean GSZ expression signatures of stemness-related transcriptional programs which act via epigenetic programming (PRC2 targets, repressive and bivalent chromatin marks). (B) Expression heatmap of chromatin modifying enzymes in the cell systems studied. The gene expression data of methyltransferases (MTs) and demethylases (DMs) of DNA cytosines, histone lysine and arginine side chains were assigned to the expression spot-cluster A–E according to their expression profiles. This suggests their involvement in the regulation of chromatin structure as writers and erasers of methylation marks at histone lysine and arginine side chains and affecting DNA methylation. Notably, a very strong variability was observed for KDM6a (alias UTX), a constituent of the SWI/SNF ATP-dependent chromatin remodeling machinery. (C) Expression of KDM6a directly relates to stemness programs (F) and inversely relates to programs repressing stemness functions (part A of the Figure). See also .

RV impairs the cellular adhesion capacity of iPSCs. (A) Gene signatures related to focal adhesion in mock- and RV-infected iPSCs. (B) Expression heatmap of selected marker genes involved in cellular adhesion. (C) Assessment of 3D stability through embryoid body (EB) formation. Shown are images before and after cultivation in suspension. (D) To verify viability, EBs were stained with calcein after cultivation for two weeks.

RV exerts lineage-specific effects on developmental signaling pathways and alters expression of transcription and growth factors. Pathway signal flow (PSF) activity plot of (A) the TGF-β signaling pathway in endodermal cells (highlighted are genes with higher (NODAL, ACVR2A, Myc) and lower (RBL1 and E2F4) activity after RV infection) and (B) of the Wnt signaling pathway in mesodermal cells (highlighted are genes with lower (the CSNK1E/AXIN1E and the LEF1/CCND1 axis) activity after RV infection). The calculation of the activity of the nodes was based on the PSF algorithm using the respective gene expression values and the wirings between the nodes []. (C) Selected genes within pathways that were specifically affected by RV infection are illustrated in the heatmap of the transcriptome of mock- and RV-infected iPSCs and iPSC-derived lineages. (D) For qRT-PCR expression analysis, the 2^−∆∆Ct method based on normalization to HPRT1 gene was used. Values are given as means ± SD (n = 4 for Wb-12-infected ectoderm, n = 2 for HPV77-infected ectoderm, otherwise n = 3). See also .

Graphical summary of the identified molecular alterations induced by RV during directed differentiation of iPSCs into the three embryonic germ layer cells. The data was set in a possible relation to the prevailing symptoms of congenital rubella embryopathy. Especially endodermal cells were characterized by profound alterations in their gene expression landscape, including the expression of markers for definitive endoderm and epigenetic factors. This could impair the crosstalk between endodermal and mesodermal cells during differentiation.