**Teratogenic Rubella Virus Alters the Endodermal Di** ff**erentiation Capacity of Human Induced Pluripotent Stem Cells**

**Nicole C. Bilz 1,**†**, Edith Willscher 2,**†**, Hans Binder 2, Janik Böhnke 1,**‡**, Megan L. Stanifer 3, Denise Hübner 1, Steeve Boulant 3,4, Uwe G. Liebert 1 and Claudia Claus 1,\***


Received: 14 July 2019; Accepted: 7 August 2019; Published: 10 August 2019

**Abstract:** The study of congenital virus infections in humans requires suitable ex vivo platforms for the species-specific events during embryonal development. A prominent example for these infections is rubella virus (RV) which most commonly leads to defects in ear, heart, and eye development. We applied teratogenic RV to human induced pluripotent stem cells (iPSCs) followed by di fferentiation into cells of the three embryonic lineages (ecto-, meso-, and endoderm) as a cell culture model for blastocyst- and gastrulation-like stages. In the presence of RV, lineage-specific di fferentiation markers were expressed, indicating that lineage identity was maintained. However, portrait analysis of the transcriptomic expression signatures of all samples revealed that mock- and RV-infected endodermal cells were less related to each other than their ecto- and mesodermal counterparts. Markers for definitive endoderm were increased during RV infection. Profound alterations of the epigenetic landscape including the expression level of components of the chromatin remodeling complexes and an induction of type III interferons were found, especially after endodermal di fferentiation of RV-infected iPSCs. Moreover, the eye field transcription factors RAX and SIX3 and components of the gene set vasculogenesis were identified as dysregulated transcripts. Although iPSC morphology was maintained, the formation of embryoid bodies as three-dimensional cell aggregates and as such cellular adhesion capacity was impaired during RV infection. The correlation of the molecular alterations induced by RV during di fferentiation of iPSCs with the clinical signs of congenital rubella syndrome suggests mechanisms of viral impairment of human development.

**Keywords:** ectoderm; mesoderm; human development; embryogenesis; interferon response; interferon-induced genes; self-organizing map (SOM) data portrayal; epigenetic signature; embryoid body; TGF-β and Wnt/β-catenin pathway
