Multi-omics analysis reveals a crucial role for Retinoic Acid in promoting epigenetic and transcriptional competence of anin vitromodel of human Pharyngeal Endoderm

ABSTRACT In vitro differentiation of human Pluripotent Stem Cells (hPSCs) into different cell types has enabled the study of developmental processes that are impossible to dissect in vivo . This innovation has allowed for the derivation of therapeutically relevant cell types that can be used for downstream applications and studies. The Pharyngeal Endoderm (PE) is considered an extremely relevant developmental tissue since it acts as a precursor to a plethora of organ systems such as Esophagus, Parathyroids, Thyroids, Lung, and Thymus. While several studies have highlighted the importance of these cells, an in vitro platform to generate human PE cells is still missing. Here we fill this knowledge gap, by providing a novel in vitro protocol for the derivation of bona fide PE cells from hPSCs. We demonstrated that our PE cells robustly express Pharyngeal Endoderm markers, they are transcriptionally similar to PE cells isolated from in vivo mouse development and represent a transcriptionally homogeneous population. Importantly, we elucidated the contribution of Retinoic Acid in promoting a transcriptional and epigenetic rewiring of PE cells. In addition, we defined the epigenetic landscape of PE cells by combining ATAC-Seq and ChIP-Seq of histone modifications. The integration of these data led to the identification of new putative regulatory regions and to the generation of a gene regulatory network orchestrating the development of PE cells. By combining hPSCs differentiation with computational genomics, our work reveals the epigenetic dynamics that occur during human PE differentiation, providing a solid resource and foundation for research focused on the development of PE derivatives and modeling of their developmental defects in genetic syndromes.