Rapid and robust directed differentiation of mouse epiblast stem cells into definitive endoderm and forebrain organoids I

Mouse is the pre-eminent model system for studying genetic regulation of embryonic development. Directed differentiation of pluripotent stem cells (PSCs) has emerged as a powerful complement to in vivo studies of development, allowing for examination of developmental mechanisms at the molecular and cellular level. However, the limited availability of high-quality protocols for directed differentiation of mouse PSCs into defined lineages and organoids has prevented the widespread application of mouse PSC directed differentiation models. Here, we examine the potential of mouse epiblast stem cells (EpiSCs) cultured in media containing Wnt pathway inhibitors (primed ground state conditions) as a starting point for directed differentiation. First, we systematically optimized conditions to generate definitive endoderm that are significantly faster and more efficient than current state-of-the-art protocols for mouse PSCs. Second, we developed a robust protocol for generation of forebrain-patterned organoids. These new models can complement and empower in vivo studies in the mouse and can inform human PSC-based studies of development, which cannot readily be compared to analogous stages in vivo. Examination of chromatin accessibility profile and bulk transcriptome in mouse epiblast stem cells (mEpiSCs) in culture and mouse definitive endoderm samples (DE) obtained after differentiation of mEpiSCs during 46h and MACS-based sorting of the CXCR4 positive cells. Additionally, we compare the transcriptome and chromatin accesbility profile of the EpiSCs across different genetic backgrounds