GSE132811
Recapitulating the Human Segmentation Clock with Pluripotent Stem Cells - RNAseq analysis of in vitro induced murine presomitic mesoderm (PSM) and identification of components of the in vitro mouse segmentation clock [Oscillation_Mm]
Pluripotent stem cells (PSCs) have increasingly been used to model different aspects of embryogenesis and organ formation. Despite recent advances in the in vitro induction of major mesodermal lineages and mesoderm-derived cell types, experimental model systems that can recapitulate more complex biological features of human mesoderm development and patterning are largely missing. Here, we utilized induced pluripotent stem cells (iPSCs) for the stepwise in vitro induction of presomitic mesoderm (PSM) and its derivatives to model distinct aspects of human somitogenesis. We focused initially on modeling the human segmentation clock, a major biological concept believed to underlie the rhythmic and controlled emergence of somites, which give rise to the segmental pattern of the vertebrate axial skeleton. We succeeded to observe oscillatory expression of core segmentation clock genes, including HES7 and DKK1, and identified novel oscillatory genes in human and mouse PSC-derived PSM. We furthermore determined the period of the human segmentation clock to be around five hours and showed the presence of dynamic traveling wave-like gene expression within in vitro induced human PSM. Utilizing CRISPR/Cas9-based genome editing technology, we then targeted genes, for which mutations in patients with abnormal axial skeletal development such as spondylocostal dysostosis (HES7, LFNG and DLL3)  have been reported. Subsequent analysis of patient-like iPSC knock-out lines as well as patient-derived iPSCs together with their genetically corrected isogenic controls revealed gene-specific alterations in oscillation, synchronization or differentiation properties, validating the overall utility of our model system, to recapitulate not only key features of human somitogenesis but also to provide novel insights into diseases associated with the formation and patterning of the human axial skeleton. Overall design: In order to identify the molecular components of the mouse segmentation clock, mouse epiblast stem cell (EpiSCs)-derived presomitic mesoderm (PSM) samples were collected in timely manner utilizing a luciferase reporter line of the key oscillatory segmentation clock gene Hes7 (Hes7-reporter). Two independent sets of 16 samples each of entrained oscillating mouse in vitro induced PSM samples were analyzed using RNAseq.