GSE116934
Recapitulating the Human Segmentation Clock with Pluripotent Stem Cells - RNAseq-based comparison of patient and isogenic rescue/healthy control iPSC lines during in vitro differentiation of human presomitic mesoderm and its derivatives
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, 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. We furthermore identified and compared oscillatory genes in human and murine PSC-derived PSM, which revealed species-specific as well as common molecular components and novel pathways associated with the mouse and human segmentation clocks. Utilizing CRISPR/Cas9-based genome editing technology, we then targeted genes, for which mutations in patients with segmentation defects of vertebrae (SDV) such as spondylocostal dysostosis (SCD) have been reported (e.g. HES7, LFNG, DLL3 and MESP2 DLL3). Subsequent analysis of patient-like knock-out and point-mutation 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 provide novel insights into the human segmentation clock as well as diseases associated with the formation of the human axial skeleton. Overall design: All stages of our stepwise in vitro induction and differentiation protocol were analyzed using spondylocostal dysostosis (SCD) patient iPSC lines (clones SCDP1-A and SCDP1-F) together with their corresponding isogenic controls (SCDP1-resA and SCDP1-resF) or healthy control iPSCs (201B7); identified heterozygous compound mutations in MESP2 of SCDP1-patient line were corrected via CRISPR/Cas9-based allele-specific genome editing. Two patient clones were utilized (SCDP1-A and SCDP1-F) together with three rescue clones (SCDP1-resA1, SCDP1-resF2 and SCDP1resF3). All stages of the induction protocol were targeted (induced pluripotent stem cell (iPSC), primitive streak (PS), presomitic mesoderm (PSM), somitic mesoderm (SM), sclerotome (SCL) and dermomyotome (DM))