GSE116928
Recapitulating the Human Segmentation Clock with Pluripotent Stem Cells - RNAseq analysis of step-wise in vitro induction/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: Induced pluripotent stem cells (iPSCs) of healthy controls (201B7 and 1231A3) were differentiated under defined feeder-free conditions in a step-wise manner into paraxial mesoderm and its major derivatives. Initially, iPSCs were induced into human primitive streak (PS), and subsequently into presomitic mesoderm (PSM) and somitic mesoderm (SM), followed by the induction of the two major derivatives of somitic mesoderm, sclerotome (SCL), which gives rise to bone and cartilage of the axial skeleton and dermomyotome (DM), which gives rise to skeletal muscle and dermis of the emerging embryo. Transcriptomes of three independent samples of each developmental stage derived from two different human healthy control iPSC lines were analyzed using RNAseq.