Modeling the Human Segmentation Clock with Pluripotent Stem Cells

Abstract

32 33 Keywords: segmentation clock, somitogenesis, human mesoderm development, induced 34 pluripotent stem cells, step-wise in vitro mesoderm differentiation, disease modeling 35 Pluripotent stem cells (PSCs) have increasingly been used to model different aspects 36 of embryogenesis and organ formation 1 . Despite recent advances in the in vitro 37 induction of major mesodermal lineages and mesoderm-derived cell types 2,3 , 38 experimental model systems that can recapitulate more complex biological features 39 of human mesoderm development and patterning are largely missing. Here, we 40 utilized induced pluripotent stem cells (iPSCs) for the stepwise in vitro induction of 41 presomitic mesoderm (PSM) and its derivatives to model distinct aspects of human 42 somitogenesis. We focused initially on modeling the human segmentation clock, a 43 major biological concept believed to underlie the rhythmic and controlled 44 emergence of somites, which give rise to the segmental pattern of the vertebrate 45 axial skeleton. We succeeded to observe oscillatory expression of core segmentation 46 clock genes, including HES7 and DKK1, and identified novel oscillatory genes in 47 human iPSC-derived PSM. We furthermore determined the period of the human 48 segmentation clock to be around five hours and showed the presence of dynamic 49 traveling wave-like gene expression within in vitro induced human PSM. Utilizing 50 CRISPR/Cas9-based genome editing technology, we then targeted genes, for which 51 mutations in patients with abnormal axial skeletal development such as 52 spondylocostal dysostosis (SCD) (HES7, LFNG and DLL3) or spondylothoracic 53 dysostosis (STD) (MESP2) have been reported. Subsequent analysis of patient-like 54iPSC knock-out lines as well as patient-derived iPSCs together with their genetically 55 corrected isogenic controls revealed gene-specific alterations in oscillation, 56 synchronization or differentiation properties, validating the overall utility of our 57 model system, to recapitulate not only key features of human somitogenesis but also 58 to provide novel insights into diseases associated with the formation and patterning 59 of the human axial skeleton. 60 We initially aimed to mimic and recreate in vitro the signaling events responsible 61for the step-wise emergence of PSM and its derivatives during embryonic development, 62as also recently attempted by others 2,4,5 , via selective activation or inhibition of 63 appropriate signaling pathways, using human iPSCs as starting material (Fig. 1a). We 64 characterized the ability of our in vitro induced human PSM cells to differentiate into 65 somitic mesoderm and its two main derivatives, sclerotome and dermomyotome, which 66give rise to bone and cartilage of the axial skeleton and skeletal muscle and dermis of the 67 emerging embryo respectively. RNA-sequencing (RNA-seq) analysis and subsequent 68 characterization of in vitro derived human PSM samples revealed, that at each step of our 69 induction and differentiation protocol, markers expected to be pr...

Systematic analysis of protein stability associated with species-specific developmental tempo

“…Treatment of mouse iPSM with 2DG decelerated the segmentation clock in a dose-dependent manner as described previously 34 (Fig. 3a,b): the oscillation period of mouse iPSM treated with 10 mM 2DG was 191 ± 1 min (mean ± SD), which was between the untreated mouse iPSM period (147 ± 4 min) and the reported human iPSM period (322 min) 6 .…”

“…We next investigated the effect of metabolism on the protein stability profile. While both glycolytic activity and electron transport chain activity are involved in the segmentation clock and somitogenesis 12,18,[20][21][22][23][24] , we have recently found that a glycolysis inhibitor, 2-Deoxy-D-glucose (2DG), and an electron transport chain inhibitor, sodium azide, decelerate the segmentation clock through different mechanisms: 2DG decelerates HES7 protein degradation whereas sodium azide elongates HES7 intron processing delay 34 .…”

Species-specific oscillation periods of human and mouse segmentation clocks are due to cell autonomous differences in biochemical reaction parameters

“…1a), as other groups have recently reported 7–12 . In essence, our PSM induction protocol is based on activation of WNT and FGF signaling as well as inhibition of TGFβ and BMP signaling 9,12 . Prior to the PSM induction, mouse ESCs, which are in the naïve pluripotent state, were pretreated with ACTIVIN A and bFGF and converted to mouse epiblast-like cells (EpiLCs) that possess primed pluripotency as human iPSCs do.…”

“…Then the cells were cultured in CDMi containing SB431542 (10 μM), DMH1 (2 μM), CHIR99021 (10 μM), and bFGF (20 ng/ml) for another 3.5 days to induce human PSM cells. For the degradation assay, our 2 step induction protocol 12 was used. Human iPSCs were seeded on a 35 mm dish coated with iMatrix-511 silk and cultured for 5 days.…”

“…To compare murine and human segmentation clocks in vitro , we induced PSM-like cells from mouse embryonic stem cells (ESCs) and human induced pluripotent stem cells (iPSCs) (Fig. 1a), as other groups have recently reported 7–12 . In essence, our PSM induction protocol is based on activation of WNT and FGF signaling as well as inhibition of TGFβ and BMP signaling 9,12 .…”