1While the mechanisms of embryonic development are similar between mouse and human, 2 the tempo is in general slower in human. The cause of interspecies differences in 3 developmental time remains a mystery partly due to lack of an appropriate model system 1 . 4 Since murine and human embryos differ in their sizes, geometries, and nutrients, we use 5 in vitro differentiation of pluripotent stem cells (PSCs) to compare the same type of cells 6 between the species in similar culture conditions. As an example of well-defined 7 developmental time, we focus on the segmentation clock, oscillatory gene expression that 8 regulates the timing of sequential formation of body segments [2][3][4] . In this way we 9 recapitulate the murine and human segmentation clocks in vitro, showing that the species-10 specific oscillation periods are derived from cell autonomous differences in the speeds of 11 biochemical reactions. Presomitic mesoderm (PSM)-like cells induced from murine and 12 human PSCs displayed the oscillatory expression of HES7, the core gene of the 13 segmentation clock 5,6 , with oscillation periods of 2-3 hours (mouse PSM) and 5-6 hours 14 (human PSM). Swapping HES7 loci between murine and human genomes did not change 15 the oscillation periods dramatically, denying the possibility that interspecies differences 16 in the sequences of HES7 loci might be the cause of the observed period difference.
17Instead, we found that the biochemical reactions that determine the oscillation period, 18 such as the degradation of HES7 and delays in its expression, are slower in human PSM 19 compared with those in mouse PSM. With the measured biochemical parameters, our 20 mathematical model successfully accounted for the 2-3-fold period difference between 21 mouse and human. We further demonstrate that the concept of slower biochemical 22 reactions in human cells is generalizable to several other genes, as well as to another cell 23 type. These results collectively indicate that differences in the speeds of biochemical 24 reactions between murine and human cells give rise to the interspecies period difference 25 of the segmentation clock and may contribute to other interspecies differences in 26 developmental time.27 28 Main 29 To compare murine and human segmentation clocks in vitro, we induced PSM-like cells 30 from mouse embryonic stem cells (ESCs) and human induced pluripotent stem cells 31 (iPSCs) (Fig. 1a), as other groups have recently reported 7-12 . In essence, our PSM 32 induction protocol is based on activation of WNT and FGF signaling as well as inhibition 33 of TGFβ and BMP signaling 9,12 . Prior to the PSM induction, mouse ESCs, which are in 34 the naïve pluripotent state, were pretreated with ACTIVIN A and bFGF and converted to 35 mouse epiblast-like cells (EpiLCs) that possess primed pluripotency as human iPSCs do.36 3To visualize the segmentation clock in the induced PSM, we introduced a HES7 promoter-1 luciferase reporter 13,14 , detecting clear synchronized oscillations of HES7 expression in 2 both murine a...