Extended Data Fig. 1: Differentiation of mouse and human pluripotent stem cells towards PSM fate for the characterization of the segmentation clock in vitro. | Nature

Extended Data Fig. 1: Differentiation of mouse and human pluripotent stem cells towards PSM fate for the characterization of the segmentation clock in vitro.

From: In vitro characterization of the human segmentation clock

Extended Data Fig. 1

a, Scheme illustrating the maturation stages of paraxial mesoderm. DF, determination front; pPSM. b, Top, immunofluorescence staining for the cadherins CDH1 and CDH2 (top), and the pluripotency factor POU5F1 (bottom) in differentiating mouse ES cells (ESCs) (left) and human iPS cells (iPSCs) (right). n = 4 independent experiments. Scale bar, 100 μm. c, qRT–PCR for the epiblast marker Fgf5, the neuromesodermal progenitor or mesodermal marker T, and the mesodermal precursor cell and PSM markers Tbx6, Msgn1 and Rspo3 on days 2–6 of mouse ES cell differentiation. Relative expression is shown as the fold change relative to ES cells at day 0. Mean ± s.d. n = 3 biological replicates. d, Percentage induction of the mouse (m)ES cell pMsgn1-Venus reporter and the human (h)iPS cell MSGN1-Venus reporter, as determined by fluorescence-activated cell sorting (FACS). Mean ± s.d. n = 12 independent experiments (mouse ES cell), n = 8 independent experiments (human iPS cell). e, Gating strategy and representative FACS plots for quantification of pMsgn1-Venus or MSGN1-Venus induction. f, qRT–PCR for cyclic genes (HES7 and LFNG), posterior-PSM markers (MSGN1, TBX6 and RSPO3), determination-front markers (MESP2 and RIPPLY2) and anterior-PSM markers (MEST and FOXC2) on days 1–4 of human iPS cell (iPSC) differentiation. Relative expression is shown as the fold change relative to iPS cells at day 0. Mean ± s.d. n = 3 biological replicates. g, Diagram outlining the targeting strategy used to generate Hes7-Achilles and HES7-Achilles knock-in reporter lines in mouse ES cells and human iPS cells, respectively. h, Normalized HES7–Achilles fluorescence intensity for three PSM cells derived from mouse ES cells, imaged in CL medium on day 4 of differentiation. n = 4 independent experiments. i, Representative Fourier transform of HES7–Achilles oscillations in PSM cells derived from mouse ES cells, indicating the predominant period. n = 19 cells. j, Total time spent in the oscillatory state for Hes7-Achilles PSM cells derived from mouse ES cells, cultured in CL or CLFBR medium from day 4 onwards. The middle hinge corresponds to median, the lower and upper hinges correspond to the first and third quartiles, respectively, and the lower and upper whiskers correspond to the minimum and maximum, respectively. n = 8 (CL), n = 12 (CLFBR) independent experiments. k, qRT–PCR comparing relative expression levels of Msgn1, Lfng, T and Tbx6 in PSM cells derived from mouse ES cells, cultured in CL or CLFBR medium from day 4 onwards. Relative expression is shown as the fold change relative to ES cells at day 0. Mean ± s.d. n = 3 biological replicates. l, Snapshots of HES7–Achilles fluorescence in PSM cells derived from human iPS cells, showing peaks and troughs over the course of 13.5 h in CL medium on day 2 of differentiation. n = 25 independent experiments. Scale bar, 100 μm. m, Representative quantification of HES7–Achilles fluorescence intensity in a small ROI from day 2 to day 3 of human iPS cell differentiation. n = 25 independent experiments. n, Representative Fourier transform of HES7-Achilles oscillations, indicating the predominant period in PSM cells derived from human iPS cells, in CL medium on day 2. n = 25 independent experiments. o, Representative instantaneous frequency in Hertz (calculated by Hilbert transformation) of HES7-Achilles oscillations in PSM cells derived from human iPS cells, from day 2 to day 3 of differentiation in CL medium. n = 25 independent experiments. p, Representative instantaneous frequency in Hertz (calculated by Hilbert transformation) of HES7-Achilles oscillations in PSM cells derived from human iPS cells, from day 2 to day 3 of differentiation in CLFBR medium. n = 33 independent experiments. q, Quantification of HES7–Achilles fluorescence in human iPS cells differentiated for 48 h without the BMP inhibitor LDN93189 (CHIR99021-only medium). n = 3 independent experiments. r, Total number of HES7-Achilles oscillations for PSM cells derived from human iPS cells, cultured in CL or CLFBR medium from day 2 onwards. Mean ± s.d. n = 15 independent experiments. s, qRT–PCR comparing relative expression levels of HES7, LFNG, TBX6 and MSGN1 in PSM cells derived from human iPS cells, cultured in CL or CLFBR medium from day 2 onwards. Relative expression is shown as the fold change relative to iPS cells on day 0. Mean ± s.d. n = 3 biological replicates.

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