Extended Data Figure 3: mES cells initiate polarization in naive conditions in a 3D Matrigel culture.
From: Pluripotent state transitions coordinate morphogenesis in mouse and human embryos
a, Experimental set-up. A, analysis. b, Immunostaining of mES cells cultured as indicated in a. Arrows indicate lumens. Scale bars, 5 μm. c, Lumen formation in cells from b. n = 31 (2 cells, gelatin +2i/LIF 48 h, Matrigel −2i/LIF 24 h), 30 (4 cells, gelatin +2i/LIF 48 h, Matrigel −2i/LIF 24 h), 31 (2 cells, gelatin −2i/LIF 48 h, Matrigel −2i/LIF 24 h) and 31 (4 cells, gelatin −2i/LIF 48 h, Matrigel −2i/LIF 24 h) spheroids. χ2 test, ***P < 0.0001. d, Internuclear distance in cells from Fig. 2b. n = 31 (+2i/LIF) and 30 (−2i/LIF) spheroids. Mann–Whitney U-test; NS, not significant. e–g, Immunostaining of mES cells cultured in Matrigel with or without 2i/LIF. Squares indicate magnified regions (e). Scale bars, 10 μm (e and f) and 5 μm (g). h–j, Representative frames from time-lapse experiments using different naive reporter mES cell lines, cultured in either gelatin or Matrigel in the presence of 2i/LIF. Time is indicated as h:min. Scale bars, 50 μm. k–m, Intensity of Nanog–YFP, ΔPE–Oct4–GFP and Rex1::GFPd2 mES cells analysed by time-lapse microscopy from h–j. AU, arbitrary units. n = 65 Nanog–YFP, 55 ΔPE–Oct4–GFP and 43 Rex1::GFPd2 mES cell colonies (gelatin); and n = 61 Nanog–YFP, 84 ΔPE–Oct4–GFP and 61 Rex1::GFPd2 mES cell spheroids (Matrigel). n, Experimental set-up. o, Immunostaining of mES cells cultured as indicated in n. Squares indicate magnified regions. Dotted lines demarcate the border of the colonies. The nucleus–centrosome angle (α) measured in p is indicated. Scale bars, 10 μm. p, Histogram of the angle between the centrosome–nucleus axis and the basal–apical axis of cells in the border of the colonies shown in o. Data are shown as relative frequencies (%). n = 76 (Matrigel +2i/LIF cultured in gelatin), 61 (Matrigel −2i/LIF cultured in gelatin), 62 (Matrigel +2i/LIF) and 60 (Matrigel −2i/LIF) centrosomes. Kruskal–Wallis test; ***P < 0.0001; NS, not significant. q, Immunostaining of control and Dgcr8 knockout mES cells (with or without 2i/LIF). Scale bars, 5 μm. r, Angle between the nucleus–centrosome axis and the nuclear–nuclear axis in cells from q. Each dot represents an individual centrosome. n = 60 control +2i/LIF, 62 control −2i/LIF, 56 Dgcr8 knockout +2i/LIF and 58 Dgcr8 knockout −2i/LIF centrosomes. Kruskal–Wallis test; NS, not significant. s, Internuclear distance in cells from q. n = 30 control +2i/LIF, 31 control −2i/LIF, 28 Dgcr8 knockout +2i/LIF and 29 Dgcr8 knockout −2i/LIF spheroids. Kruskal–Wallis test. t, Nanog intensity in cells from q. n = 30 control +2i/LIF, 31 control −2i/LIF, 28 Dgcr8 knockout +2i/LIF and 30 Dgcr8 knockout −2i/LIF spheroids. Kruskal–Wallis test; ***P < 0.0001. u, Immunostaining of mES cells cultured in Matrigel with or without Gö6983. Scale bars, 10 μm. v, Internuclear distance in cells from Fig. 2i. n = 26 (+Gö6983) and 28 (−Gö6983) spheroids. Mann–Whitney U-test; NS, not significant. G, gelatin; M, Matrigel.
