Extended Data Fig. 3: Further characterization of elongated cortical assembloids.
a-b) Representative images of elongated assembloids (8 div) properly embedded maintaining elongated shape (a) compared to elongated assembloids from the same batch without matrigel, shrinking without spatial constrain (b). c) Length of elongated assembloids after different days in vitro (div, div1 n = 8, div7 n = 6, div30 n = 4, div60 n = 24, div120 n = 22 assembloids grown from 3 independent clones). P-values resulting from one-way ANOVA Tukey’s multiple comparisons tests) among the different lines are: div1 vs div7, p = 0.0045; div1 vs div30, p = 0.9999; div7 vs div30, p = 0.0366; div7 vs div60, p = 0.9998; div7 vs div120, p = 0.51; div30 vs div60, p = 0.0049; div60 vs div120, p = 0.1825; div1 vs div60, div1 vs div120, div30 vs div120, p < 0.0001. d) RT-qPCR analysis of neural markers (SOX2, SOX1, SP8 and LMO4), ventral marker NXK2.1 and differentiation markers SNAI1 and T-BRA, in elongated assembloids (eOrg) compared to round organoids (rOrg), after treatment with high FGF8 as described in Fig. 1. Values are mean ± SD, n = 3 in three lines (hESC H9, H1 and iPSC 178/5). P-values resulting from one-way ANOVA (Tukey’s multiple comparisons tests) are: eORGSOX2 d10 vs. rORGSOX2 d10, p = 0.0024; eORGSOX2 d30 vs. rORGSOX2 d30, p = 0.3144; eORGSOX2 d60 vs. rORGSOX2 d60, p = 0.9434; eORGSOX1 d10 vs. rORGSOX1 d10, p = 0.0009; eORGSOX1 d30 vs. rORGSOX1 d30, p = 0.1573; eORGSOX1 d60 vs. rORGSOX1 d60, p = 0.7266; eORGSP8 d10 vs. rORGSP8 d10, p = 0.0006; eORGSP8 d30 vs. rORGSP8 d30, p = 0.1891; eORGSP8 d60 vs. rORGSP8 d60, p = 0.8004; eORGLMO4 d10 vs. rORGLMO4 d10, p = 0.0114; eORGLMO4 d30 vs. rORGLMO4 d30, p = 0.8378; eORGLMO4 d60 vs. rORGLMO4 d60, p = 0.9821; eORGNKX2.1 d10 vs. rORGNKX2.1 d10, p = 0.0008; eORGNKX2.1 d30 vs. rORGNKX2.1 d30, p = 0.1345; eORGNKX2.1 d60 vs. rORGNKX2.1 d60, p = 0.9036; eORGSNAI1 d10 vs. rORGSNAI1 d10, p = 0.0026; eORGSNAI1 d30 vs. rORGSNAI1 d30, p = 0.6125; eORGSNAI1 d60 vs. rORGSNAI1 d60, p = 0.96; eORGT-BRA d10 vs. rORGT-BRA d10, p = 0.6815; eORGT-BRA d30 vs. rORGT-BRA d30, p = 0.3026; eORGT-BRA d60 vs. rORGT-BRA d60, p = 0.1297. e) Fraction of TUNEL+ cells normalized to total DAPI+ cells in elongated versus conventional round organoids (div, days in vitro). N = 36 insets for 4 div elongated, n = 33 for 4 div Round, n = 122 for 7 div elongated, n = 37 for 7 div Round, n = 81 for 30 div elongated, n = 82 for 30 div Round organoids from 3 lines per condition. P-values resulting from one-way ANOVA (Tukey’s multiple comparisons tests) are: 4 div eOrg vs. 4 div rOrg, p < 0.0001; 7 div eOrg vs. 7 div rOrg, p = 0.31; 30 div eOrg vs. 30 div rOrg, p = 0.0115. f) Box plots showing the fraction of TUNEL+ cells normalized to total DAPI+ cells in proximal and distal insets of elongated assembloids (n = 18 insets for 4 div Proximal and Distal, n = 32 for 7 div Proximal, n = 33 for 7 div Distal, n = 40 for 30 div Proximal, n = 42 for 30 div Distal; organoids are grown from 3 lines per condition). P-values resulting from one-way ANOVA (Tukey’s multiple comparisons tests) are: 4 div proximal vs. 4 div distal, p = 0.6097; 7 div proximal vs. 7 div distal, p = 0.6559; 30 div proximal vs. 30 div distal, p = 0.9897. g) Representative images of the OrEB localization throughout elongated assembloids growth quantified in Fig. 2c (scale bars 500 µm). h) Top, representative images of proximal and distal CTIP2 staining (scale bars 50 µm). Bottom, fraction of CTIP2+ cells normalized to total DAPI+ cells in proximal and distal insets of controls (conCA) and polCAs at 60 div (n = 50 insets for P and D conCAs, n = 40 insets for P and D polCAs from 3 organoids). P-values resulting from one-way ANOVA (Tukey’s multiple comparisons tests) are: proximal conCA vs. distal conCA, p = 0.8204; proximal polCA vs. distal polCA, p < 0.0001. i) Top, representative images of proximal and distal LMO4 staining (scale bars 50 µm). Bottom, fraction of LMO4+ cells normalized to total DAPI+ cells in proximal and distal insets of control(tdt) and polCAs at 60 div (n = 61 insets for P and D from 6 conCAs, n = 51 insets for P and D from 4 polCA). P-values resulting from one-way ANOVA (Tukey’s multiple comparisons tests) are: proximal conCA vs. distal conCA, p > 0.9999; proximal polCA vs. distal polCA, p < 0.0001. ns, non-significant. All whiskers are min to max, boxes represent the 25th to 75th percentiles (Q1 to Q3) and lines indicate median. j-j’) Images of immunostained longitudinal sections of elongated assembloids at 60 div (scale bars 500 µm).
