Fig 1: Neuroepithelial polarization can be driven by Matrigel or by endogenous self‐organization of ECM proteins A–DOrganoids generated with 80% WT H9 ESCs and 20% SOX2::EGFP were used to visualize the organization of neural progenitors. (A) Immunostaining for PKCζ and GFP at D10. (B) Quantification of the number of cavitation spots per cross section, using 45 organoids (see results for individual organoids of all cell lines in Appendix Fig S2A). Immunostaining for PKCζ and GFP at D13 (C), and D20 (D). Bottom panels: magnification of inset. Arrowheads mark the location of PKCζ lining the organoid outer surface (empty arrowheads) or the ventricular zone of neural rosettes (filled arrowheads).ESchematic representation of the radial organization of neural progenitors in neural rosettes, seen in all conditions at D20.FQuantification of the number of neural rosettes per cross section at D13 (110 organoids), D16 (160 organoids), and D20 (148 organoids; see results for individual organoids of all cell lines and timepoints in Appendix Fig S2B–D). Boxplots mark the median value; the two hinges correspond to the first and third quartiles (the 25th and 75th percentiles); and the whiskers extend from the hinge to the highest/lowest value no further than 1.5*IQR from the hinge (where IQR is the inter‐quartile range, or distance between the first and third quartiles). Statistical tests are analysis of variance (ANOVA); 0 ≤ P < 0.001, ***; 0.001 ≤ P < 0.01, **; 0.01 ≤ P < 0.05, *; P ≥ 0.05, ns (see results of statistical tests in Appendix Table S1).GImmunostaining for PKCζ and Ms/h‐FN, marking the apical and basal domains of neural rosettes, respectively. For a zoomed‐in view and representative images of all cell lines from D13 to D20, see Appendix Fig S3B.HImmunostaining for ECM proteins. The Ms‐LAMA1 antibody can be used to identify mouse‐derived ECM (Matrigel). At D20, MG+D, and MG+L organoids show a coating of Ms‐LAMA1 originating from Matrigel, which co‐localizes with Ms/h‐FN, LAMA1, and Perlecan (white arrowheads); also, FN+ but Ms‐LAMA1− speckles are seen within the tissue, indicating endogenously produced FN (green arrowheads). ExECM− organoids show abundant endogenously produced ECM surrounding neural rosettes (green arrowheads).IQuantification of the area around rosettes covered by Matrigel‐derived (Ms‐LAMA1+) and endogenously derived ECM (Ms‐LAMA1−Ms/h‐FN+), using 88 organoids and 276 rosettes (see methodology and results for individual organoids of all cell lines in Appendix Fig S4). The whiskers represent the positive standard deviation from the mean value (mean + SD).JExperimental paradigm to test the effect of liquid embedding with purified Laminin or Collagen IV.KBrightfield imaging and co‐staining of Ms‐LAMA1 (magenta arrowheads) and Ms/h‐LAMA1 (green arrowheads) of Laminin+L and Coll.IV+L organoids at D20. For representative images at D13‐20, see Appendix Fig S6. Source data are available online for this figure.