Extended Data Fig. 11: Matrigel influences organoid regional patterning.

a) Overview of the protocol used to generate organoids to test the influence of matrix on brain organoids development (Cerebral organoid, Protocol II). b) Time-course images of organoids generated from mTagRFP–T-Tubulin-alpha1b iPSC line, showing merged brightfield and RFP expression in the presence (n = 4) and absence (n = 2) of Matrigel. Imaging was done with a widefield Nikon Ti2 microscope. c) UMAP embeddings of scRNA-seq data performed at day 16 with cells labeled by treatment (left) or colored by cluster and labeled by cell population (right). d) Feature plots showing normalized expression of selected marker genes. e) barplot showing cluster proportions of the cell populations in Matrigel and no-matrix organoids. f) Volcano plot showing differentially expressed genes upregulated in Matrigel (right) and upregulated in no-matrix conditions (left), calculated using a non-parametric Wilcoxon Rank Sum test with p-value cutoff using adjusted p-value. The p-value adjustment was performed using Bonferroni correction based on the total number of genes in the dataset. g-k) Images show example organoids stained whole-mount with fluorescence in situ hybridization chain reaction (HCR) showing regionalized expression of marker genes (false color, merged using Fiji) between organoids grown with Matrigel or with no-matrix. Three organoids were used for staining in each case. The n indicates the number of organoids imaged. g) Images show maximum intensity projections of organoids treated with Matrigel (top, n = 2) and or with no-matrix (bottom, n = 1) on day 15. Organoids were generated with protocol II, shown in a. Scale, 100 micrometers. h-k) Cross section images of HCR stainings. h) Day 7, Matrigel n = 1 and no-matrix n = 2. i) Day 11, n = 3 for both. j) Day 15 n = 3 for both. k) Day 15 no-matrix n = 2 and Matrigel n = 3. Organoids were generated with protocol shown in Fig. 1a. Scale, 250 micrometers.