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. 2023 Aug 9;3(8):100548. doi: 10.1016/j.crmeth.2023.100548

Figure 4.

Figure 4

Day 63 organoids demonstrate continued perturbation by signaling modulation

(A) UMAP with cell-type annotations from individually hashed D63 organoids exposed to BMP, CHIR, and SU5402:CHIR treatments. RPCprolif, proliferating RPC; RPCproneural, proneural RPC; ONHprog, optic nerve head progenitor; CN, cortical neuron; RG, radial glia; CP, choroid plexus.

(B)Heatmap of fold change in cell-type abundance compared with BMP. The data was modeled using a beta-binomial distribution and significance was determined by a Wald test. An asterisk (∗) in the center of the box indicates a q value of less than 0.05 after Benjamini-Hochberg correction.

(C) Violin plots of size-factor-normalized cell counts for day 63 organoids. Dark gray, control treatment in (B); light gray, no significant change; teal, significant decrease in abundance (p < 0.05); orange, significant increase in abundance (p < 0.05). Significance was determined as in (B).

(D) Stacked bar plots, each bar represents an individual organoid. Bars are colored by the cell-type composition. Right: cell-type color legend.

(E) Immunostaining of cryosectioned day 63 organoids with OTX2 (photoreceptors/RPE/CP, blue), MAP2 (retinal and cortical neurons, green), POU4F2 (RGC, red), and DAPI (gray). Arrow, non-retinal neurons; filled arrowhead, Otx2+ retinal tissue; empty arrowhead, Pou4f2+ retinal tissue. Scale bar represents 100 μm.

(F) Violin plots of percentage of area measurements for RPE as calculated from bright-phase microscopy images of day 62 organoids. Paired t tests followed by Bonferroni correction were used to determine significance (p < 0.05). Plots colored as in (C).

(G) Violin plots of size-factor-normalized RPE cell counts for day 63 organoids. Paired t tests followed by Bonferroni correction were used to determine significance (p < 0.05). Plots colored as in (C).

(H) Representative bright-field images of day 62 organoids from each group quantified in (F) and (G). Arrow, non-retinal tissue; filled arrowhead, retinal tissue; empty arrowhead, RPE. Scale bar represents 200 μm.