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. 2022 Sep 8;2(9):100289. doi: 10.1016/j.crmeth.2022.100289

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© 2022 The Author(s)

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

PMC Copyright notice

A FGF2 concentration in feeder-free iPSCs contributes to forebrain identity acquisition in EBs

(A) Schematic illustration of generating EBs and neuroepithelia from feeder-free iPSCs with high (100 ng/mL, high-FGF2 iPSCs) or low (10 ng/mL, low-FGF2 iPSCs) concentrations of FGF2.

(B) Representative images of high- or low-FGF2 iPSCs (201B7), EBs, and neuroepithelia. n > 30. Scale bar, 300 μm.

(C) Diameter of high- or low-FGF2 EBs generated from high- or low-FGF2 iPSCs cultured for 3, 4, and 10 weeks. Greater than 6 EBs from three different batches.

(D) Volcano plot of differential gene expression in high- versus low-FGF2 iPSCs by DEG analysis. The expression difference is considered significant for a log2 fold change of >1 (outer pink dash vertical line) and a p value of <0.00124 corresponding to 0.05 in adjusted p value (pink dash horizontal line). See also Table S1.

(E) Volcano plot of differential gene expression in high- versus low-FGF2 EBs by DEG analysis. The expression difference is considered significant for a log2 fold change of >1 (outer pink dash vertical line) and a p value of <0.00242 corresponding to 0.05 in adjusted p value (pink dash horizontal line). See also Table S1.

(F) Enrichment analysis for GO biological process of significant up- or down-regulated genes between high- and low-FGF2 iPSCs. Six top terms are shown. See also Figure S1A and Table S1.

(G) Enrichment analysis for GO biological process of significant up- or down-regulated genes between high- and low-FGF2 EBs. Six top terms are shown. See also Figure S1B and Table S1.

(H) Heatmap of neural stem/progenitor cell (NS/PC), forebrain, midbrain, hindbrain, and spinal cord marker gene expression in high- and low-FGF2 iPSCs (n = 3), and high- and low-FGF2 EBs on day 7 (n = 5). See also Figure S2A.

(I) PCA map showing PC1 and PC3 of high- and low-FGF2 iPSCs and high- and low-FGF2 EBs (pink triangle: high-FGF2 iPSCs; green circle: low-FGF2 iPSCs; purple triangle: high-FGF2 EBs; blue circle: low-FGF2 EBs). See also Figure S2B.

(J) Immunohistochemical staining of the forebrain marker FOXG1 and the neural progenitor marker SOX2 in high- (left panel) or low-FGF2 (right panel) neuroepithelia derived from 201B7 iPSC line on day 14 (n = 3). The nuclei were stained with Hoechst 33258. Scale bar, 50 μm. See also Figures S2C–S2F.