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. 2025 Feb 11;31:101561. doi: 10.1016/j.mtbio.2025.101561

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© 2025 The Authors

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

PMC Copyright notice

Fig. 3 — Extended gel embedding enlarges the neural epithelium of SCOs and affects Rostro-Caudal/Dorsal-Ventral pattern in SCOs.

a) Bright field and pseudo-color images (Below) of SCOs. Scale bar = 500 μm.

b) Immunofluorescence in SCOs cryo-sections at day 20 of differentiation showing expression of neural progenitors (Nestin/Ki-67/Tuji) markers. Scale bar = 500 μm.

c) Expansion area of the SCOs sphere at day 20(n = 8). Data are presented as the mean ± SD.

d) The area ratio of the light and dark field of the SCOs sphere. Data are presented as mean ± SD.

e) Calculate the area of the neuroepithelial region (Nestin⁺) of the SCOs using immunofluorescence staining images of the neuroepithelium(n = 6). Data are presented as the mean ± SD.

f) GO enrichment of differential genes (up-regulated) in SCOs transcriptomics at day 20 of differentiation. The mRNA library data were collected from an independent differentiation of the ESC-H9 cell line.

g) Gene expression analysis of neural progenitor domains mitiotic markers in SCOs. Data are expressed as fold changes to that of D6[<0.05 (∗), <0.01 (∗∗), <0.001 (∗∗∗), <0.0001 (∗∗∗∗)].

h) Gene expression analysis of genes expressed along the dorso-ventral axes at day 20 of differentiation. Data are presented as the mean. Each cell represents two independent differentiations of a single cell line[Kruskal-Wallis test p = 0.1461(ESC9), p = 0.0285(iPSC2) for LHX1, p = 0.6053(ESC9), p = 0.0406(iPSC2) for EVX1, p = 0.7571(ESC9), p = 0.0714(iPSC2) for EN1, p = 0.0175(ESC9), p = 0.0026(iPSC2) for CHX10, p = 0.1506(ESC9), p = 0.1675(iPSC2) for GATA3, p = 0.4849(ESC9), p = 0.0463(iPSC2) for HB9, P < 0.0001(ESC9), p = 0.0110(iPSC2) for ISL1, p = 0.9784(ESC9), p = 0.0500(iPSC2) for SIM1].

i) Gene expression analysis of genes expressed along the rostro-caudal axes at day 20 of differentiation. Data are presented as the mean. Each cell represents two independent differentiations of a single cell line[Kruskal-Wallis test p = 0.0068(ESC9), p = 0.0370(iPSC2) for HOXA2, p = 0.0024(ESC9), p = 0.0074(iPSC2) for HOXB4, p = 0.1187(ESC9), p = 0.1879(iPSC2) for HOXC5, p = 0.2393(ESC9), p = 0.6186(iPSC2) for HOXC6, p = 0.0072(ESC9), p = 0.9075(iPSC2) for HOXC9].

j) Representative Immunofluorescence images of neural tube. Scale bar = 200 μm.