Fig. 2.

Gel embedding affects the expansion scale of neural epithelium and the neural progenitor's identity in nascent spinal cord organoids (SCOs).

a) Schematic illustrating the differentiation conditions used to obtain SCOs with gel embedding. EB: Embryoid body; NMP: Neuromesodermal Progenitors; NP: Neural; ROCKi: ROCK inhibitor.

b) Brightfield images of SCOs. On the 6th day, SCOs was embedded in Matrigel (M-gel) or spinal cord matrix gel (S-gel), with no gel embedding serving as the control (CTR). Pseudo-color images and bright field images (Upper right) at day 12 were presented. Scale bar = 200 μm.

c) Immunofluorescence staining in SCOs cryo-sections at day 6 of in vitro differentiation showing the expression of NMP(T/SOX2) and neuroepithelial cell (NE, ZO-1/SOX2) markers. Scale bar = 50 μm.

d) Expansion area of the SCOs sphere at day 1,6, and 12(n = 8 at each time point). Data are presented as the mean ± SD.

e) The area ratio of the light and dark field of the SCOs sphere at day 12. The light field and dark field were defined following the established threshold of 16 bit color map of pseudo-color image (n = 6).

f) Immunofluorescence staining in SCOs cryo-sections at day 12 of in vitro differentiation showing expression of NE marker. Scale bar = 200 μm.

g) Gene expression analysis of neural progenitor domains mitiotic markers in SCOs at day 12 of in vitro differentiation in 4 independent differentiations (n = 3, N = 12). Data are expressed as fold changes relative to that of D6[<0.05 (∗), <0.01 (∗∗), <0.001 (∗∗∗), <0.0001 (∗∗∗∗)].