Fig. 2: Generation of layer-specific neural cells.
From: Integration of 3D-printed cerebral cortical tissue into an ex vivo lesioned brain slice
a Schematic showing the differentiation timeline of hiPSCs to DNs and UNs, via the corresponding progenitors: DNPs and UNPs. Abbreviations: Day in vitro (DIV); Neural Induction Medium (NIM); Neural Maintenance Medium (NMM), Neural Terminal Medium (NTM); Growth Factors (GFs). DIV 0–8: hiPSCs induction, committing to a neural ectoderm lineage; DIV 8–19: neural ectoderm cells differentiating into DNPs; DIV 19–40: DNPs differentiating to UNPs during extended culture in NMM supplemented with growth factors (FGF-2, EGF and BDNF). DIV 19–29+ and DIV 40–50 + , DNPs and UNPs maturing as DNs and UNs, respectively. b Bright-field images (top row) and immunocytochemistry images (bottom row) of cells at different stages of differentiation: DIV 0, 31, 50+, correspond to GFP-labelled hiPSCs, RFP-labelled NPs and RFP-labelled UNs respectively. c Immunocytochemistry analysis of the young neuron marker β3-tubulin (labelled with the TUJ1 antibody), the neural stem cell marker (SOX2), middle-upper layer marker (SATB2), upper layer markers (CUX1 and BRN2) and the deep layer marker (CTIP2) expression in differentiated RFP-labelled UNs at DIV50+. See Supplementary Fig. 4b for further immunocytochemistry analysis of DNs. d Quantification of marker expression in c (n = 3 biological replicates; mean ± SEM; one-way ANOVA test). e Quantitative RT-PCR analysis of upper layer markers (CUX1, CUX2 and BRN2), deep layer marker (CTIP2), neurofilament marker (NESTIN) and neuroectoderm marker (PAX6). Marker expression of indicated cell types relative to RFP-labelled hiPSCs. (n = 3 biological replicates; mean ± SEM; one-way ANOVA test). For b and c, scale bar, 50 µm. For both d and e, ns not significant. *P < 0.05, **P < 0.01 and ***P < 0.001.
