Fig 2: Examples of successful organoid differentiation from EBs.Brain organoids were formed using guided and unguided differentiation protocols to induce telencephalic differentiation. Regardless of the use of U- or V-bottom wells, the EBs demonstrated budding neuroepithelia at the periphery, hinting at successful organoid formation. Examples of EBs produced in a (A) a U-bottom well without centrifugation, (B) a U-bottom well with centrifugation, (C) a V-bottom well without centrifugation, and (D) a V-bottom well with centrifugation. Scale bar: 500 µm. (E-F) Successful organoid differentiation at 30 daysof culture. Dotted lines indicate the rosette formations in the periphery of the organoid that show the undifferentiated neuroepithelial marker SOX2 and the neural identity markers of ß3-tubulin and MAP2. EBs cultured under (E) guided (F) and unguided differentiation protocols showed similar structural locations of the expressed markers, suggesting the neural predestination of the neuroepithelial tissue. (E) Guided protocols yielded a better-defined rosette and exhibited matured ß-tubulin and MAP2 expression, while (F) the unguided protocols showed initial signs of axonal (ß-tubulin) growth. Scale bar: 200 µm.

Fig 3: Protein expression analysis of HFSC spheres and adherent cells? Adherent HFSC and emerging spheres were stained with pluripotency and neural crest typical markers. Except for Nanog, OCT-4 and ZO-1 both adherent cells and spheres stained positively for SOX-2, SSEA-4, Tra-1–60, CD271, Nestin, ZO-1 and Connexin. Nuclei were stained with DAPI (for isotype control see supplementary data). Scale bars represent 50 µm

Fig 4: Elp2H206R impairs neurogenesis in mice.a Immunostaining of sagittal brain sections of adult wild-type and Elp2H206R mice, for layer-specific markers Cux1 (upper layers, II-IV) and Ctip2 (lower layers, V and VI). Cux1+ and Ctip2+ cell number, thickness (white lines), and cell density are shown. b–d Immunofluorescence staining of coronal cortical sections of 14.5 days post-coitum (dpc) wild-type and Elp2H206R embryos using Dcx (differentiating neurons), Tbr2 (intermediate progenitors) and Sox2 (apical progenitors) markers. Sections were counterstained with DAPI. b Dcx thickness (white lines) and Tbr2+ and Sox2+ cell number. c Dcx to Sox2 layer ratio and number of newborn intermediate progenitors (Sox2+Tbr2+). d Apoptosis (cleaved caspase-3, CC3) of Sox2+ cells and cell cycle exit (percentage of BrdU+Ki67- cells out of total BrdU+ cells). e Neurosphere assay of progenitor cells derived from cortices of 14.5 dpc embryos. Average number of neurospheres and sphere diameter are shown in three consecutive passages. For (a–e) n = 3 animals per genotype and 3 sections per animal (a–d); representative images are shown. f Differential expression (DE) of genes in the forebrain extracts of Elp2H206R vs. wild-type 14.5 dpc embryos (n = 6 per genotype). g Transformation of the DE data from (f) using TRIAGE. Top downregulated transcripts of genes involved in early neurogenesis are depicted. The vertical dotted lines demarcate p = 0.01 in (f), and p-value divided by the repressive tendency score (RTS) of 0.01 in (g). The horizontal dotted lines show RTS = 0.03 used as a threshold in the previous study56. Down- (blue) and upregulated (red) genes in Elp2H206R are shown. h Gene ontology (GO) analysis of DE genes showing down- and upregulated (arrows) functionally related genes in Elp2H206R. The color bar denotes fold change of up/downregulated GO terms. Scale bars, 100 µm. Statistical analysis: unpaired two-tailed t-test (a = 0.05) with Welch’s correction. Holm-Sidak correction was applied to adjust for multiple comparisons (a and e). Statistically significant differences are indicated (*p = 0.05; **p = 0.01; ***p = 0.001; ns not significant). Data represent mean ± SEM. CP cortical plate, SVZ sub-ventricular zone, VZ ventricular zone. Source data are provided as a Source Data file.

Fig 5: Comparison of stem cell potency associated gene expression between spheres and adherent cells. The relative normalized gene expression of Nanog, POU5F1, SOX-2 and KlF4 was examined for 3D spheres compared to single adhering cells. Gene expression data were normalized using the housekeeping genes GAPDH and B2M. Error bars represent three replicates. Significance in comparison to control was analyzed by one way anova testing. * indicates p < 0.05, ** indicates p < 0.01 and *** indicates p < 0.001