Fig 2: Analysis of neural marker expression in the 3D bioprinted construct. (A) Phase contrast images of cells within the 3D bioprinted construct (“3D printed” panels), at the indicated days post printing, and cells in conventional monolayer conditions (“2D” panels) or resuspended in the bioink (“3D bulk” panels) and maintained for the same time of differentiation. (B) RT-PCR analysis of neuronal progenitor markers (PAX6, FOXG1, TBR2), a cortical neuron marker (TBR1) and an astrocyte marker (GFAP). GAPDH was used as a housekeeping control. (C) Immunostaining analysis of bioprinted cells at DPP7. MAP2 (green), TBR1 (red) and DAPI (blue) signals are shown. Scalebar: 150 µm. (D) Mosaic reconstruction of confocal images of bioprinted neural cells at DPP70, showing the entire sample, stained with MAP2 (green), TBR1 (white) and GFAP (red) antibodies. Scalebar: 2 mm. (D’) Mosaic reconstruction of confocal images of the region inside the white box in panel D, acquired at higher resolution. Scalebar: 300 µm.

Fig 3: Characterization of iPSCs during neural induction and differentiation. (A) Fluorescence microscopy images of Vimentin (green), Pax6 (red), Oct4 (magenta), and DAPI (blue) in euploid and trisomic iPSCs at 6 days post-neural induction. (B) Fluorescence microscopy images of DCX (green) and DAPI (blue) in euploid and trisomic iPSCs at 18 days post neural induction. (C) Fluorescence microscopy images of Tuj1 (red), MAP2 (green), and DAPI (blue) in euploid and trisomic iPSCs following neuronal differentiation

Fig 4: Patient presentations and iPSC characterization.(A) Table of presentations for ECHS1 patient at clinical visits. (B) Brain MRI scan of the ECHS1 patient showing T2 hyperintensity in deep white matter of the frontal and parietal lobes and basal ganglia (marked by arrows). (C) The genomic DNA of the iPSC clones were evaluated using target-specific Sanger sequencing to confirm mutations (n = 3). (D) Verification of trilineage differentiation potential of iPSC was carried out using spontaneous embryoid body (EB) culture. iPSC colonies were suspended in non-ES cell medium and allowed to form rotund structures in low attachment plates. The cells were allowed to spontaneously differentiate and plated onto gelatin-coated culture dishes on day 8. Immunostaining revealed cells from all three lineages: mesoderm, alpha-smooth muscle actin (SMA); endoderm, Sox17; and ectoderm, Otx2. Scale bars, 5 mm (EB), 10 µm (mesoderm), 100 µm (endoderm), and 15 µm (ectoderm) (n = 3). DAPI, 4',6-diamidino-2-phenylindole. (E) iPSC differentiation to NPCs (Pax6 and Nestin), fibroblasts (HSP47 and FSP), CMs (Nkx2.5 and cardiac troponin), and neurons (Map2 and NeuN). Scale bars, 100 µm (NPCs), 100 µm (fibroblasts), 10 µm (CMs), and 50 µm (neurons) (n = 3). The “n” is representative of the biological replicates. The data are representative of three independent experiments.

Fig 5: Immunocytochemistry (ICC) / Immunofluorescence analysis at Day 13To assess the self-patterning stage beyond the characterization by brightfield microscopy, immunofluorescence imaging analysis was performed at day 13.(A) Cells at this stage show abundant expression of the structural cytoskeletal protein expressed in early neurons, beta-3 tubulin (TUBB3 in red) commonly used as an immature neuronal marker (A; DAPI in blue).(B and C) Additionally, cells are immunostained for the early telencephalic marker FOXG1 (B in green; DAPI in blue), and PAX6 (C in red; DAPI in blue).(D) SOX2 is also highly expressed at this stage (D in red) and the progenitor cells also express the intermediate filament NESTIN (D in green). Scale bar corresponds to 20 µm.