Fig 2: Comparison of four protocols P−, P+, G−, and G+ in terms of neuron-astrocyte ratio. (A) Immunostaining of MAP2 in neuron-astrocyte cultures on DIV28 that received different medium compositions. (B) Same cultures as in (A), but with astrocyte marker GFAP stained in red. (C/D) Same as (A/B), but cultures received half medium refreshments and P+ and G+ conditions only received CNTF during the first four medium changes. Scale bar: 100 μm.

Fig 3: SZ-related MAP2-IR decreases occur across regions on a within-subject basis. Interregional correlation matrix of loge(MAP2-IR) pairwise ratios (SZ:NPC). Points in the lower left quadrant indicate that the SZ subject had lower MAP2-IR than the paired NPC subject in both cortical areas. N = 18 pairs. AU = arbitrary units

Fig 4: Elimination of lipofuscin auto-fluorescence from the MAP2-IR signal. Lipofuscin (a) was imaged by excitation at 405 nm with emissions collected at 668 nm. Signal was subjected to thresholding at a value manually selected from an intensity histogram to derive a binary mask (c). Ridler–Calvard thresholding was used to convert 647 channel emissions (b) into a mask (d), from which the 405 channel mask was subtracted to yield a final mask (e) for intensity analysis. Arrowheads indicate sites of mask subtraction. Scale bars = 10 µm

Fig 5: Differentiation protocol of iPSCs into a co-culture of neurons and astrocytes. (A) Protocol of neural differentiation as published in de Leeuw et al. (2020). (B) Bright-field images of the neuronal differentiation process. (C) Characterization of cell types present in the NPC stage. NPCs were positive for neural progenitor marker NES, neuronal TUBB3, and MAP2, and to some extent for neural rosette marker ZO-1. Neural progenitor marker PAX6 and stem cell markers SSEA4 and ECAD were absent at this stage. (D) Characterization of cell types present in the neuronal differentiation stage. Neurons expressed TUBB3, mature marker NeuN, and axonal TAU and dendritic MAP2 in specific regions of the cells. Astrocyte marker GFAP and markers for synaptic signaling SYNPR and PSD95 were also abundantly present on DIV14 and DIV28. Insert on the third image from the left shows SYNPR staining only for clarity. (E) Relative gene expression changes in iPSCs, NPCs, and the neuron-astrocyte co-culture of cell markers for stem cells (POU5F1 and NANOG), ectodermal differentiation (NEUROG1 and NES), neurons (TUBB3 and MAP2), astrocytes (GFAP), synaptic markers (SYNPR and DLG4), and vesicle transporters (SLC17A6 and SLC32A1). Sample size: iPSCs/neurons day 14 n = 4, NPCs n = 3, neurons day 7/21/28 n = 5. For clarity reasons, the reader is referred to the supplementary data for mean differences and significance values between experimental groups. Scale bar: 100 μm. CNTF, ciliary neurotrophic factor; EB, embryoid body; G−/+, medium from Gunhanlar et al. (2018) without (−) and with (+) CNTF; NI, STEMdiff™ Neural Induction Medium; NP, STEMdiff Neural Progenitor Medium; P−/P+, neural differentiation medium adopted from Pistollato et al. (2017) without (−) and with (+) addition of CNTF; NPC, neural progenitor cell.