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. 2023 Jan 18;18:5. doi: 10.1186/s13024-022-00591-3

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© The Author(s) 2023

Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

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Fig. 1 — Generation and characterisation of functional hiPSC-derived astrocytes. A Overview of the astrocyte differentiation protocol. hiPSCs were dissociated at day 0 (d0) and cultured for the induction phase as embryoid bodies (EBs) before they were plated for neural rosette formation at day 7 (d7). The neural rosettes underwent expansion to generate neural progenitor cells (NPCs) until day 16 (d16). NPCs were then differentiated into astrocyte progenitor cells (APCs, d25/d+0) after which the APCs were matured into astrocytes for an additional 4 weeks (d + 28). B Representative confocal images of astrocytes at week 4 (d + 28) of maturation stained with astrocytic markers S100β, AQP4, ALDH1L1 and SOX9 in addition to neuronal marker MAP2. Nuclei stained with DAPI. Scale bar: 75 μm. C Quantification of the number of cells positive for astrocyte and neuronal markers. Mean ± s.e.m. of 3 biological replicates (n = 15 images). D Heatmap of the expression of cell type-specific genes in 4 weeks mature astrocytes from RNAseq experiment performed in 3 biological replicates. Dark blue cells: FPKM > 100. E Functional assessment of spontaneous calcium transients in 6 weeks mature astrocytes loaded with the Fluo-4 dye. Y-axis range: ΔF/F₀ = 50%. F Quantification of peak Fluo-4 intensity. Mann–Whitney test. ****p < 0.0001. G Percentage of active astrocytes. Inactive astrocyte cut-off: Fluo-4 intensity increase < 100. Unpaired t test. *p < 0.05. H Volcano-plots of up- (red) and down- (blue) regulated genes in week 4 mature astrocytes. Green lines indicate thresholds: log₂FC < -1.0 with –log₁₀(FDR) > 2.0 were considered downregulated, and log₂FC > 1.0 with –log₁₀(FDR) > 2.0 were considered upregulated. I Comparison of differentially expressed genes between mutation sets. Graphs in panels (F and G) show mean ± s.e.m. of 3 biological replicates with each 2–6 technical replicates. Cell illustrations are modified from Smart Servier Medical Art licensed under a Creative Commons Attribution 3.0 Unported License (https://creativecommons.org/licenses/by/3.0/). See also Suppl. Figure 1–3, Additional file 2, as well as Additional files 3, 4, 5 and 6