Generation and validation of CTL and DS astrocytes from fibroblasts and hiPSCs. (A) Production workflow of hiPSCs, NPCs and astrocytes. (B) Karyotypes of CTL and DS lines. (C) Expression of pluripotency markers in hiPSCs (TRA-1-60 and Oct4), and selective markers in neuronal precursor cell (NPC) markers (Sox1 and Nestin) and astrocyte cultures (GFAP and Sox9). Scale = 20 μm in TRA-1-60/Oct4 image and 50 μm in all others. (D) At 90 days, astrocytes express markers GFAP, S100B and Sox9 and low levels of the NPC marker Sox1. Scale = 50 μm. (E) Astrocyte differentiation causes a reduction of the NPC marker Sox1 and upregulation of astrocytic markers S100B, GFAP and Sox9. (F) CTL and DS astrocytes express equal levels of astrocyte-related genes GFAP, EAAT1, glutamine synthetase (GS), SOX9 and ALDH1L1 (shown by qPCR). (G) Chromosome 21 genes are upregulated in DS astrocytes (shown by qPCR). Data are represented as mean ± SEM. Two-tailed, unpaired t-tests were performed *P ≤ 0.05, n = 3 (three experiments each performed in the three DS and three CTL cell lines). FBS: fetal bovine serum; NIM: neural induction medium; NPC: neural precursor cells.