Journal: Cell Reports Methods

Article Title: Wnt activation and dual SMAD inhibition for induction and maintenance of hindbrain-like neural stem cell from hiPSCs

doi: 10.1016/j.crmeth.2026.101372

Figure Lengend Snippet: Induction of Hb-LiNSCs (A) Schematic of the method used to induce human induced pluripotent stem cells (iPSCs) into Hb-LiNSCs. (B) Phase contrast images of iPSC and Hb-LiNSCs colonies, and differentiated neurons from left to right. Scale bars, 100 μm. Representative images ( n = 3, n represents the number of independent induction experiments). (C) E-cadherin ( CDH1 ), N-cadherin ( CDH2 ), SOX1 , SOX2 , PAX6 , and POU5F1 gene expression in iNSCs during the induction phase relative to that on day 0 (iPS cells). Bars indicate means; error bars as SD ( n = 3, n represents the number of independent induction experiments). (D) Immunocytochemical staining for SOX1, SOX2, NESTIN, PAX6, NANOG, POU5F1, and that of nuclei with DAPI in Hb-LiNSCs at day 7 after induction of iPSCs. Scale bars, 50 μm. Representative images ( n = 3, n represents the number of independent induction experiments). (E) Heatmap of selected genes representing pluripotency, NSCs, differentiated neurons, and canonical markers for the hindbrain region, with hierarchical clustering of genes and samples. Three samples of 1231A3 iPS (as control) and three samples of 1231A3 Hb-LiNSCs at PN0 day 7 were used. Normalized gene expression data are represented by the color intensity of the row Z scores. (F) Log fold change values ( x axis) are plotted against significance (negative log 10 of p -adjusted value, y axis) for PN0 day 7 Hb-LiNSC samples (three samples) vs. 1231A3 iPS control (three samples). Red color dots indicate significantly differentially expressed genes. Vertical dashed lines indicate the threshold of 1.5 log fold change, and the horizontal dashed lines indicates the significance threshold ( p = 0.05). (G) Gene enrichment analysis of the significantly ( p < 0.05) upregulated (log 2 fold change ≥1.5) genes in the PN0 Day 7 Hb-LiNSCs vs. iPSC control comparison for selected datasets. Dot size indicates the number of genes overlapping with the dataset, color intensity indicates the significance (top 10 terms ordered by p values), and the x axis indicates the combined score calculated using Enrichr. Relevant terms are highlighted in red.

Article Snippet: Hb-LiNSCs and/or attached neurospheres were fixed with 4% paraformaldehyde and then stained with antibodies against SOX1 (Cell Signaling, 4194S), SOX2 (R&D systems, MAB2018), NESTIN (R&D systems, MAB1259), NANOG (R&D systems, AF1997), POU5F1 (Santa Cruz Biotechnology, sc-5279), PAX6 (Abcam, EPR15858 ), TUBB3 (GeneTex, GTX85469), OLIG2 (GeneTex, GTX132732), or GFAP (Santa Cruz Biotechnology, sc-33673), and with DAPI for the nuclei.

Techniques: Gene Expression, Staining, Control, Comparison

Journal: Cell Reports Methods

Article Title: Wnt activation and dual SMAD inhibition for induction and maintenance of hindbrain-like neural stem cell from hiPSCs

doi: 10.1016/j.crmeth.2026.101372

Figure Lengend Snippet: Long-term maintenance of Hb-LiNSCs (A) Schematic of the maintenance plan (on the left). Hb-LiNSCs were passaged every week, and thereafter, at every fifth passage, one clone of cells was cryopreserved, one was used to extract bulk RNA, and one was used for maintaining the culture. The grouping into early, mid, and late samples according to the passage number is shown on the right. (B) Karyotyping of Hb-LiNSCs at PN53 derived from the 1231A3 human iPSC line. (C) Immunocytochemical staining of Hb-LiNSCs for TUBB3, SOX1, NESTIN, PAX6, NANOG, and SOX2, and that of nuclei with DAPI at PN60 (60 weeks) after induction of iPSCs. Scale bars, 50 μm. Representative images ( n = 3, n represents the number of independent induction experiments). (D) Heatmap of selected genes representing pluripotency, NSC, neural differentiation, and canonical markers for different brain regions, with hierarchical clustering of genes. The colored bar on the top indicates the samples. Samples PN5_1 to PN5_3 and PN60_1 to PN60_3 were derived from the 1231A3 iPSC line. Samples PN5_4 and PN5_5 were derived from HLAKO and SgT5 iPSC lines, respectively. “_diff” stands for differentiated neurons. Normalized gene expression data are represented by the color intensity of the row Z scores. (E) PCA plots of the first and second (PC1 and PC2) components for the iPSC control are indicated in red, and Hb-LiNSCs and their differentiated cells at early-, mid-, and late-passage numbers are indicated in green, purple, and blue, respectively. (F) Gene enrichment analysis of the significantly ( p < 0.05) upregulated (log 2 fold change ≥ 1.5) genes in the late PN group vs. iPSC control comparison for selected datasets. Dot size indicates the number of genes overlapping with the dataset, color intensity indicates the significance (top 10 terms ordered by p values), and the x axis indicates the combined score calculated using Enrichr. Relevant terms are highlighted in red. (G) Pairwise correlation heatmap showing the relationships among samples. The color intensity in the heatmap indicates the correlation, ranging from 1 (positive correlation) in red through 0 (no correlation) in white to −1 (negative correlation) in green.

Article Snippet: Hb-LiNSCs and/or attached neurospheres were fixed with 4% paraformaldehyde and then stained with antibodies against SOX1 (Cell Signaling, 4194S), SOX2 (R&D systems, MAB2018), NESTIN (R&D systems, MAB1259), NANOG (R&D systems, AF1997), POU5F1 (Santa Cruz Biotechnology, sc-5279), PAX6 (Abcam, EPR15858 ), TUBB3 (GeneTex, GTX85469), OLIG2 (GeneTex, GTX132732), or GFAP (Santa Cruz Biotechnology, sc-33673), and with DAPI for the nuclei.

Techniques: Derivative Assay, Staining, Gene Expression, Control, Comparison