Journal: Nature Neuroscience

Article Title: Single-cell genotyping and transcriptomic profiling of mosaic focal cortical dysplasia

doi: 10.1038/s41593-025-01936-z

Figure Lengend Snippet: a , UMAP visualization comparing age distribution of subjects between the reference Velmeshev et al. postmortem control dataset (V19, left) and the complete integrated dataset from this study (right). Color gradient indicates subject age in years. b , Relative proportion of major cell types across individual samples. Cell types include glutamatergic neurons (GluN and GluL2-6), GABAergic interneurons (IN-MGE and IN-CGE), glial cells (astrocytes, oligodendrocytes, OPCs) and other cell types (microglia, endothelial cells). Abbreviations: Glu, glutamatergic; N, neurons; L, layer; CC, cortico-cortical projection neurons; IN-MGE/CGE, interneurons originating from the medial/caudal ganglionic eminence; OPC, oligodendrocyte precursor cells. c , Individual UMAP plots showing nucleus distribution for each patient and control. d , Quality metrics for snRNA-seq data across cell types and individuals: total count of unique molecular identifiers (UMIs) per nucleus (N counts), mean number of unique genes (N genes) detected per nucleus and percentage (%) of transcripts from mitochondrial genes.

Article Snippet: Nuclei were resuspended in staining buffer (2% bovine serum albumin, 1 mM EDTA and phosphate-buffered saline) and immunostained overnight at 4 °C with the following primary antibodies: conjugated anti-NEUN-PE for neurons (1:1,000, Milli-Mark, cat. no. FCMAB317PE) , conjugated anti-PU.1-AF647 for microglia (1:100, Cell Signaling Technology, cat. no. 2240S conjugate) , , unconjugated anti-OLIG2 for oligodendrocytes (1:500, Abcam, cat. no. ab109186) , conjugated anti-PAX6-APC for astrocytes (1:1,000, Novus Biologicals, cat. no. NBP2-34705APC) and rabbit unconjugated anti-TBR1 for excitatory neurons (1:1,000, Abcam, cat. no. ab31940) .

Techniques: Control

Journal: Nature Neuroscience

Article Title: Single-cell genotyping and transcriptomic profiling of mosaic focal cortical dysplasia

doi: 10.1038/s41593-025-01936-z

Figure Lengend Snippet: a , Quantitative assessment of differentially expressed genes (DEGs) between patients and controls (log 2 (FC) > 0.4, p-value < 0.05, Fig. ). Left: Correlation between DEG count and total number of nuclei per cell type. Right: Correlation between DEG count and mean number of genes detected per nucleus in each cell type. Pearson correlation coefficients (R) and p-values are shown. Abbreviations: Astro, astrocytes; Glu, glutamatergic; N, neurons; L, layer; IN-MGE/CGE, interneurons originating from the medial/caudal ganglionic eminence; Micro, microglia; Oligo, oligodendrocytes; OPC, oligodendrocyte precursor cells. b-f , Gene Ontology (GO) pathway analysis of DEGs comparing focal FCDII patients to controls for interneurons ( b ), glutamatergic neurons ( c ), astrocytes ( d ), oligodendrocytes ( e ) and microglia ( f ). Top 10 significantly enriched GO terms with adjusted (adj.) p-value < 0.05 are shown per cell type.

Article Snippet: Nuclei were resuspended in staining buffer (2% bovine serum albumin, 1 mM EDTA and phosphate-buffered saline) and immunostained overnight at 4 °C with the following primary antibodies: conjugated anti-NEUN-PE for neurons (1:1,000, Milli-Mark, cat. no. FCMAB317PE) , conjugated anti-PU.1-AF647 for microglia (1:100, Cell Signaling Technology, cat. no. 2240S conjugate) , , unconjugated anti-OLIG2 for oligodendrocytes (1:500, Abcam, cat. no. ab109186) , conjugated anti-PAX6-APC for astrocytes (1:1,000, Novus Biologicals, cat. no. NBP2-34705APC) and rabbit unconjugated anti-TBR1 for excitatory neurons (1:1,000, Abcam, cat. no. ab31940) .

Techniques:

Journal: Nature Neuroscience

Article Title: Single-cell genotyping and transcriptomic profiling of mosaic focal cortical dysplasia

doi: 10.1038/s41593-025-01936-z

Figure Lengend Snippet: a , Distribution of 808 genotyped nuclei in UMAP space: 117 were classified as Mut. (pt10 = 89, pt9 = 25, pt7 = 2, pt6 = 1) or as Ref. Right, Mut. nuclei percentages per cell type (top) and across cell types (bottom). b , Representative images of co-immunofluorescence staining on formalin-fixed paraffin-embedded sections ( n = 1 per patient) showing mTOR-hyperactive (pS6 + ) neurons (NEUN + , pt2), astrocytes (GFAP + , pt2), oligodendrocytes (OLIG2 + , pt2) and microglia (IBA1 + , pt10). Nuclei (in blue) are labeled with DAPI. Scale bars, 20 µm. All patients included in this experiment are detailed in Supplementary Table . c , Cytomegalic cells representing a minor fraction of mutated cells. Left, representative immunostaining of SMI311 + DNs and VIM + BCs on frozen brain tissue from pt5. Nuclei (in blue) are labeled with DAPI for total cell counting. Scale bar, 25 µm. Right, mutated cell percentage (inferred by the detected VAF) and proportion of DNs or BCs identified in each patient ( n = 1 section/patient/staining was analyzed). d , Schematic of the distribution of mutated cells across cell types and the fraction of mutated cytomegalic cells in pt10. Astro, astrocytes; Endo, endothelial cells; Hemi, hemispherical; Oligo, oligodendrocytes; Micro, microglia.

Article Snippet: Nuclei were resuspended in staining buffer (2% bovine serum albumin, 1 mM EDTA and phosphate-buffered saline) and immunostained overnight at 4 °C with the following primary antibodies: conjugated anti-NEUN-PE for neurons (1:1,000, Milli-Mark, cat. no. FCMAB317PE) , conjugated anti-PU.1-AF647 for microglia (1:100, Cell Signaling Technology, cat. no. 2240S conjugate) , , unconjugated anti-OLIG2 for oligodendrocytes (1:500, Abcam, cat. no. ab109186) , conjugated anti-PAX6-APC for astrocytes (1:1,000, Novus Biologicals, cat. no. NBP2-34705APC) and rabbit unconjugated anti-TBR1 for excitatory neurons (1:1,000, Abcam, cat. no. ab31940) .

Techniques: Immunofluorescence, Staining, Formalin-fixed Paraffin-Embedded, Labeling, Immunostaining, Cell Counting

Journal: Nature Neuroscience

Article Title: Single-cell genotyping and transcriptomic profiling of mosaic focal cortical dysplasia

doi: 10.1038/s41593-025-01936-z

Figure Lengend Snippet: a , Expression analysis of cell-type-specific markers in mutation detected (Mut.) and reference detected (Ref.) nuclei. Dot size represents the proportion of nuclei expressing each marker; color intensity indicates average normalized expression level. Abbreviations: N, neurons; Glut.N, glutamatergic neurons; IN, interneurons; Astro, astrocytes; Oligo, oligodendrocytes; Micro, microglia. b , Fluorescence-activated nuclei sorting (FANS) gating strategy for cell population enrichment. Representative gating from pt9 is shown. Sequential gating begins with initial selection based on DAPI nuclear staining, followed by separation of neuronal (NEUN+) and non-neuronal populations. Cell-type-specific enrichment was then achieved using TBR1 for glutamatergic neurons, PAX6+/NEUN- for astrocytes, OLIG2+/NEUN- for oligodendrocytes, and PU.1 + /NEUN- for microglia. Note: TBR1 subpopulation analysis was only performed for pt9 due to tissue constraints. c , Quantification of somatic mutations across FANS-enriched cell populations was performed using two complementary approaches: ddPCR detection for MTOR and PIK3CA variants, and deep targeted amplicon sequencing (TAS) for the RHEB variant in pt9. Mutation-positive bulk brain DNA and mutation-negative blood DNA served as controls. ddPCR detection limits (LOD) were >3 FAM+ mutated droplets for MTOR , >6 FAM+ mutated droplets for PIK3CA in ddPCR analysis. Results are presented as mean ± SD where technical replication was feasible. Due to limited tissue availability, biological replicates could not be performed, and technical replicates were not possible for specific cell populations in patients pt4 (PAX6+/NEUN- and OLIG2+/NEUN-), pt9 (none), pt12 (PU.1+/NEUN-), and pt14 (PU.1+/NEUN-).

Article Snippet: Nuclei were resuspended in staining buffer (2% bovine serum albumin, 1 mM EDTA and phosphate-buffered saline) and immunostained overnight at 4 °C with the following primary antibodies: conjugated anti-NEUN-PE for neurons (1:1,000, Milli-Mark, cat. no. FCMAB317PE) , conjugated anti-PU.1-AF647 for microglia (1:100, Cell Signaling Technology, cat. no. 2240S conjugate) , , unconjugated anti-OLIG2 for oligodendrocytes (1:500, Abcam, cat. no. ab109186) , conjugated anti-PAX6-APC for astrocytes (1:1,000, Novus Biologicals, cat. no. NBP2-34705APC) and rabbit unconjugated anti-TBR1 for excitatory neurons (1:1,000, Abcam, cat. no. ab31940) .

Techniques: Expressing, Mutagenesis, Marker, Fluorescence, Selection, Staining, Amplification, Sequencing, Variant Assay

Journal: Nature Neuroscience

Article Title: Single-cell genotyping and transcriptomic profiling of mosaic focal cortical dysplasia

doi: 10.1038/s41593-025-01936-z

Figure Lengend Snippet: a-e , Validation of transcriptional changes (that is genes with absolute log 2 (FC) > 0.3) between mutation detected (Mut.) and reference detected (Ref.) glutamatergic neurons (GluN) and astrocytes (Astro). Box plots depict the median and interquartile range, with whiskers indicating minimum and maximum values. a-b , Differential expression analysis using 50 random subset comparisons of n = 29 GluN ( a ) or n = 17 astrocytes ( b ) amongst Mut. and Ref. nuclei of pt9 and pt10 compared to the rest of GluN and astrocytes, respectively. Left, proportion of shared dysregulated genes in ‘random’ vs ‘observed’ Mut. vs Ref. comparisons. Right, Jaccard similarity index. Wilcoxon signed rank test with continuity correction confirms significant differences between ‘random’ and ‘observed’ dysregulated genes in both GluN and astrocytes. c , Linear regression model using ‘random’ dysregulated genes from iteration n.1 of GluN and Astro cannot predict Mut. vs Ref. nuclei. Box plots depict the median and interquartile range, with whiskers indicating minimum and maximum values. d , Linear regression model using observed dysregulated genes successfully discriminates Mut. from Ref. nuclei for both GluN and Astro, with potential false negatives identified in 14% (1/7) of pt9 and 9% (8/85) of pt10 Ref. GluN nuclei (indicated by black dotted box above red threshold line). Box plots depict the median and interquartile range, with whiskers indicating minimum and maximum values. e , Expression patterns of GluN and Astro ‘observed’ dysregulated genes across patients (pt9-10) and controls (ct1-3). Analysis restricted to samples with >10 Mut./Ref. nuclei. Statistical analysis of average gene expression in Mut. vs. Ref. GluN by individual using Kruskal-Wallis test shows significant mutation effects (genes upregulated in Mut.: H statistics = 340.50, FDR-adjusted p-value: 9.92×10 − 76; genes downregulated in Mut.: H statistics = 30.59, FDR-adjusted p-value: 3.19×10 − 8). No statistical test was performed for astrocytes since only one patient with > 10 Mut. nuclei was available. f , Top 10 GO terms for Mut. vs. Ref. dysregulated genes in astrocytes. g , Mean expression of GluN dysregulated genes between Ref. nuclei from patients vs. controls in Mut., Ref. and control GluN from pt9-10 and ct1-2. Left, all dysregulated genes. Right, epilepsy-related dysregulated genes. h , Top 10 GO terms for patient Ref. vs. control. nuclei dysregulated genes. Only significant GO terms (adjusted p-value < 0.05) are shown.

Article Snippet: Nuclei were resuspended in staining buffer (2% bovine serum albumin, 1 mM EDTA and phosphate-buffered saline) and immunostained overnight at 4 °C with the following primary antibodies: conjugated anti-NEUN-PE for neurons (1:1,000, Milli-Mark, cat. no. FCMAB317PE) , conjugated anti-PU.1-AF647 for microglia (1:100, Cell Signaling Technology, cat. no. 2240S conjugate) , , unconjugated anti-OLIG2 for oligodendrocytes (1:500, Abcam, cat. no. ab109186) , conjugated anti-PAX6-APC for astrocytes (1:1,000, Novus Biologicals, cat. no. NBP2-34705APC) and rabbit unconjugated anti-TBR1 for excitatory neurons (1:1,000, Abcam, cat. no. ab31940) .

Techniques: Biomarker Discovery, Mutagenesis, Quantitative Proteomics, Expressing, Gene Expression, Control

Journal: Nature Neuroscience

Article Title: Single-cell genotyping and transcriptomic profiling of mosaic focal cortical dysplasia

doi: 10.1038/s41593-025-01936-z

Figure Lengend Snippet: a , Left, LCM–seq workflow for capturing pools of DNs, BCs and NNs from eight patients (pt1–5 and pt7–9). Right, heatmap of NEFM and VIM normalized expression with unsupervised hierarchical clustering. b , Label transfer of LCM–seq samples on to the snRNA-seq UMAP space showing NNs or DNs matching with GluNs and BCs with astrocytes. c , Left, NRGN and GFAP normalized expression heatmap with unsupervised hierarchical clustering. Right, co-immunofluorescence showing NRGN in pS6 + /SMI311 + DNs and GFAP in pS6 + /VIM + BCs (pt5) ( n = 1 section/patient/staining analyzed). GFAP-pS6 and VIM-pS6 double stainings were performed on two consecutive sections and the same BC was recognized in both sections. Nuclei (in blue) are labeled with DAPI. Scale bars, 50 µm. d , Visium spatial transcriptomics showing intermingled spots containing DNs and BCs across the tissue (pt5). Magnified images show representative DN- and BC-containing spots after hematoxylin and eosin staining ( n = 1 section per patient analyzed). Scale bars, 1.5 mm; insets = 55 µm. e , Top markers of DN- and BC-containing spots (pt5). Known histological markers for DNs ( NEFM ) and BCs ( CRYAB ) are enriched in spots with DNs and BCs. f , Spatial semi-supervised clustering of Visium spots showing clusters enriched in GluNs, astrocytes and oligodendrocytes (pt5) with top marker genes in parentheses. g , Distinct clusters for DNs, BCs, astrocytes (Astros) and GluNs from single cells (pt5 and pt9) of the MERSCOPE UMAP space. h , Heatmap of the top ten DN or BC markers with representative MERSCOPE images (pt5). DNs are identified as pS6 + /NEUN + and BCs as pS6 + /NEUN − ( n = 1 section per patient analyzed). Scale bars, 50 µm. i , Left, number of shared dysregulated genes across Mut. versus Ref. GluNs (snRNA-seq), DNs versus NNs (LCM–seq) and DN-containing spots (Visium). Right, top GO terms of DN upregulated genes. Ribo-nt., ribonucleotides; metab., metabolic; proc., process; Ribo-ns., ribonucleosides; RP., ribosomal proteins; rNTP, ribonucleoside triphosphates. j , Representative images of strong VDAC1 immunostaining in pS6 + DNs (pt2) ( n = 1 section/patient/staining analyzed). Scale bars, 50 µm. k , Electron microscopy of DNs (pt5) showing an accumulation of vesicular, swollen, damaged mitochondria (black circles) ( n = 1 section per patient analyzed). Scale bar, 2.5 µm. Detailed sample information for each experiment and analysis is provided in Supplementary Table . expr., expression; max., maximum; min., minimum.

Article Snippet: Nuclei were resuspended in staining buffer (2% bovine serum albumin, 1 mM EDTA and phosphate-buffered saline) and immunostained overnight at 4 °C with the following primary antibodies: conjugated anti-NEUN-PE for neurons (1:1,000, Milli-Mark, cat. no. FCMAB317PE) , conjugated anti-PU.1-AF647 for microglia (1:100, Cell Signaling Technology, cat. no. 2240S conjugate) , , unconjugated anti-OLIG2 for oligodendrocytes (1:500, Abcam, cat. no. ab109186) , conjugated anti-PAX6-APC for astrocytes (1:1,000, Novus Biologicals, cat. no. NBP2-34705APC) and rabbit unconjugated anti-TBR1 for excitatory neurons (1:1,000, Abcam, cat. no. ab31940) .

Techniques: Expressing, Immunofluorescence, Staining, Labeling, Marker, Immunostaining, Electron Microscopy

Journal: Nature Neuroscience

Article Title: Single-cell genotyping and transcriptomic profiling of mosaic focal cortical dysplasia

doi: 10.1038/s41593-025-01936-z

Figure Lengend Snippet: a , Semi-supervised clustering of Visium data from patients pt2, pt5 and pt9. Clusters (highlighted in red) are annotated based on predominant marker genes. To differentiate clusters enriched for the same cell type, the top expressed marker was added to the cluster annotation. Clusters lacking clear cell-type enrichment in pt9 are labeled ‘Unknown’. Abbreviations: N., neurons; IN, interneurons; NF high, cluster with high levels of neurofilament genes; Astro, astrocytes; Oligo, oligodendrocytes; Micro, microglia. On the right: corresponding hematoxylin and eosin (HE) -stained section. Scale bars = 1.5 mm. b , Spatial distribution of DN/BC-containing spots in pt2 and pt9 showing distribution across both anatomical space and transcriptionally-defined clusters. To differentiate clusters enriched for the same cell type, the top discriminating marker expressed was added to the cluster annotation. Scale bars = 1.5 mm. c , Expression analysis of cluster-defining markers in each Visium sample. Dot size indicates percentage of expressing spots; color intensity shows average normalized expression. Abbreviations: Pct. Expr., percentage of spots expressing the genes; Avg. Exprs., average normalized gene expression per group. d , Spatial distribution of cortical layer marker expression scores calculated using previously defined gene sets (Maynard et al., 2021) . Higher scores indicate increased expression of the gene set.

Article Snippet: Nuclei were resuspended in staining buffer (2% bovine serum albumin, 1 mM EDTA and phosphate-buffered saline) and immunostained overnight at 4 °C with the following primary antibodies: conjugated anti-NEUN-PE for neurons (1:1,000, Milli-Mark, cat. no. FCMAB317PE) , conjugated anti-PU.1-AF647 for microglia (1:100, Cell Signaling Technology, cat. no. 2240S conjugate) , , unconjugated anti-OLIG2 for oligodendrocytes (1:500, Abcam, cat. no. ab109186) , conjugated anti-PAX6-APC for astrocytes (1:1,000, Novus Biologicals, cat. no. NBP2-34705APC) and rabbit unconjugated anti-TBR1 for excitatory neurons (1:1,000, Abcam, cat. no. ab31940) .

Techniques: Marker, Labeling, Staining, Expressing, Gene Expression

Journal: Molecular Vision

Article Title: Embryonic stem cell–derived photoreceptor precursor cells differentiated by coculture with RPE cells

doi:

Figure Lengend Snippet: Primary antibody information.

Article Snippet: PAX6 , 1:100 , Novus biologicals , , Littleton, CO.

Techniques:

Journal: Advanced Science

Article Title: TTNPB Promotes Human Pluripotent Stem Cell‐to‐Neural Stem Cell Transition via Modulation of Chromatin Accessibility and the S‐(5′‐adenosyl)‐L‐homocysteine/Choline Metabolic Network

doi: 10.1002/advs.202515648

Figure Lengend Snippet: Synergistic induction of advanced neural stem cells (ANSCs) from PSCs by TTNPB and CHIR99021. (A), Schematic of the generation of advanced neural stem cells (ANSCs) from pluripotent stem cells (PSCs). (B), Morphology and alkaline phosphatase staining of PSCs (passage 20) and ANSCs (passage 50). Scale bar: 100 µm. (C), Immunofluorescence staining of pluripotency markers ( OCT4, SOX2, NANOG ) and lineage markers ( SOX17, TBXT, PAX6 ) of PSCs and ANSCs. Scale bar: 50 µm. (D), Volcano plot showing differentially expressed genes (DEGs) between ANSCs and PSCs (|log 2 FC| > 1, P < 0.05). (E), Heatmap illustrating the expression levels of pluripotency genes, neuroectodermal genes, genes associated with retinoic acid (RA) signaling, and downstream target genes of the RA signaling pathway in ANSCs and PSCs. (F), Hierarchical clustering of transcriptomic profiles from PSCs, NSCs, and ANSCs (distance metric: 1‐ Spearman correlation coefficient). (G), Schematic and morphology on day 9 of ANSCs during spontaneous differentiation in N2B27 medium. Scale bar: 100 µm. (H), RT‐qPCR analysis of SOX2, SOX1, SOX10, HOXA1, PAX6, TUBB3 , and NESTIN expression in ANSCs before (Day 0) and after spontaneous differentiation (Day 9). Data were normalized to GAPDH . Error bars represent mean ± SD. (n = 3 biological replicates). P values were determined using two‐tailed Student's t ‐tests. (I), Representative images of mouse 8‐cell embryos at 24 and 48 h post‐injection with ANSCs or PSCs. Yellow arrows indicate the injected cells carrying the tdTomato fluorescent protein. Scale bar: 100 µm. (J), Cell counts of ANSCs and PSCs contributing to mouse embryos were performed separately. (K), Representative images of ANSCs treated with TTNPB alone (CHIR99021 withdrawal). T: TTNPB; CHIR: CHIR99021. Scale bars: 100 µm. (L), Representative images of NSCs treated with LIF alone (CHIR99021 withdrawal). L: LIF (leukemia inhibitory factor). Scale bars: 100 µm. (M), Representative images of NSCs treated with CHIR99021 alone (LIF withdrawal). C: CHIR99021. Scale bars: 100 µm.

Article Snippet: The primary antibodies used included: rabbit polyclonal OCT4 (Novus Biologicals, #NBP2‐15053, 1:200), rabbit polyclonal NANOG (PeproTech, #P236, 1:200), goat polyclonal SOX2 (R&D Systems, #AF2018, 1:200), rabbit monoclonal NESTIN (Boster, #PB9874, 1:200), rabbit polyclonal PAX6 (Elabscience, #E‐AB‐61653, 1:200), rabbit polyclonal N‐Cadherin (Abcam, #ab76057, 1:200), goat polyclonal Brachyury (R&D Systems, #AF2085, 1:100), goat polyclonal SOX17 (R&D Systems, #AF1924, 1:200), mouse monoclonal NeuN (CST, #93972, 1:100), mouse monoclonal TUBB3 (Bioss, #BSM‐33177M, 1:200), rabbit polyclonal GFAP (Bioss, #bs‐0199R, 1:200), and rabbit monoclonal CDX2 (Biogenex, #Cdx2‐88).

Techniques: Staining, Immunofluorescence, Expressing, Quantitative RT-PCR, Two Tailed Test, Injection

Journal: Advanced Science

Article Title: TTNPB Promotes Human Pluripotent Stem Cell‐to‐Neural Stem Cell Transition via Modulation of Chromatin Accessibility and the S‐(5′‐adenosyl)‐L‐homocysteine/Choline Metabolic Network

doi: 10.1002/advs.202515648

Figure Lengend Snippet: ANSCs exhibit distinct chromatin accessibility landscapes. (A), ATAC‐seq analysis of PSCs, NSCs, and ANSCs. ATAC‐seq signals at Refseq genes as normalized CPM (counts per million). Heatmaps showing the landscapes of peaks around the transcription start sites (TSSs). (B), Signal intensity distribution of differential chromatin accessibility between ANSCs and PSCs (sigUp: significantly upregulated; sigDown: significantly downregulated; nonSig: not significant). (C), Signal intensity distribution of differential chromatin accessibility between ANSCs and NSCs (categories as in B). (D), Integrative Genomics Viewer (IGV) snapshots showing ATAC‐seq signals at pluripotency‐ and neuroectodermal‐ associated gene loci. (E), Genomic distribution of differentially enriched ATAC‐seq peaks (ANSCs vs. PSCs). (F), Venn diagram illustrating the overlap of upregulated genes in ANSCs vs. PSCs and H9‐ANSCs vs. H9‐PSCs from ATAC‐seq data (upper panel). Venn diagram illustrating the overlap of upregulated genes in NSCs vs. PSCs and H9‐NSCs vs. H9‐PSCs from ATAC‐seq data (lower panel). (G), Scatter plot of chromatin accessibility activity scores for ANSCs vs. PSCs. (H), Gene Ontology (GO) enrichment analysis of genes showing increased chromatin accessibility in ANSCs compared to PSCs. BP: biological process; CC: cellular component; MF: molecular function. (I), Chromatin footprints of pluripotent and neuroectodermal transcription factors in ANSCs vs. PSCs. Red: ANSCs; Blue: PSCs. (J), Venn diagram showing the overlap between upregulated genes from ATAC‐seq and RNA‐seq (ANSCs vs. PSCs). (K), Heatmap showing neuroectodermal gene expression in ANSCs and PSCs. (L), IGV snapshots showing ATAC‐seq and RNA‐seq signals at the PAX6 locus.

Article Snippet: The primary antibodies used included: rabbit polyclonal OCT4 (Novus Biologicals, #NBP2‐15053, 1:200), rabbit polyclonal NANOG (PeproTech, #P236, 1:200), goat polyclonal SOX2 (R&D Systems, #AF2018, 1:200), rabbit monoclonal NESTIN (Boster, #PB9874, 1:200), rabbit polyclonal PAX6 (Elabscience, #E‐AB‐61653, 1:200), rabbit polyclonal N‐Cadherin (Abcam, #ab76057, 1:200), goat polyclonal Brachyury (R&D Systems, #AF2085, 1:100), goat polyclonal SOX17 (R&D Systems, #AF1924, 1:200), mouse monoclonal NeuN (CST, #93972, 1:100), mouse monoclonal TUBB3 (Bioss, #BSM‐33177M, 1:200), rabbit polyclonal GFAP (Bioss, #bs‐0199R, 1:200), and rabbit monoclonal CDX2 (Biogenex, #Cdx2‐88).

Techniques: Activity Assay, RNA Sequencing, Gene Expression

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Generation, transcriptome profiling, and functional validation of cone-rich human retinal organoids.

doi: 10.1073/pnas.1901572116

Figure Lengend Snippet: Fig. 1. Generation of cone-rich retinal organoids with elongated inner/ outer segments in cone photoreceptors. n > 3 experiments. (A) A scheme for the generation of cone-rich retinal organoids. (B) Adherent cultures on day 15 highly expressed retinal progenitor markers VSX2 and PAX6 (n = 6/6 wells of cultures). (C–H) Retinal organoids on day 32 continuously expressed ret- inal progenitor markers PAX6, VSX2, SIX3, and RAX and started to express RGC marker POU4F2 and photoreceptor, bipolar, and horizontal cell marker OTX2 (31) (n = 6/6 retinal organoids). (I and J) Retinal organoids on day 105 expressed VSX2, PAX6, RCVRN, and OTX2 in a laminar manner (n = 3/ 4 retinal organoids). (K–N) Retinal organoids on day 215 expressed cone photoreceptor marker OPN1MW/OPN1LW and rod photoreceptor RHO in the protruding hair-like structures (n = 6/6 retinal organoids). (O) OPN1MW/ LW-positive cone photoreceptors were more abundant than RHO-positive rod photoreceptors. Represents six stacks of optical sections in four retinal organoids. One representative optical section in the stacks is shown (K–M). C/R, the ratio of cones to rods. (P and Q) EM of photoreceptors in 181- d retinal organoids. Basal body and maturing disk membrane are shown (arrowheads in P and Q, respectively). [Scale bars, 200 μm (B), 100 μm (C and I), 50 μm (K), 70 μm (N), 1 μm (P), 0.2 μm (Q).]

Article Snippet: The primary antibodies are L/M-opsin (1:2,000; from J. Nathans, Johns Hopkins University, Baltimore), OTX2 (1:1,500; R&D AF1979), PAX6 (1:500; Covance PRB-278P), POU4F2 (Santa Cruz; SC-6026, 1:200), RAX (1:500; Abcam ab86210), RCVN (1:2,000; Millipore AB5585), RHO (ab98887; 1:500; Abcam), SIX3 (1:500; Rockland), and VSX2 (1:500; Millipore AB9016).

Techniques: Marker, Membrane