Journal: Brain

Article Title: Schizophrenia-associated changes in neuronal subpopulations in the human midbrain

doi: 10.1093/brain/awae321

Figure Lengend Snippet: Unsupervised clustering identifies 18 neuronal populations in the human midbrain . ( A ) Experimental design of the study . A schematic representation of the rostral midbrain at the level of the red nucleus and third cranial nerves (created with biorender.com ). The lines indicate where the superior colliculus (SC) and cerebral peduncles (CP) were removed before nuclei isolation, FANS and snRNAseq. ( B ) Unsupervised clustering of 31 669 neuronal nuclei from 14 schizophrenia (SZ) cases and 14 controls ( and ). Each dot indicates a nucleus, colours indicate clusters. ( C ) Average expression per cluster of selected neurotransmitter-, neuropeptide- or interneuron-related genes. ( D ) Bar plot depicting the total number of nuclei obtained per population, with the percentage of the total NEUN+ population enclosed in parentheses. ( E ) Violin plots depicting log-normalized expression levels of cluster markers (log 2 fold-change > 0.25, P -adjusted < 0.05) . ( F ) Violin plot depicting log-normalized expression level of DLX1 , a cluster marker for inh-CCK. ( G ) A schematic representation of the rostral midbrain indicating the periaqueductal grey and the third cranial nerve nucleus with a box and the reticular formation with a dotted box. In these regions the immunohistochemical staings, shown in H , were performed. ( H ) Representative images of immunohistochemical stainings for DLX1 (brown) and GFAP (black) in control midbrain ( n = 5). Scale bar = 50 μm. avg. = average; expr. = expression; FANS = fluorescence-activated nuclear sorting; log-norm. = log-normalized; snRNAseq = single nucleus RNA-sequencing; UMAP = Uniform Manifold Approximation and Projection.

Article Snippet: Briefly, after sucrose density centrifugation, nuclei were incubated with fluorescently-conjugated antibodies directed against neuronal marker NEUN (RBFOX3/NEUN 1B7 AF647 mouse mAB, Novus Biologicals, NBP1-92693AF647) and the transcription factor OLIG2 for the oligodendrocyte lineage (Anti-OLIG2 clone 211F1.1 AF488 mouse mAB, Merck Millipore, MABN50A4).

Techniques: Isolation, Expressing, Marker, Immunohistochemical staining, Control, Fluorescence, RNA Sequencing

Journal: Brain

Article Title: Schizophrenia-associated changes in neuronal subpopulations in the human midbrain

doi: 10.1093/brain/awae321

Figure Lengend Snippet: Schizophrenia-associated shifts in proportions of midbrain neuronal populations and subpopulations . ( A ) Uniform Manifold Approximation and Projection (UMAP) of the clusters split by diagnosis. The colour legend indicates the number of nuclei obtained per population and the percentage of the total NEUN+ population enclosed in brackets. ( B ) Box plot depicting the percentage of exc-VGLUT1 and 2 nuclei detected in midbrain samples of schizophrenia and control cases ( n = 14 per group; ). Outlier cases are labelled with grey diamond shapes, each dot represents a case, and horizontal lines indicate the median. Outliers were defined as proportions outside of the third quartile plus 1.5 times the interquartile range. ( C ) UMAP of the subclusters split by diagnosis. The colour legend indicates the number of nuclei obtained per population and the percentage of the total NEUN+ population enclosed in brackets. ( D ) Box plots depicting the percentage of nuclei detected per case in subclusters that had significantly altered proportions in schizophrenia; and . Outlier cases are labelled with grey diamond shapes and were defined as data points outside the third quartile + 1.5 × interquartile range. ( E ) Total count of differentially expressed genes (DEGs) between schizophrenia and controls within a neuronal population. Numbers above the bar indicate the total count of increased plus decreased gene expression changes in schizophrenia. ( F ) Volcano plot depicting differentially expressed genes (absolute log 2 fold-change > 0.25, P -adjusted < 0.05) between schizophrenia and controls within the neuronal population ‘inh-CCK’. The corresponding information on DEGs is available in . ( G ) Total count of differentially expressed genes between schizophrenia and controls within a neuronal subpopulation. Numbers above the bar indicate the total count of increased plus decreased gene expression changes in schizophrenia. ( H ) Volcano plot depicting DEGs (absolute log 2 fold-change > 0.25, P -adjusted < 0.05) between schizophrenia and controls within the neuronal subpopulation ‘inh-C’. The number of gene labels was reduced to avoid overlapping text; the full information on DEGs is available in . ( I ) Heat map with the number indicating the detected schizophrenia susceptibility genes per neuronal population . The colour indicates the normalized ratio of detected susceptibility genes over the total number of expressed genes per neuronal population. High-confidence schizophrenia susceptibility genes derived from Wang et al .

Article Snippet: Briefly, after sucrose density centrifugation, nuclei were incubated with fluorescently-conjugated antibodies directed against neuronal marker NEUN (RBFOX3/NEUN 1B7 AF647 mouse mAB, Novus Biologicals, NBP1-92693AF647) and the transcription factor OLIG2 for the oligodendrocyte lineage (Anti-OLIG2 clone 211F1.1 AF488 mouse mAB, Merck Millipore, MABN50A4).

Techniques: Biomarker Discovery, Control, Gene Expression, Derivative Assay

Journal: bioRxiv

Article Title: Modeling cerebral development in vitro with L- MYC -immortalized human neural stem cell-derived organoids

doi: 10.1101/2025.02.12.637976

Figure Lengend Snippet: (A) Brightfield images of LMNSC01 organoids from an initial seed of 1000 cells/organoid (D0). Organoids progressively increase in confluency between D0 and D47. At D15 the organoids appear more homogeneous; internal structure becomes evident at D30. (B) Reconstructed confocal images of enhanced green fluorescent protein (eGFP)-expressing LMNSC01 cells illustrating expansion, folding, and internal architectural complexity that becomes evident at D30. Some organoids curl (D60), while most remain roughly spherical (D90). (C) Increase in numbers of cells per organoid over 120 days in culture: rapid growth over the first 30 days, a characteristic dip at D60, followed by stabilization at D90 to D120 (*: p < 0.05; **: p < 0.01; ****: p < 0.001). Organoids were dissociated, and cells counted by flow cytometry. Cell viability after organoid dissociation: D15, 96.6 ± 1.0%; D30, 92.0 ± 2.6%; D60, 88.2 ± 2.4%; D90, 95.2 ± 0.3%; D120, 91.4 ± 0.5% (mean ± s.d., number of organoids as indicated on the figure). (D) Eccentricity of organoid shape assessed as major axis/minor axis, illustrating that most organoids were smooth and ovoid (green bars). Grey bars denote organoids identified by eye as not smoothly spherical or ovoid. By this measure, 1.0 indicates a spherical organoid. Numbers of organoids are as indicated on the figure. The majority of organoids were smooth spherical/ovoid: D15, 66%; D30, 94%, D60, 100%, D90, 82%. Statistical tests are between smooth/ovoid populations. (E) Organoid cross-sectional areas over time in culture. This value remained relatively stable at around 2 mm 2 for both spherical/ovoid and more eccentric organoids. Mean diameters measured as average of major and minor axis (mm): D15, 1.7 ± 0.2; D30, 1.6 ± 0.4; D60, 1.6 ± 0.4; D90, 1.3 ± 0.1 (mean ± s.d.); p < 0.05 for all days relative to D90, all other comparisons not significant; numbers of organoids as indicated on the figure. (F) Expression (NanoString) of genes in the pathway “Tissue Integrity and Maintenance” ( Supplemental Table 1 ), ordered by expression level at D120. (i) Genes that show their highest expression at D30, and whose expression becomes minimal at D120 (blue time arrow). (ii) Genes with higher expression in organoids between D34-D120 and whose expression is maintained at D120. (G) D30 organoid. Series of images of organoid edge at increasing depth (schematic), as described in the text, showing stratification of GFAP POS , SOX2 POS , and β-tubulin III POS cells. (H) D30 organoid. Neurons (NeuN POS and MAP2 POS ) are internal to GFAP POS glia near the organoid surface. (I) D30 organoid. PAX6 POS cortically fated cells surrounding a ventricle-like cell-free area, internal to a surround of β-tubulin III POS immature neurons.

Article Snippet: Antibodies for flow cytometry: NeuN:PE (Novus Biologicals NBP1-92693PE); SOX2:APC (R&D Systems IC2018R-100UG); GFAP:PerCP (Novus Biologicals NBP2-33184PCP); β-tubulin III:PE:Cy7 (Biolegend 801218).

Techniques: Expressing, Flow Cytometry

Journal: bioRxiv

Article Title: Modeling cerebral development in vitro with L- MYC -immortalized human neural stem cell-derived organoids

doi: 10.1101/2025.02.12.637976

Figure Lengend Snippet: (A1) 3D reconstruction of organoid immunofluorescence; SOX2-expressing stem and progenitor cells remain distributed throughout organoids over the period D15-D90. (A2) Percentage of SOX2 POS cells over the in vitro period (D15-D120), as measured by organoid dissociation and flow cytometry. *: p < 0.05, n = 4 at each time point. (B,C) Paired images of entire organoids (3D reconstruction, above) and detail (single optical section, below). (B) FoxG1 immunofluorescence marking forebrain-fated cortical progenitor cells. (C) Persistence of Wnt receptor Fdz9, a marker of hippocampal fate, D30-D90, also illustrating changes in cell morphology. (D) Expression of TTR (a marker of choroid plexus fate) by immunofluorescence. D30 organoid. (E) Heatmap of cell type clustering by expression profiling within fetal cortex (BrainSpan, in weeks post-conception) and organoids (NanoString, in weeks in vitro ). (F) Dendrogram constructed from NanoString expression profiling of cell type markers (from E ), indicating the closest relatedness of organoids to cortex and distance from other regions.

Article Snippet: Antibodies for flow cytometry: NeuN:PE (Novus Biologicals NBP1-92693PE); SOX2:APC (R&D Systems IC2018R-100UG); GFAP:PerCP (Novus Biologicals NBP2-33184PCP); β-tubulin III:PE:Cy7 (Biolegend 801218).

Techniques: Immunofluorescence, Expressing, In Vitro, Flow Cytometry, Marker, Construct

Journal: bioRxiv

Article Title: Modeling cerebral development in vitro with L- MYC -immortalized human neural stem cell-derived organoids

doi: 10.1101/2025.02.12.637976

Figure Lengend Snippet: Diagonal lines indicate image or data not available . (A1-E1) Immunofluorescence image pairs of representative organoids. Above, confocal 3D reconstructions of entire organoids; below, detailed higher resolution single optical sections (below). (A2-E2) Expression of markers, by percentage of total positive cells (by dissociation and flow cytometry; blue), and by mRNA levels in organoids (NanoString; red) and fetal cortex (BrainSpan; black). Data are plotted at 70% transparency; the inset illustrates increasing density of dots with overlap of multiple points. (A2) Transcription factor T-box brain1 (Tbr1) , a marker of postmitotic neurons , see text. (B1, B2) Doublecortin (DCX) , a marker of migrating early neurons, see text. (C1, C2) β-Tubulin III , a marker of maturing neurons ; see text. (D1, D2) MAP2 , a neural differentiation marker ; see text (E1, E2) NeuN , a nuclear marker of mature neurons ; see text.

Article Snippet: Antibodies for flow cytometry: NeuN:PE (Novus Biologicals NBP1-92693PE); SOX2:APC (R&D Systems IC2018R-100UG); GFAP:PerCP (Novus Biologicals NBP2-33184PCP); β-tubulin III:PE:Cy7 (Biolegend 801218).

Techniques: Immunofluorescence, Expressing, Flow Cytometry, Marker

Journal: bioRxiv

Article Title: Modeling cerebral development in vitro with L- MYC -immortalized human neural stem cell-derived organoids

doi: 10.1101/2025.02.12.637976

Figure Lengend Snippet: In this figure, each dot represents a data point from an organoid (NanoString) or a cortical specimen (BrainSpan). The horizontal lines indicate the mean of the data. The lines are included to aid in viewing trends in the data . (A) Expression of SOX9 by immunofluorescence, often in the nuclei of vimentin-expressing neuroprogenitor cells (white arrow heads). (B) Expression of oligodendrocyte marker O1 by immunofluorescence. [C] Expression of phospho-vimentin in cell bodies of mitotic GFAP-expressing cells by immunofluorescence. (Da, i-iii) Maintained expression of SOX9 in organoids of increasing age by immunofluorescence. (Da, iv-vi) Expression of GFAP in organoids of increasing age by immunofluorescence. (Db) Almost parallel expression of GFAP as percent expressing cells (flow cytometry) and mRNA (NanoString), with a transient decrease around D60. (Dc) Expression of SOX9 mRNA in organoids (red, NanoString) and fetal cortex (black, BrainSpan). (Dd) Expression of GFAP mRNA in organoids (red, NanoString) and fetal cortex (black, BrainSpan). Numbers of organoids expression profiled by NanoString for days in culture (D); numbers of organoids/number of cohorts: D34, 4/1; D60, 10/2; D65, 4/1; D90, 7/2; D94, 4/1; D120, 4/1. Numbers of cortical region fetal samples as reported by BrainSpan for weeks post-conception (W); numbers of samples/number of donors: W8, 9/1; W9, 7/1; W12, 31/3; W13, 32/3; W16, 28/3; W17, 9/1; W19, 8/1; W21, 11/2; W24, 11/1; W25, 1/1; W26, 3/1; W35, 1/1; W37, 11/1.

Article Snippet: Antibodies for flow cytometry: NeuN:PE (Novus Biologicals NBP1-92693PE); SOX2:APC (R&D Systems IC2018R-100UG); GFAP:PerCP (Novus Biologicals NBP2-33184PCP); β-tubulin III:PE:Cy7 (Biolegend 801218).

Techniques: Expressing, Immunofluorescence, Marker, Flow Cytometry