Journal: PLoS ONE

Article Title: Mesenchymal Transition and PDGFRA Amplification/Mutation Are Key Distinct Oncogenic Events in Pediatric Diffuse Intrinsic Pontine Gliomas

doi: 10.1371/journal.pone.0030313

Figure Lengend Snippet: Panel A: Radial plot showing the expression of oligodendroglial markers in the two groups of DIPG, in log2 ratios related each other. The red circle represent the median expression level of the whole population of DIPG. Group 1 expresses higher levels of oligodendroglial markers than group 2 DIPG. Panel B: Morphological oligodendroglial differenciation in group 1 tumors (HES staining, ×40). Panel C: Morphological astrocytic differenciation in group 2 tumors (HES staining ×40). Panel D: Olig2 immunohistochemistry in a group 1 DIPG showing that probably not all cells in the biopsy are tumoral (×40). Panel E: Dual immunohistochemistry for Olig2 and GFAP showing that tumor cells in mitosis are GFAP negative but Olig2 positive (×100). Panel F: Overall survival of 55 DIPG according to the presence (red) or absence (blue) of oligodendroglial differenciation. Median OS was shorter in patients with oligodendroglial type of tumors (7.73 vs 12.37, p = 0.045, log rank test).

Article Snippet: Sections were then incubated with various commercial monoclonal primary antibodies against Olig2 (AF 2418, 1/150, R/D system, CA, USA), P53 (DO-1, 1/1, Ventana) and MIB-1 (1/100; Dako, Glostrup, Denmark).

Techniques: Expressing, Staining, Immunohistochemistry

Journal: bioRxiv

Article Title: Spatial epigenomic niches underlie glioblastoma cell state plasticity

doi: 10.1101/2025.05.09.653178

Figure Lengend Snippet: (A) Overview of NicheCompass workflow. (B) Spatial plots of NicheCompass-defined cellular niches with regions of interest highlighted. (D) Spatial plot of SOX10 targets gene program across all samples. (E) Spatial plot of VEGFA combined gene program score. (F) Volcano plot of gene activity scores for pixels belonging to niches 3 and 7 (OPC-like) versus niche 0 (MES-like). (G – H) Representative spatial plots of SOX10 and VEGFA gene activity scores highlighting regions of OPC-like niches surrounded by MES-like niches (I) Representative microscopy image from PhenoCycler data showing the presence of MES-like (white arrows) and OPC-like cells (magenta arrowheads) within the core of a GBM tumor. (J) Quantification of cellular identity in PhenoCycler data. MES is defined as VIM positive, OPC as OLIG2 positive, and intermediate as positive for both markers.

Article Snippet: Primary antibody staining was performed overnight at 4 °C using anti-OLIG2 (Cell Signaling, 65915) diluted 1:400 in DPBS supplemented with 1% Bovine Serum Albumin (BSA) and 0.3% Triton™ X-100 (pH 8.0).

Techniques: Activity Assay, Microscopy

Journal: bioRxiv

Article Title: Spatial epigenomic niches underlie glioblastoma cell state plasticity

doi: 10.1101/2025.05.09.653178

Figure Lengend Snippet: (A) Schematic demonstrating how data are portrayed for panels B and C. (B – C) Signaling programs as defined by NicheCompass demonstrating PDGFB signaling within the OPC-like niche and GPI signaling from the MES-like niche to the OPC-like niche. (D) Heatmap of signaling probabilities as defined by CellChat based on snATAC data of malignant GBM cells. (E) Top ligand-receptor pairs contributing to BMP signaling pathway. (F) Violin plots of BMP7 expression in single cell RNA sequencing data from Couturier and colleagues. (G) Violin plot of gene activity score from snATAC dataset. Note the relatively modest difference in gene activity. (H) Coverage plot of the BMP7 gene. Note relatively similar chromatin landscape. (I) Fluorescence microscopy images of GBM cell culture (GSC016) under control conditions and after treatment for 72 hours with recombinant BMP7 (n = 2 – 4 wells per condition). Scale bar indicates 100 µm. (J) Quantification of percent OLIG2 positive cells in (I).

Article Snippet: Primary antibody staining was performed overnight at 4 °C using anti-OLIG2 (Cell Signaling, 65915) diluted 1:400 in DPBS supplemented with 1% Bovine Serum Albumin (BSA) and 0.3% Triton™ X-100 (pH 8.0).

Techniques: Expressing, RNA Sequencing, Activity Assay, Fluorescence, Microscopy, Cell Culture, Control, Recombinant

Journal: Nature Communications

Article Title: Nfat/calcineurin signaling promotes oligodendrocyte differentiation and myelination by transcription factor network tuning

doi: 10.1038/s41467-018-03336-3

Figure Lengend Snippet: Nfat/calcineurin signaling is required for oligodendroglial differentiation in vivo. a – f Analysis of oligodendroglial marker expression in 18.5 dpc-old embryos of wildtype (WT) and CnB1 ΔSox10 mice by immunohistochemistry and in situ hybridization. Antibodies were directed against Olig2 ( a ), Pdgfra ( b ), Nkx2.2 ( c ), and Myrf ( d ). Riboprobes recognized Mbp ( e ) and Plp1 ( f ) mRNA. Scale bars, 100 µm. g – n From these and similar stainings for Ki67 ( m ) and cleaved caspase 3 ( n ) quantifications were performed on three or six embryos for each genotype ( n = 3 for g – l and n , n = 6 for m ) counting three separate sections. Presentations are as absolute ( g – l ) or relative ( m , n ) numbers (values: Olig2: 483 ± 46 for WT and 465 ± 11 for CnB1 ΔSox10 ; Pdgfra: 211 ± 18 for WT and 175 ± 13 for CnB1 ΔSox10 ; Nkx2.2: 37 ± 3 for WT and 21 ± 1 for CnB1 ΔSox10 ; Myrf: 54 ± 3 for WT and 26 ± 4 for CnB1 ΔSox10 ; Mbp : 72 ± 11 for WT and 34 ± 5 for CnB1 ΔSox10 ; Plp1 : 66 ± 7 for WT and 29 ± 4 for CnB1 ΔSox10 ; Ki67: 100 ± 6.4 for WT and 80.8 ± 7.7 for CnB1 ΔSox10 ; cleaved caspase 3: 100 ± 4.5 for WT and 102.7 ± 4.8 for CnB1 ΔSox10 ). Statistical significance was determined by two-tailed Student’s t -test ( *P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001)

Article Snippet: After antigen retrieval, slides were incubated overnight with mouse anti-NFATc1 monoclonal (Santa Cruz, clone H-10, #sc-17834, Lot# D0110, 1:50 dilution), rabbit anti-NFATc2 antiserum (Sigma, #HPA008789, Lot# A33084, 1:50 dilution), rabbit anti-NFATc3 antiserum (Sigma, #HPA023844, Lot# A83120, 1:50 dilution), rabbit anti-NFATc4 antiserum (Santa Cruz, clone L-9, #sc-32985, Lot# H0306, 1:50 dilution), rabbit anti-OLIG2 antiserum (IBL, #18953, Lot# 1B-327, 1:150 dilution), mouse anti-OLIG2 monoclonal (Medac # 387M-16, Lot# 1308812B, 1:100 dilution), mouse anti-NOGOA monoclonal (Clone 11c7, gift from M.E.

Techniques: In Vivo, Marker, Expressing, Immunohistochemistry, In Situ Hybridization, Two Tailed Test

Journal: Nature Communications

Article Title: Nfat/calcineurin signaling promotes oligodendrocyte differentiation and myelination by transcription factor network tuning

doi: 10.1038/s41467-018-03336-3

Figure Lengend Snippet: Sox10 activates oligodendroglial Nkx2.2 expression via ECR19. a QrtPCR analysis of Nkx2.2 expression in mouse oligodendroglial cultures after 6 and 24 h differentiation in absence (Ctr, open bars) or presence of 1 µM VIVIT (grey bars) ( n = 3). Nkx2.2 amounts under control conditions after 6 h were set to 1 (values: 1 ± 0.20 for Ctr and 0.65 ± 0.14 for VIVIT after 6 h, 3.21 ± 0.12 for Ctr and 1.92 ± 0.25 for VIVIT after 24 h). b Rat Nkx2.2 locus with exons (solid pink boxes), ChIP-Seq peaks (GEO accession numbers GSE64703 and GSE42447) for Olig2 (green boxes) and Sox10 (blue boxes) in ECRs (open pink boxes) at −115, −19, +5, and +45 kilobases relative to the transcriptional start site (arrow) and ChIP control regions (Ctr1 and Ctr2, grey boxes). c N2a cell transfections with luciferase reporters carrying Nkx2.2 ECRs in absence (−) or presence of low (+, light grey bars) and high Sox10 amounts (++, dark grey bars) ( n = 3). Sox10-dependent fold inductions ± SEM were determined after 48 h with activities in the absence of Sox10 set to 1 (ECR115: 1.6 ± 0.3 at both concentrations; ECR19: 32.9 ± 7.1 and 198.3 ± 26.0; ECR5: 44.6 ± 16.4 and 107.3 ± 10.7; ECR45: 4.5 ± 2.1 and 10.6 ± 0.9). d ChIP on differentiating rat oligodendrocytes after 4 days ( n = 3) using rabbit pre-immune (pre IS, open bars) and anti-Sox10 (αSox10, grey bars) antiserum. Amounts of immunoprecipitated ECR19 and control region (Ctr1) were qPCR-determined and are presented as percent of input (ECR19: 0.012 ± 0.004 for pre IS and 0.073 ± 0.013 for αSox10; Ctr1: 0.016 ± 0.005 for pre IS and 0.021 ± 0.005 for αSox10). e Localization of binding sites for Sox10 (putative, light blue; EMSA-confirmed, dark blue), Nfat (brown) and Olig2 (green) in ECR19. Numbers on left and right correspond to mouse ECR19 positions relative to transcriptional start site. For EMSA and sequences, see Suppl. Fig. . f N2a cell transfections with wildtype (WT) or mutant ECR19 luciferase reporters with inactivated Sox10 binding sites (S4m, S7/7am, and S4/7/7am) in absence (−) or presence (+, grey bars) of Sox10 ( n = 3). Sox10-dependent fold inductions ± SEM were determined 48 h post transfection (WT: 106.7 ± 0.7; S4m: 54.6 ± 9.1; S7/7am 23.4 ± 0.9; S4/7/7am 14.6 ± 2.3). g Sox10 immunohistochemistry (green) on cortical slices 7 days after transduction with retroviruses carrying tdTomato reporters (red) under control of a minimal promoter (control) or minimal promoter and ECR19 (ECR19). Co-expression is in yellow, DAPI counterstain in blue. Scale bar, 50 µm. Statistical significance was determined by two-tailed Student’s t-test (* P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001)

Article Snippet: After antigen retrieval, slides were incubated overnight with mouse anti-NFATc1 monoclonal (Santa Cruz, clone H-10, #sc-17834, Lot# D0110, 1:50 dilution), rabbit anti-NFATc2 antiserum (Sigma, #HPA008789, Lot# A33084, 1:50 dilution), rabbit anti-NFATc3 antiserum (Sigma, #HPA023844, Lot# A83120, 1:50 dilution), rabbit anti-NFATc4 antiserum (Santa Cruz, clone L-9, #sc-32985, Lot# H0306, 1:50 dilution), rabbit anti-OLIG2 antiserum (IBL, #18953, Lot# 1B-327, 1:150 dilution), mouse anti-OLIG2 monoclonal (Medac # 387M-16, Lot# 1308812B, 1:100 dilution), mouse anti-NOGOA monoclonal (Clone 11c7, gift from M.E.

Techniques: Expressing, ChIP-sequencing, Transfection, Luciferase, Immunoprecipitation, Binding Assay, Mutagenesis, Immunohistochemistry, Transduction, Two Tailed Test

Journal: Nature Communications

Article Title: Nfat/calcineurin signaling promotes oligodendrocyte differentiation and myelination by transcription factor network tuning

doi: 10.1038/s41467-018-03336-3

Figure Lengend Snippet: Sox10-dependent enhancer activation is stimulated by Nfatc2 and inhibited by Olig2 and Nkx2.2. a Transient transfections of N2a cells with luciferase reporters carrying Nkx2.2 ECR19 in the absence (−) or presence (+) of Sox10 and constitutively active Nfatc2 as effectors ( n = 3). Activation of reporter gene expression was determined in extracts 48 h post transfection and is presented as fold inductions ± SEM with transfections in the absence of effectors arbitrarily set to 1 (58.5 ± 4.0 for Sox10, 0.3 ± 0.0 for Nfatc2, and 130.5 ± 11.6 for Sox10 in combination with Nfatc2). b , c Neural tube electroporations in HH11-stage chicken embryos ( n = 3). The relative number of electroporated cells, in which Nkx2.2 expression was induced 48 h after neural tube electroporation of Sox10 and Nfatc2, was quantified ( b : 0 for control, 19.5 ± 4.9 for Sox10, 25.9 ± 4.1 for Nfatc2, and 39.0 ± 4.6 for Sox10 in combination with Nfatc2). Transverse sections were used for quantifications ( c ). Sections were probed for the occurrence of Nkx2.2 (white in upper row, red in lower row). Electroporated cells on the left side all express GFP (green) and to variable extent Nkx2.2 (white in upper row, red in lower row). Nuclei are counterstained by DAPI (blue). Scale bar, 50 µm. d , e Transient transfections of N2a cells with luciferase reporters carrying ECR19 ( d ) or OLEa ( e ) in the absence of added transcription factors (−) or in the presence (+) of Sox10, Olig2, Nkx2.2, and combinations thereof ( n = 3). Activation of reporter gene expression was determined in extracts 48 h post transfection and is presented as fold inductions ± SEM with transfections in the absence of added transcription factors being arbitrarily set to 1 (ECR19: 55.3 ± 7.7 in the presence of Sox10, 0.7 ± 0.1 in the presence of Olig2 and 13.1 ± 2.0 in the presence of Sox10 and Olig2; OLEa: 21.6 ± 1.3 in the presence of Sox10, 2.2 ± 0.1 in the presence of Nkx2.2 and 8.8 ± 1.2 in the presence of Sox10 and Nkx2.2). Statistical significance was determined by two-tailed Student’s t -test (* P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001)

Article Snippet: After antigen retrieval, slides were incubated overnight with mouse anti-NFATc1 monoclonal (Santa Cruz, clone H-10, #sc-17834, Lot# D0110, 1:50 dilution), rabbit anti-NFATc2 antiserum (Sigma, #HPA008789, Lot# A33084, 1:50 dilution), rabbit anti-NFATc3 antiserum (Sigma, #HPA023844, Lot# A83120, 1:50 dilution), rabbit anti-NFATc4 antiserum (Santa Cruz, clone L-9, #sc-32985, Lot# H0306, 1:50 dilution), rabbit anti-OLIG2 antiserum (IBL, #18953, Lot# 1B-327, 1:150 dilution), mouse anti-OLIG2 monoclonal (Medac # 387M-16, Lot# 1308812B, 1:100 dilution), mouse anti-NOGOA monoclonal (Clone 11c7, gift from M.E.

Techniques: Activation Assay, Transfection, Luciferase, Expressing, Electroporation, Two Tailed Test

Journal: Nature Communications

Article Title: Nfat/calcineurin signaling promotes oligodendrocyte differentiation and myelination by transcription factor network tuning

doi: 10.1038/s41467-018-03336-3

Figure Lengend Snippet: Nfat proteins overcome cross-repression of Olig2 and Nkx2.2 in cooperation with Sox10. a TetSox10 , Brn4::Cre and Rosa26 stopflox-tTA alleles for CNS overexpression of Sox10. Arrows mark transcription start sites, triangles loxP sites. 9mycSox10, myc-tagged Sox10 coding sequences; bGI β-globin intron, Brn4p Brn4 promoter, Cre Cre coding sequences, EGFP enhanced GFP coding sequences, neo neomycin resistance cassette, pA polyadenylation site, PGK phosphoglycerate kinase promoter, SA splice acceptor, tetO bidirectional tetracycline-responsive promoter, tTA tetracycline-controlled transactivator coding sequences. b Immunohistochemistry for Sox10, Olig2, and Nkx2.2 on transverse spinal cord sections of wildtype (wt) and Sox10 overexpressing ( TetSox10 Brn4 ) embryos at 12.5 dpc. Right panels are magnifications of boxed areas with Olig2 in green and Nkx2.2 in red. Scale bars, 50 µm. c N2a cell transfections with ECR19 luciferase reporter in absence (−) or presence (+) of Sox10, constitutively active CnA (CnA ca ) and Olig2 ( n = 3). Fold inductions ± SEM were determined after 48 h with reporter activity in the absence of effectors set to 1 (values: 13.1 ± 1.4 for Sox10, 0.9 ± 0.2 for Olig2, 1.8 ± 0.5 for CnA ca , 6.0 ± 0.5 for Sox10 and Olig2, 13.9 ± 2.4 for Sox10, Olig2 and CnA ca , and 42.9 ± 7.9 for Sox10 and CnA ca ). d N2a cell transfections with Olig2 OLEa luciferase reporter in absence (−) or presence (+) of Sox10 and Nfatc2 ( n = 3) (14.6 ± 1.7 for Sox10, 0.7 ± 0.0 for Nfatc2 and 14.9 ± 2.0 for Sox10 and Nfatc2) or ( e ) in absence (−) or presence (+) of Sox10, CnA ca and Nkx2.2 ( n = 3) (values: 19.0 ± 2.3 for Sox10, 1.8 ± 0.1 for Nkx2.2, 1.0 ± 0.1 for CnA ca , 7.3 ± 0.3 for Sox10 and Nkx2.2, 16.9 ± 0.6 for Sox10, Nkx2.2 and CnA ca , and 23.1 ± 3.3 for Sox10 and CnA ca ). f Neural tube electroporations in HH11-stage chicken embryos ( n = 3) and quantification of the relative number of electroporated cells, in which joint expression of Nkx2.2 and Olig2 was induced after 48 h (values: 0 for control, 8.4 ± 3.5 for Sox10, 11.5 ± 2.0 for Nfatc2, and 20.9 ± 5.4 for Sox10 and Nfatc2). g Immunohistochemical determination of the percentage of Mbp-expressing rat oligodendroglia transduced with GFP-expressing control (−) or Nkx2.2-overexpressing retrovirus (+), after 6 days of differentiation in absence (Ctr, open bars) or presence of 1 µM FK506 (grey bars) ( n = 3 separate cultures). (Values: 84.0 ± 3.6% in GFP-transduced untreated cultures; 89.0 ± 1.7% in Nkx2.2-transduced untreated cultures; 53.7 ± 13.7% in GFP-transduced FK506-treated cultures; 84.1 ± 11.7% in Nkx2.2-transduced FK506-treated cultures). Statistical significance was determined by two-tailed Student’s t -test ( c – f ) and Bonferroni-corrected one-way ANOVA ( g ) (* P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001)

Article Snippet: After antigen retrieval, slides were incubated overnight with mouse anti-NFATc1 monoclonal (Santa Cruz, clone H-10, #sc-17834, Lot# D0110, 1:50 dilution), rabbit anti-NFATc2 antiserum (Sigma, #HPA008789, Lot# A33084, 1:50 dilution), rabbit anti-NFATc3 antiserum (Sigma, #HPA023844, Lot# A83120, 1:50 dilution), rabbit anti-NFATc4 antiserum (Santa Cruz, clone L-9, #sc-32985, Lot# H0306, 1:50 dilution), rabbit anti-OLIG2 antiserum (IBL, #18953, Lot# 1B-327, 1:150 dilution), mouse anti-OLIG2 monoclonal (Medac # 387M-16, Lot# 1308812B, 1:100 dilution), mouse anti-NOGOA monoclonal (Clone 11c7, gift from M.E.

Techniques: Over Expression, Immunohistochemistry, Transfection, Luciferase, Activity Assay, Expressing, Immunohistochemical staining, Transduction, Two Tailed Test

Journal: Nature Communications

Article Title: Metformin alters mitochondria-related metabolism and enhances human oligodendrocyte function

doi: 10.1038/s41467-025-63279-4

Figure Lengend Snippet: A Schematic representation of hESC-derived monolayer oligodendroglia differentiation protocol. B Illustrative immunofluorescence image of hESC-derived oligodendroglia at day 70 of in vitro differentiation (DIV) showing PDGFRα + oligodendrocyte precursor cells (OPCs) (magenta) and MBP + oligodendrocytes (grey) co-expressing OLIG2 (cyan). Scale bar = 100 μm. C Fold change (FC) difference of OLIG2 + MBP + oligodendrocytes after treatment with metformin or clemastine compared to their respective vehicle-treated controls (ddH2O or DMSO). n = 5 for metformin and vehicle control, n = 6 for clemastine and vehicle control where n = number of differentiations with 4 technical repeats. Kolmogorov-Smirnov normality test with Dallal-Wilkinson-Lillie for p -value, two-tailed unpaired t test. Mean ± SEM. D UMAP representation of clustered oligodendroglia. E Dot plot of selected marker genes showing cluster segregation. F Integration with adult human post-mortem spinal cord oligodendroglia snRNAseq dataset . G Cosine similarity heatmap showing similarities between the hESC-derived monolayer oligodendroglia (target, labelled mono_) dataset and the adult human spinal cord oligodendroglia (source) dataset. H Integration with the second and third trimester foetal OPC snRNAseq dataset . Source data are provided in the Source Data file. Created in BioRender. Swire, M. (2025) https://BioRender.com/j633z18 .

Article Snippet: Sections underwent H 2 O 2 treatment for 10 min at RT, washed in ddH 2 O and incubated for target retrieval for 5 min at 100 o C. Slides were washed again in ddh2O, followed by 0.1% PBS-Tween and incubated with anti-Olig2 primary antibody (Atlas antibodies, HPA003254) overnight at 4 o C. Sections were washed in 0.1% PBS-Tween and post-fixed in 4% PFA at room temperature for 30 min.

Techniques: Derivative Assay, Immunofluorescence, In Vitro, Expressing, Control, Two Tailed Test, Marker

Journal: Nature Communications

Article Title: Metformin alters mitochondria-related metabolism and enhances human oligodendrocyte function

doi: 10.1038/s41467-025-63279-4

Figure Lengend Snippet: A Schematic representation of hESC-derived cortical brain organoid oligodendroglia differentiation protocol. B Illustrative immunofluorescence images of hESC-derived oligodendroglia in organoids at day 67 of in vitro differentiation (DIV), showing PDGFRα + oligodendrocyte precursor cells (OPCs) (red) co-expressing OLIG2 (cyan) and of day 112 showing MBP + oligodendrocytes (yellow) co-expressing OLIG2 (cyan). Scale bars = 100 μm or 50 μm (zoom). C MBP + area of immunofluorescence per total organoid area after treatment with metformin compared to vehicle-treated controls (ddH2O). n = 5 biological replicates. Kolmogorov-Smirnov normality test with Dallal-Wilkinson-Lillie for p -value, two-tailed unpaired t test. Mean ± SEM. D UMAP representation of clustered organoid-derived oligodendroglia integrated with scRNAseq organoid data from (Marton et al. ). E Dot plot of selected marker genes showing cluster segregation. F Integration with adult human post-mortem cortical hemisphere oligodendroglia snRNAseq dataset . G Cosine similarity heatmap showing similarities between the hESC-derived brain organoid oligodendroglia (target, labelled with co_) dataset and the adult human cortical oligodendroglia (source) dataset. Source data are provided in the Source Data file. Created in BioRender. Swire, M. (2025) https://BioRender.com/cl7w2km .

Article Snippet: Sections underwent H 2 O 2 treatment for 10 min at RT, washed in ddH 2 O and incubated for target retrieval for 5 min at 100 o C. Slides were washed again in ddh2O, followed by 0.1% PBS-Tween and incubated with anti-Olig2 primary antibody (Atlas antibodies, HPA003254) overnight at 4 o C. Sections were washed in 0.1% PBS-Tween and post-fixed in 4% PFA at room temperature for 30 min.

Techniques: Derivative Assay, Immunofluorescence, In Vitro, Expressing, Two Tailed Test, Marker

Journal: Nature Communications

Article Title: Metformin alters mitochondria-related metabolism and enhances human oligodendrocyte function

doi: 10.1038/s41467-025-63279-4

Figure Lengend Snippet: A Schematic representation of green fluorescent protein-positive (GFP + ) human embryonic stem cell (hESC)-derived PDGFRA + oligodendrocyte precursor cells (OPCs) transplantation into the corpus callosum of Rag2 -/- : Shi/Shi P2-P4 mice. B Illustrative immunofluorescence and electron micrograph (EM) images of chimeric corpus callosum at 70 days post transplantation showing HuNu + cells (cyan)(left) or MBP + oligodendrocytes (cyan) co-localising with OLIG2 (magenta)(middle) and myelinated rodent axons (right). Scale bar = 100 μm (fluorescence) or 5 μm (EM). For control untreated mice, a mean of 16.15% ± 1.88 SEM rodent axons were myelinated. n = 11 animals. C Quantification of myelinated axons showed a significant increase after metformin treatment compared to the vehicle-treated (ddH 2 O) controls. Each dot represents an animal, n = 5 animals. Kolmogorov-Smirnov normality test with Dallal-Wilkinson-Lillie for p -value, two-tailed unpaired t test. Mean ± SEM. D UMAP representation of clustered oligodendroglia. E Dot plot of selected marker genes showing cluster segregation. F Integration with adult human post-mortem oligodendroglia snRNAseq dataset (brain and spinal cord) . G Cosine similarity heatmap showing similarities between the hESC-derived chimeric oligodendroglia (target, labelled with chi_) dataset and the adult human oligodendroglia (source) dataset. Source data are provided in the Source Data file. Created in BioRender. Swire, M. (2025) https://BioRender.com/ghxthad .

Article Snippet: Sections underwent H 2 O 2 treatment for 10 min at RT, washed in ddH 2 O and incubated for target retrieval for 5 min at 100 o C. Slides were washed again in ddh2O, followed by 0.1% PBS-Tween and incubated with anti-Olig2 primary antibody (Atlas antibodies, HPA003254) overnight at 4 o C. Sections were washed in 0.1% PBS-Tween and post-fixed in 4% PFA at room temperature for 30 min.

Techniques: Derivative Assay, Transplantation Assay, Immunofluorescence, Fluorescence, Control, Two Tailed Test, Marker

Journal: Nature Communications

Article Title: Metformin alters mitochondria-related metabolism and enhances human oligodendrocyte function

doi: 10.1038/s41467-025-63279-4

Figure Lengend Snippet: A Electron microscopy (EM) of myelinated chimeric animal axons showing a significant increase of mitochondrial area after metformin treatment compared to vehicle-treated controls, quantified ( B ) as the average of myelinated axons measured per mouse or number per individual axon measured to indicate the spread. n = 5 metformin- and n = 4 vehicle-treated animals. Kolmogorov-Smirnov normality test with Dallal-Wilkinson-Lillie for p -value, two-tailed unpaired t test. Mean ± SEM. Boxes in plots visualise median and 25th and 75th percentiles, and whiskers mark range up to 1.5 * inter-quartile ranges to show potential outliers. See Supplementary Data for exact summary statistics. Scale bar = 1 μm. C EM of chimeric animal corpus callosum showing glia with increased area of mitochondria after metformin treatment compared to vehicle, quantified ( D ) as average of glia measured per mouse or number per individual glial cytoplasm area measured to indicate the spread. Kolmogorov-Smirnov normality test with Dallal-Wilkinson-Lillie for p -value, two-tailed unpaired t test. n = 6 metformin- and n = 3 vehicle-treated animals. Mean ± SEM. Boxes in plots visualise median and 25th and 75th percentiles, and whiskers mark range up to 1.5 * inter-quartile ranges to show potential outliers. See Supplementary Data for exact summary statistics. Scale bar = 1 μm. E Gene Ontology (GO) analysis of differentially expressed genes between human embryonic stem cell (hESC)-derived oligodendroglia from metformin- and vehicle-treated chimeric animals. F Volcano plot of significantly differentially expressed genes between hESC-derived oligodendroglia from metformin- and vehicle-treated chimeric animals. pval < 0.05 and logFC > 0.5. DGE analysis was performed using MAST within Seurat, a two-sided statistical test that accounts for detection rate and zero inflation typical of single-cell RNA-seq data. Latent variables to correct for batch effects were included. P-values were adjusted for multiple comparisons using the Benjamini-Hochberg false discovery rate (FDR) method. G Violin plot of the most highly expressed specific genes in hESC-derived oligodendrocytes in metformin-treated chimeric animals. H Quantification of in situ hybridisation using NDUFA11 and EIF1 RNA probes and immunofluorescence for OLIG2 on human cells in chimera tissue with and without metformin treatment. n = 3 metformin- and 3 vehicle-treated animals with 2 ROIs of high-density HuNu + nuclei analysed per animal. Composite score = (fraction of OLIG2 + cells) x (mean number of RNA probe puncta). Kolmogorov-Smirnov normality test with Dallal-Wilkinson-Lillie for p -value, two-tailed unpaired t test. Mean ± SEM. I Western blot showing TOMM20 and CHCHD2 levels, compared to housekeeping B-TUBULIN in hESC-derived oligodendroglial monocultures treated for 7 days with metformin versus vehicle, with quantification. Mean ± SEM, two-tailed paired t test. M = metformin treated, C = vehicle treated controls. n = 3 separate differentiations. J Violin plot showing expression of EIF1 in multiple sclerosis (MS) donor oligodendrocytes with and without metformin (met) treatment versus controls (CTR). Source data are provided in the Source Data file.

Article Snippet: Sections underwent H 2 O 2 treatment for 10 min at RT, washed in ddH 2 O and incubated for target retrieval for 5 min at 100 o C. Slides were washed again in ddh2O, followed by 0.1% PBS-Tween and incubated with anti-Olig2 primary antibody (Atlas antibodies, HPA003254) overnight at 4 o C. Sections were washed in 0.1% PBS-Tween and post-fixed in 4% PFA at room temperature for 30 min.

Techniques: Electron Microscopy, Two Tailed Test, Derivative Assay, RNA Sequencing, In Situ, Hybridization, Immunofluorescence, Western Blot, Expressing