nsc-100  (Viavi Solutions)

Journal: Nature Communications

Article Title: Chi3l3 induces oligodendrogenesis in an experimental model of autoimmune neuroinflammation

doi: 10.1038/s41467-018-08140-7

Figure Lengend Snippet: Chi3l3 directly promotes oligodendrogenesis in vitro. Representative confocal image (above) and quantification (below) of neural stem cells (NSCs) cultured in the presence of PBS (control) or Chi3l3 (100 ng/ml) for 3 days a – d , 5 days e – h , or primary OPCs cultured in the presence of PBS (control) or Chi3l3 (500 ng/ml) for 7 days i . Cells were treated with the nuclear stain TO-PRO-3 (blue) and immunostained for early progenitor markers NG2 ( a ; oligodendrocyte precursor cells, green), GFAP ( b ; astrocytes, green), Dcx ( c ; neuroblasts, green), late progenitor markers O4 ( e ; oligodendrocytes, green), GFAP ( f ; astrocytes, green) and microtubule-associated protein 2 ( g ; Map2, neurons, green), the neural stem cell marker Sox2 ( d , h ; green) and the myelin protein MBP ( i , oligodendrocytes, green). Exposure of differentiating NSCs to Chi3l3 led to significant increase in oligodendrocyte precursor cells and oligodendrocytes, significant decrease in astrocytes, neuroblasts, and neurons and a significant increase in Sox2 + neural stem cells. Scale bar, 50 μm. Inserts show representative cells. Scale bar, 20 μm. j – n Gene expression of Cspg4 (NG2; j ), Gfap k , and Map2 ( l ; 3 days) and Ccnd1 and Ccnd2 ( m , n ; 24 h) mRNA in PBS (control) or Chi3l3-treated differentiating NSCs. Values were normalized against Gapdh (AU, arbitrary unit; n.s., not significant;). Number o and size p of neurospheres from NSCs exposed to Chi3l3 or PBS (control). (n.s., not significant; two-tailed Student’s t test; data are representative of three independent experiments with n = 5 replicates a , c , d , i , n = 9 (Control) and 10 (Chi3l3) replicates b , n = 12 (Control) and 9 (Chi3l3) replicates e , n = 12 replicates f , g , n = 3 replicates h , j , l , m , n ), n = 3 (Control) and 2 (Chi3l3) replicates k , n = 4 replicates o , n = 8 (control) and four (Chi3l3) replicates p ). mean ± s.e.m. * p < 0.05; ** p < 0.01; *** p < 0.005

Article Snippet: For analysis of hCHI3L1- or hCHIT1-dependent human neural stem cell differentiation, human NSCs (H9, N7800–100, Thermofisher scientific) were dissociated, washed and plated at 5 × 10 5 cells on glass coverslips (diameter: 18 mm, Thermofisher scientific) precoated with poly- l -lysine (0.01% solution, Sigma) and cultured for 1 or 2 weeks in NSC differentiation medium containing NeuroBasal media, 2% B27 supplement, Glutamax, and antibiotic–antimycotic (Thermofisher scientific) in the presence of 250 ng/ml recombinant human CHI3L1 and CHIT1 (all R&D Systems Inc.).

Techniques: In Vitro, Cell Culture, Control, Staining, Marker, Gene Expression, Two Tailed Test

Journal: Nature Communications

Article Title: Chi3l3 induces oligodendrogenesis in an experimental model of autoimmune neuroinflammation

doi: 10.1038/s41467-018-08140-7

Figure Lengend Snippet: Chi3l3 promotes oligodendrogenesis by inducing EGFR signaling. a ingenuity pathway analysis showing membrane-bound receptors (EGFR, FGFR1, DLG4 (PSD95), NTRK2, FYN) upstream of Chi3l3-induced MAPK signaling (Ptk2b (Pyk2), MAPK14 (p38MAPK), PLCG2 (PLCγ2), RAF1 (c-Raf), ERK1/2, PI3K p85) and pro-oligodendrogenic transcriptional pattern ( Olig1, Olig2, Sox10, Hes5 , Id2 , Id4 ). b Mean fluorescence intensity (MFI) quantification of individual neural stem cells (NSCs), activated by Chi3l3, EGF, or PBS (control) for 15 min, and immunostaining for phospho (p)-specific EGFR (Tyr1068). One-way-ANOVA with Dunn’s multiple comparison test; c Quantification of NSC-derived NG2 + oligodendrocyte precursor cells after 3 days of differentiation in the presence of Chi3l3 ( + ) or PBS (−) and EGFR kinase inhibitor AZD8931 ( + , left) and Gefitinib ( + , right) or DMSO (−). One-way ANOVA with Bonferroni’s multiple comparison test; d Quantification of NG2 + oligodendrocyte precursor cells derived from differentiating NSCs, transfected with control shLVPs or EGFR shLVPs and treated with Chi3l3 ( + ) or PBS (−). Deficiency in EGFR signaling significantly reduced Chi3l3-induced oligodendrogenesis. One-way ANOVA with Bonferroni’s multiple comparison test; e Mean fluorescence intensity (MFI) quantification of NSCs, activated by Chi3l3 ( + ), EGF ( + , positive control) or PBS (−, negative control) in the presence of EGFR inhibitor AZD8931 for 15 min, and immunostaining for phospho (p)-specific Pyk2 (Tyr402). One-way ANOVA with Bonferroni’s ( d , neuroblasts, red) test; f Mean fluorescence intensity (MFI) quantification of NSCs transfected with control shLVPs or EGFR shLVPs, activated by Chi3l3 ( + ), EGF ( + , positive control) or PBS (−, negative control for 15 min, and immunostained for phospho (p)-specific Pyk2 (Tyr402). Deficiency in EGFR signaling reduced Pyk2 phosphorylation in neural stem cells. All values are expressed as percent change to PBS-treated NSCs. One-way ANOVA with Bonferroni’s multiple comparison test; mean ± s.e.m; * p < 0.05; ** p < 0.01; *** p < 0.005

Article Snippet: For analysis of hCHI3L1- or hCHIT1-dependent human neural stem cell differentiation, human NSCs (H9, N7800–100, Thermofisher scientific) were dissociated, washed and plated at 5 × 10 5 cells on glass coverslips (diameter: 18 mm, Thermofisher scientific) precoated with poly- l -lysine (0.01% solution, Sigma) and cultured for 1 or 2 weeks in NSC differentiation medium containing NeuroBasal media, 2% B27 supplement, Glutamax, and antibiotic–antimycotic (Thermofisher scientific) in the presence of 250 ng/ml recombinant human CHI3L1 and CHIT1 (all R&D Systems Inc.).

Techniques: Membrane, Fluorescence, Control, Immunostaining, Comparison, Derivative Assay, Transfection, Positive Control, Negative Control, Phospho-proteomics

Journal: Nature Communications

Article Title: Chi3l3 induces oligodendrogenesis in an experimental model of autoimmune neuroinflammation

doi: 10.1038/s41467-018-08140-7

Figure Lengend Snippet: Chi3l3 infusion induces pro-oligodendrogenic fate of subventricular zone neural stem cells in vivo. a Protocol of BrdU labeling via intraperitoneal (i.p.) injection and Chi3l3 administration. b – f Representative confocal images b , d and quantification c , e , f of the ipsilateral (IL) or contralateral (CL) subventricular zone (SVZ) of naive mice infused with Chi3l3 or PBS (vehicle) and immunostained for BrdU (green) and NG2 ( b , oligodendrocyte precursor cells, red) or Dcx ( d , neuroblasts, red). IL SVZ of Chi3l3-treated mice were compared with IL SVZ of vehicle-treated mice as well as the CL site of the same mouse. Dashed lines mark the ventricular wall of the right or left lateral ventricle (RLV; LLV). Scale bar (left), 50 μm. Double-positive cells are indicated by yellow and white arrowheads and representative cells (box) are shown as high magnification images (right). Scale bar (right), 5 μm. Quantifications show a significantly higher percentage of BrdU/NG2 double-positive cells c and reduced percentage of BrdU + /Dcx + cells e in the ipsilateral SVZ of Chi3l3 infused mice indicating pro-oligodendrogenic modulation of SVZ stem cell fate choice in vivo. f Quantification of the total number of BrdU labeled cells/mm 2 in the SVZ showed no significant difference between Chi3l3 or vehicle infusion in the ipsilateral or contralateral SVZ. (One-way ANOVA with Turkey’s post hoc test; c n = 2 mice (Control IL, CL), n = 4 (Chi3l3 IL), and n = 3 (chi3l3 CL); e n = 2 mice per group, n = 4 mice per group; mean ± s.e.m. * p < 0.05; ** p < 0.01; *** p < 0.005)

Article Snippet: For analysis of hCHI3L1- or hCHIT1-dependent human neural stem cell differentiation, human NSCs (H9, N7800–100, Thermofisher scientific) were dissociated, washed and plated at 5 × 10 5 cells on glass coverslips (diameter: 18 mm, Thermofisher scientific) precoated with poly- l -lysine (0.01% solution, Sigma) and cultured for 1 or 2 weeks in NSC differentiation medium containing NeuroBasal media, 2% B27 supplement, Glutamax, and antibiotic–antimycotic (Thermofisher scientific) in the presence of 250 ng/ml recombinant human CHI3L1 and CHIT1 (all R&D Systems Inc.).

Techniques: In Vivo, Labeling, Injection, Control

Journal: Nature Communications

Article Title: Chi3l3 induces oligodendrogenesis in an experimental model of autoimmune neuroinflammation

doi: 10.1038/s41467-018-08140-7

Figure Lengend Snippet: Human CLPs directly promote oligodendrogenesis. Human neural stem cells (NSCs) were cultured in the presence of PBS (control), human CHI3L1 and human CHIT1 at 250 ng/ml for 14 days. a – c Cells were immunostained for MBP ( a , oligodendrocytes, green), DCX ( a , neurons, red), NG2 ( b , OPC, green), GFAP ( c , astrocyte, red), SOX2 ( d , NSC, red) and nuclear stain DAPI (blue). Scale bar, 50 μm. e Quantification of MBP + , NG2 + , GFAP + , DCX + , and SOX2 + NSCs after exposure to human Chi3L1or human Chit1. Exposure of differentiating NSCs to CHI3L1 and CHIT1 led to significant increase in oligodendrogenesis. Values are expressed as percent change to PBS-treated NSCs (data are representative of three independent experiments. one-way ANOVA with Dunnett’s multiple comparison test; mean ± s.e.m * p < 0.05; ** p < 0.01; *** p < 0.005; N.S. = not significant

Article Snippet: For analysis of hCHI3L1- or hCHIT1-dependent human neural stem cell differentiation, human NSCs (H9, N7800–100, Thermofisher scientific) were dissociated, washed and plated at 5 × 10 5 cells on glass coverslips (diameter: 18 mm, Thermofisher scientific) precoated with poly- l -lysine (0.01% solution, Sigma) and cultured for 1 or 2 weeks in NSC differentiation medium containing NeuroBasal media, 2% B27 supplement, Glutamax, and antibiotic–antimycotic (Thermofisher scientific) in the presence of 250 ng/ml recombinant human CHI3L1 and CHIT1 (all R&D Systems Inc.).

Techniques: Cell Culture, Control, Staining, Comparison