Journal: International Journal of Molecular Sciences

Article Title: Progenitor Cells from Cartilage: Grade Specific Differences in Stem Cell Marker Expression

doi: 10.3390/ijms18081759

Figure Lengend Snippet: Comparative analysis of MSC and Chondrocyte markers in the tissue (D0) isolated from mild and severe OA regions. ( A ) The mRNA expression levels of CD105, CD166 and Notch 1 (MSC markers) in mild versus severe OA at D0; ( B ) The mRNA expression levels of Sox9, Acan and Col II (chondrocyte markers) in mild versus severe OA at D0. Mild OA is used as the control expression to normalized results.

Article Snippet: Cells were expanded for 2 weeks in DMEM/F12 supplemented with FBS 10%, PS 1% and Fungizon 0.1% (GibcoTM, Courtaboeuf, France) and then subjected to immunofluorescence for antibodies to anti-goat Notch 1 (1:50; sc-9170, Santa Cruz Biotechecnology ® , Inc., Dallas, TX, USA), anti-rabbit CD90 (1:50; sc-9163, Santa Cruz Biotechecnology ® , Inc.), anti-mouse CD166 (1:50; sc-74557, Santa Cruz Biotechecnology ® , Inc., Santa Cruz, CA, USA), anti-rabbit nucleostemin (1:100; ab 70346, Abcam ® , Paris, France), anti-rabbit CD73 (1:50; ab175396, Abcam ® , Paris, France) and R-phycoerythrin-conjugated anti-CD105 (clone SN6; IgG1; Ancell Corporation, Bayport, MN, USA).

Techniques: Isolation, Expressing, Control

Journal: International Journal of Molecular Sciences

Article Title: Progenitor Cells from Cartilage: Grade Specific Differences in Stem Cell Marker Expression

doi: 10.3390/ijms18081759

Figure Lengend Snippet: Comparative analysis of MSC and Chondrocyte markers between tissue (D0) and proliferated cells (D14) in Mild OA. ( A ) The mRNA expression levels of CD105, CD166 and Notch 1 (MSC markers) at D14 versus D0; ( B ) The mRNA expression levels of Sox9, Acan and Col II (chondrocytes markers) at D14 versus D0. D0 is used as the control expression to normalise results. ** p < 0.01, *** p < 0.001, **** p < 0.0001, Student’s t -test ( n = 8 in Mild OA, n = 6 in severe OA).

Article Snippet: Cells were expanded for 2 weeks in DMEM/F12 supplemented with FBS 10%, PS 1% and Fungizon 0.1% (GibcoTM, Courtaboeuf, France) and then subjected to immunofluorescence for antibodies to anti-goat Notch 1 (1:50; sc-9170, Santa Cruz Biotechecnology ® , Inc., Dallas, TX, USA), anti-rabbit CD90 (1:50; sc-9163, Santa Cruz Biotechecnology ® , Inc.), anti-mouse CD166 (1:50; sc-74557, Santa Cruz Biotechecnology ® , Inc., Santa Cruz, CA, USA), anti-rabbit nucleostemin (1:100; ab 70346, Abcam ® , Paris, France), anti-rabbit CD73 (1:50; ab175396, Abcam ® , Paris, France) and R-phycoerythrin-conjugated anti-CD105 (clone SN6; IgG1; Ancell Corporation, Bayport, MN, USA).

Techniques: Expressing, Control

Journal: International Journal of Molecular Sciences

Article Title: Progenitor Cells from Cartilage: Grade Specific Differences in Stem Cell Marker Expression

doi: 10.3390/ijms18081759

Figure Lengend Snippet: Comparative analysis of MSC and Chondrocytes markers between tissue (D0) and proliferated cells (D14) in severe OA. ( A ) The mRNA expression levels of CD105, CD166 and Notch 1 (MSC markers) at D14 versus D0; ( B ) The mRNA expression levels of Sox9, Acan and Col II (chondrocytes markers) at D14 versus D0. D0 is used as the control expression to normalized results. ** p < 0.01, Student’s t -test ( n = 8 in Mild OA, n = 6 in severe OA).

Article Snippet: Cells were expanded for 2 weeks in DMEM/F12 supplemented with FBS 10%, PS 1% and Fungizon 0.1% (GibcoTM, Courtaboeuf, France) and then subjected to immunofluorescence for antibodies to anti-goat Notch 1 (1:50; sc-9170, Santa Cruz Biotechecnology ® , Inc., Dallas, TX, USA), anti-rabbit CD90 (1:50; sc-9163, Santa Cruz Biotechecnology ® , Inc.), anti-mouse CD166 (1:50; sc-74557, Santa Cruz Biotechecnology ® , Inc., Santa Cruz, CA, USA), anti-rabbit nucleostemin (1:100; ab 70346, Abcam ® , Paris, France), anti-rabbit CD73 (1:50; ab175396, Abcam ® , Paris, France) and R-phycoerythrin-conjugated anti-CD105 (clone SN6; IgG1; Ancell Corporation, Bayport, MN, USA).

Techniques: Expressing, Control

Journal: International Journal of Molecular Sciences

Article Title: Progenitor Cells from Cartilage: Grade Specific Differences in Stem Cell Marker Expression

doi: 10.3390/ijms18081759

Figure Lengend Snippet: Protein expression of MSC markers in Mild and severe OA-derived cells at D14 (scale bar 20 µm). Immunofluorescence of Nucleostemin, CD105, CD166, CD73, CD90 and Notch 1 in proliferated cells (D14) isolated from areas of Mild OA ( A ) and severe OA ( B ). Markers of interest-RE staining (Red), contra staining DAPI (blue), and Isotype control + DAPI-left corner of each image.

Article Snippet: Cells were expanded for 2 weeks in DMEM/F12 supplemented with FBS 10%, PS 1% and Fungizon 0.1% (GibcoTM, Courtaboeuf, France) and then subjected to immunofluorescence for antibodies to anti-goat Notch 1 (1:50; sc-9170, Santa Cruz Biotechecnology ® , Inc., Dallas, TX, USA), anti-rabbit CD90 (1:50; sc-9163, Santa Cruz Biotechecnology ® , Inc.), anti-mouse CD166 (1:50; sc-74557, Santa Cruz Biotechecnology ® , Inc., Santa Cruz, CA, USA), anti-rabbit nucleostemin (1:100; ab 70346, Abcam ® , Paris, France), anti-rabbit CD73 (1:50; ab175396, Abcam ® , Paris, France) and R-phycoerythrin-conjugated anti-CD105 (clone SN6; IgG1; Ancell Corporation, Bayport, MN, USA).

Techniques: Expressing, Derivative Assay, Immunofluorescence, Isolation, Staining, Control

Journal: International Journal of Molecular Sciences

Article Title: Progenitor Cells from Cartilage: Grade Specific Differences in Stem Cell Marker Expression

doi: 10.3390/ijms18081759

Figure Lengend Snippet: Comparative analysis of MSC and Chondrocyte markers in proliferated cells (D14) isolated from mild and severe OA regions. ( A ) The mRNA expression levels of CD105, CD166 and Notch 1 (MSC markers) in mild versus severe OA at D14; ( B ) The mRNA expression levels of Sox9, Acan and Col II (chondrocytes markers) in mild versus severe OA at D148. Mild OA is used as the control expression to normalized results. * p < 0.05, ** p < 0.01, Student’s t -test ( n = 8 in Mild OA, n = 6 in severe OA).

Article Snippet: Cells were expanded for 2 weeks in DMEM/F12 supplemented with FBS 10%, PS 1% and Fungizon 0.1% (GibcoTM, Courtaboeuf, France) and then subjected to immunofluorescence for antibodies to anti-goat Notch 1 (1:50; sc-9170, Santa Cruz Biotechecnology ® , Inc., Dallas, TX, USA), anti-rabbit CD90 (1:50; sc-9163, Santa Cruz Biotechecnology ® , Inc.), anti-mouse CD166 (1:50; sc-74557, Santa Cruz Biotechecnology ® , Inc., Santa Cruz, CA, USA), anti-rabbit nucleostemin (1:100; ab 70346, Abcam ® , Paris, France), anti-rabbit CD73 (1:50; ab175396, Abcam ® , Paris, France) and R-phycoerythrin-conjugated anti-CD105 (clone SN6; IgG1; Ancell Corporation, Bayport, MN, USA).

Techniques: Isolation, Expressing, Control

Journal: Nature Communications

Article Title: Identification of a common mesenchymal stromal progenitor for the adult haematopoietic niche

doi: 10.1038/ncomms13095

Figure Lengend Snippet: ( a ) Representative FACS profiles of stromal cells (CD45 − Ter119 − CD31 − ) near the endosteum (bone-disassociated). They were separated based on their CD166, CD146 and Sca1 expression profiles. Numbers shown in the gate represent the percentage of total live cells. ( b ) Single-cell colony forming efficiency of BM stromal progenitors (*: P ≤0.05, three repeats of 60 cells each, student's t -test). ( c ) Sections of femur from Flk1-GFP mouse stained with antibodies against Sca1 and scanned with an iCys Research imaging cytometer. Nuclei were stained with Dapi. ( d ) Confocal maximum intensity projections of sections of femurs from Flk1-GFP mice stained with antibodies against Sca1 or CD146; scanned near the epiphyseal plate. ( e ) Confocal maximum intensity projections of sections of femur stained with antibodies against Sca1 or CD146 and laminin; scanned near epiphyseal plate. White arrows point to mesenchymal progenitors yellow arrows point to endothelial cells.

Article Snippet: Sections were blocked with 1% BSA, then probed with primary antibody at 4 °C overnight; purified rat-anti-Sca1 (1:50 BioLegend 108102), purified rat-anti-CD146 (1:50, BioLegend 134702), CD166-Fitc (1:25 R&D Systems FAB1172F), CD45-Alexa647 (1:50 BioLegend 103124) and/or rabbit-anti-laminin (1:200 Sigma-Aldrich L9393-.2ML).

Techniques: Expressing, Staining, Imaging, Cytometry

Journal: Nature Communications

Article Title: Identification of a common mesenchymal stromal progenitor for the adult haematopoietic niche

doi: 10.1038/ncomms13095

Figure Lengend Snippet: ( a – b ) FACS analysis of bone-disassociated stromal cells (CD45 − TER119 − CD31 − ) from ( a ) Col2.3-GFP or ( b ) Osx1-Cre:GFP mice. The fequency of the parent gate is displayed. ( c – d ) The frequency of Sca1+, CD146+, CD166+ and Sca1- in the GFP+ population for (mean±s.d.) ( c ) Col2.3-GFP and ( d ) Osx1-Cre:GFP obtained from FACS analysis ( n =3). ( e – f ) Sections of femurs from ( e ) Col2.3-GFP or ( f ) Osx1-Cre:GFP mice stained with antidodies to Sca1 or CD146.

Article Snippet: Sections were blocked with 1% BSA, then probed with primary antibody at 4 °C overnight; purified rat-anti-Sca1 (1:50 BioLegend 108102), purified rat-anti-CD146 (1:50, BioLegend 134702), CD166-Fitc (1:25 R&D Systems FAB1172F), CD45-Alexa647 (1:50 BioLegend 103124) and/or rabbit-anti-laminin (1:200 Sigma-Aldrich L9393-.2ML).

Techniques: Staining

Journal: Nature Communications

Article Title: Identification of a common mesenchymal stromal progenitor for the adult haematopoietic niche

doi: 10.1038/ncomms13095

Figure Lengend Snippet: ( a ) Representative qRT-PCR of 18 single cells from CD146 + , CD166 + or Sca1 + stromal populations either near the endosteum (bone-disassociated) or in the marrow (Marrow) using the Fluidigm 48.48 dynamic array. Each column within a given population represents expression in an individual cell. ( b ) Comparison of the first and second prinicpal components from a principal component analysis of single-cell PCR gene expression data from all 47 genes. ( c – e ) Expression patterns of genes that are significantly overexpressed in mean±s.d. ( c ) Sca1 + cells ( d ) Sca1 + cells and/or CD146 + cells, or ( e ) CD166 + cells. Statistical significance was calculated using one-way ANOVA analysis * P ≤0.05, ** P ≤0.005, *** P ≤0.00; n =72. ANOVA, analysis of variance.

Article Snippet: Sections were blocked with 1% BSA, then probed with primary antibody at 4 °C overnight; purified rat-anti-Sca1 (1:50 BioLegend 108102), purified rat-anti-CD146 (1:50, BioLegend 134702), CD166-Fitc (1:25 R&D Systems FAB1172F), CD45-Alexa647 (1:50 BioLegend 103124) and/or rabbit-anti-laminin (1:200 Sigma-Aldrich L9393-.2ML).

Techniques: Quantitative RT-PCR, Expressing, Comparison, Gene Expression

Journal: Nature Communications

Article Title: Identification of a common mesenchymal stromal progenitor for the adult haematopoietic niche

doi: 10.1038/ncomms13095

Figure Lengend Snippet: ( a ) Sca1 + progenitors were transduced with lentivirus as indicated and 1,000 sorted GFP + Sca1 + progenitors were co-transplanted with unmarked fetal skeletal progenitors under the kidney capsule. ( b ) Representative cross sections of the engrafted ectopic bone stained with H&E (right) or GFP (left) to identify the donor origin, harvested 1 month after transplant. ( c ) Representative FACS profiles of LSK frequencies that were pre-gated for live, CD45 + lineage − cells. ( d ) Cellularity of the ectopic niche marrow (* P <0.05, n =5, student's t -test). ( e ) Rescue of lethally irradiated mice with 2 × 10 5 marrow cells from engrafted ectopic niche generated with either mixed Sca1 + /shKITL progenitors (red) or with Sca1 + /Scrambled shRNA progenitors (yellow) or control bone marrow from host tibia (black) ( n =8). ( f ) The schematic summaries our findings such that Sca1+ cells are the most primative giving rise to CXCL12 producing stromal cells, CD146+ cells and CD166+ cells. CD146+ and CD166+ cells are osteo-progenitors. It is possible that CD146+ is an intermidiary cell fate between Sca1+ cells and CD166+ cells.

Article Snippet: Sections were blocked with 1% BSA, then probed with primary antibody at 4 °C overnight; purified rat-anti-Sca1 (1:50 BioLegend 108102), purified rat-anti-CD146 (1:50, BioLegend 134702), CD166-Fitc (1:25 R&D Systems FAB1172F), CD45-Alexa647 (1:50 BioLegend 103124) and/or rabbit-anti-laminin (1:200 Sigma-Aldrich L9393-.2ML).

Techniques: Transduction, Staining, Irradiation, Generated, shRNA, Control

Journal: Nature

Article Title: Floor plate-derived netrin-1 is dispensable for commissural axon guidance

doi: 10.1038/nature22331

Figure Lengend Snippet: Coronal cryostat sections of the hindbrain of E11 and E13 embryos and spinal cord (brachial level) of E11 embryos . a-b , In Foxg1:Cre;Netrin-1 L/+ embryos as in wild type, netrin-1 is in the hindbrain VZ (arrowhead) and commissural axons (arrow). This is not the case in Foxg1:Cre;Netrin-1 L/L mutant. The floor plate is still labelled (Fp). Note that netrin-1 is present in the vicinity of the Fp (n=6). c , Foxg1:Cre drives Cre expression in E13 (left) and E11 (right) hindbrain cells (Tomato+ cells in red) but not in the floor plate (arrowheads; n=3/3). A few Alcam+ floor plate cells are Tomato+. d, e, Netrin-1 distribution is similar in the spinal cord of Foxg1:Cre;Netrin-1 L/+ (d) and Foxg1:Cre;Netrin-1 L/L ( e ) embryos (n=5). f , In Netrin1 ßgeo/ßgeo , Foxp2+ olivary neurons fail to migrate (arrowheads) ventrally and only few of them are able to reach to the floor plate (arrowheads; n=6). g , Quantification of the size of hindbrain commissures in the different mutants compared to controls. Six embryos of each genotype and 9 sections from each were quantified. Data are normalized with the wt and are represented as the mean ± SEM (One-way Kruskal-Wallis with Mann-Whitney post-test, *P<0.05, ns, not significant). h, Netrin-1 guidance mechanisms of hindbrain commissural axons: past and current models. In the initial model, soluble netrin-1 secreted by floor plate (FP) forms a ventro-dorsal gradient, which attracts ventrally commissural axon (CN) growth cones (GC). In the revised model, pioneer CN axons form in a superficial region containing high levels of netrin-1 produced by neural progenitor cells (NPCs) extending from the ventricular zone (VZ) to the basal lamina (BL) at the surface of the hindbrain. CN axons might also capture Netrin-1 and establish a netrin-1-rich pathway guiding follower axons. Their ventral extension might be facilitated by chemorepellents (question mark) produced in the dorsal hindbrain. Scale bars, 100µm.

Article Snippet: Sections were then incubated O/N with the following primary antibodies: goat anti-human Robo3 (1:250, R&D Systems AF3076), goat anti-Dcc (1:500, Santa Cruz sc-6535), goat anti-Robo1 (1:500, R&D Systems AF1749), rat anti-mouse Netrin-1 (1:500, R&D Systems MAB1109), mouse anti-Nestin-Alexa488 (1:1000, Abcam ab197495), mouse anti Neurofilament (1:300, DSHB 2H3), goat anti-Foxp2 (1:1000, Santa Cruz sc-21069), rabbit anti-Foxp2 (1:1000, Abcam AB16046), rabbit anti-Sox2 (1:500, Abcam ab97959), rabbit anti-βgal (1:500, Cappel 55976), goat anti-human Alcam (1:500, R&D Systems AF656), rabbit anti-GFP (1:800, Life Technologies A11122), rabbit anti-Dsred (1:500, Clontech 632496) followed by 2 hours incubation in species-specific secondary antibodies directly conjugated to fluorophores (Cy-5, Cy-3, Alexa-Fluor 647 from Jackson ImmunoResearch, or from Invitrogen).

Techniques: Mutagenesis, Expressing, MANN-WHITNEY, Produced

Journal: Journal of neurochemistry

Article Title: Distribution and development of molecularly distinct perineuronal nets in visual thalamus

doi: 10.1111/jnc.14614

Figure Lengend Snippet: (A) Volcano scatter plot mapping RNAseq expression differences between vLGN and dLGN at P12 obtained from RNAseq (Monavarfeshani et al., 2018) to proteomic data. Each data point is one mRNA transcript. Green circles indicate no significant differences in protein expression level associated with that mRNA, blue triangles indicate proteins enriched in vLGN, orange triangles indicate proteins enriched in dLGN, and blank circles indicate transcripts captured in RNAseq but not in mass-spectrometry (MS) datasets. (B) Comparison of expression data for select molecules obtained with quantitative PCR, RNAseq, and proteomics. # indicates no peptides detected by MS. (C) Immunohistochemical validation of expression of select proteins in (B). (D-F) In situ hybridization validating mRNA expression of select genes and peptides in (B). (G) WB analysis validating protein expression of select genes and peptides in (B). Calb1 – Calbindin 1; Calb2 – Calbindin 2; Gad1 – Glutamic acid decarboxylase; Gad2 – Glutamic acid decarboxylase 2; Vglut1 – Vesicular glutamate transporter 1; Vglut2 – Vesicular glutamate transporter 2; Slc8a1 – Sodium/calcium exchanger 1; Pcdh1 – Protocadherin 1; Myh11 – Myosin heavy chain 11; Sez6l – Seizure related 6 homolog like; Syt1 – Synaptotagmin 1; Alcam – Activated leukocyte cell adhesion molecule; GAD67 – Glutamic acid decarboxylase 67. Scale bars: (C) 40 μm, (D-F) 200 μm (inset 40 μm). All expression studies were performed at P12, n=3 mice.

Article Snippet: After blocking PVDF membranes using 5% non-fat milk in PBS (0.05% Tween), membranes were incubated overnight with primary antibodies (VGLUT1 [Neuromab 75–066, RRID:AB_2187693] 1:400, VGLUT2 [Neuromab 75–067, RRID:AB_2239153] 1:400, GAD67 [EMD Millipore MAB5406, RRID:AB_2278725] 1:5000, GAD67/65 [Chemicon AB1511, RRID:AB_90715] 1:5000, CALB1 [Swant, CB‐38a] 1:10000, CALB2 [Chemicon AB5054] 1:5000, ALCAM [R&D System AF1172] 1:1000, SEZ6l [R&D System AF4804] 1:1000, ACAN [Millipore AB1031] 1:500, ACTIN [EMD Millipore MAB1501, RRID:AB_2223041] 1:5000), followed by HRP-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA).

Techniques: Expressing, Mass Spectrometry, Comparison, Real-time Polymerase Chain Reaction, Immunohistochemical staining, Biomarker Discovery, In Situ Hybridization