Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: GORAB promotes embryonic lung maturation through antagonizing AKT phosphorylation, versican expression and mesenchymal cell migration

doi: 10.1096/fj.201902075R

Figure Lengend Snippet: (A, B) Quantification of type I (aquaporin 5, Aqp5) and type II (prosurfactant associated protein C, proSFTPC) epithelial markers by quantitative RT-PCR (a) and western blotting (B) in control (Gorab+/+) and homozygous Gorab knockout mouse models (Gorab−/−). (C) Immunofluorescence labeling of AQP5 and proSFTPC in lung of Gorab+/+ or Gorab−/− fetuses, and quantification (n > 300 cells per three pairs of littermates). Nuclei are stained with DAPI (blue). (D - G) Evaluation of versican (Vcan) expression in the lung of control (Gorab+/+) and homozygous Gorab knockout mouse models (Gorab−/−) by in situ hybridization (D), immunofluorescence (E), quantitative RT-PCR (F), and western blotting (G). (H) Western blotting of VCAN in primary fibroblasts isolated from E18.5 Gorab+/+ and Gorab−/− fetuses. Data are shown as means ± SD (n ≥ 3 pairs of littermates). ∗∗∗ P < 0.001, NS, not statistically significant vs. corresponding controls. Scale bars, 25 μm (C, E), 50 μm (D).

Article Snippet: Mouse Vcan cDNA (V2) was obtained from Origene (MR225930, Rockville, MD) and transfected (1 μg per well of 12-well plate) to mouse primary lung fibroblasts as described for siRNA.

Techniques: Quantitative RT-PCR, Western Blot, Control, Knock-Out, Immunofluorescence, Labeling, Staining, Expressing, In Situ Hybridization, Isolation

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: GORAB promotes embryonic lung maturation through antagonizing AKT phosphorylation, versican expression and mesenchymal cell migration

doi: 10.1096/fj.201902075R

Figure Lengend Snippet: (A) Appearance of alveolar-like structure formed by co-culturing A549 epithelial cells with primary fibroblasts isolated from lungs of E18.5 Gorab+/+ or Gorab−/− fetuses for indicated durations. (B) Quantifications of the thickness (left) and height (middle) of cell ridge and the area of pockets (right) at 72 hours of co-culturing. (C) Representative distribution of cells in 72-hour co-cultures by immunofluorescence labeling of A549 (CellTracker dye CMTPX, red) and fibroblasts (vimentin, VIM, green). (D - E) Appearance (D) and quantification (E) of alveolar-like structure formed by co-culturing A549 epithelial cells with primary fibroblasts isolated from the lungs of wild type E18.5 Gorab+/+ fetuses and transfected with Vcan-V2 cDNA or Vector. Mock indicates non-transfected cells. Cells were examined 48 hours after transfection. (F - G) Appearance (F) and quantification (G) of alveolar-like structure formed by co-culturing A549 epithelial cells with primary fibroblasts isolated from the lungs of E18.5 Gorab−/− fetuses and transfected with Vcan siRNA or non-targeting (NT) siRNA. Mock indicates non-transfected cells. Cells were examined 48 hours after transfection. Data are shown as means ± SD (n ≥ 3 independent experiments). Student’s t-test and Bonferroni’s multiple comparison test, one-way ANOVA. ∗ P < 0.05 vs. corresponding controls. Scale bars, 500 μm (A), 100 μm (C), 250 μm (D, F).

Article Snippet: Mouse Vcan cDNA (V2) was obtained from Origene (MR225930, Rockville, MD) and transfected (1 μg per well of 12-well plate) to mouse primary lung fibroblasts as described for siRNA.

Techniques: Isolation, Immunofluorescence, Labeling, Transfection, Plasmid Preparation, Comparison

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: GORAB promotes embryonic lung maturation through antagonizing AKT phosphorylation, versican expression and mesenchymal cell migration

doi: 10.1096/fj.201902075R

Figure Lengend Snippet: (A) Western blotting of phospho-AKT (p-AKT S473 and S308), PDGF signaling components, and PHLPP2 in the lung of E18.5 control (Gorab+/+) and homozygous Gorab knockout mouse models (Gorab−/−). (B) Western blotting of p-AKT in primary fibroblasts isolated from the lung of E18.5 Gorab+/+ and Gorab−/− fetuses (n ≥ 3 pairs of littermates). (C) Western blotting of p-AKT in primary mesenchymal cells treated with PDGF-AA. (D) H&E staining of distal regions of the lungs biopsied from E18.5 wild type (Pdgfrα+/+) and heterozygous Pdgfrα+/J mutants. (E) Quantifications of the area of alveolar sac (left), the length of alveolar sac chord (middle), and the thickness of alveolar septum (right). (F) Western blotting of p-AKT and VCAN in the lung of E18.5 Pdgfrα+/+ and Pdgfrα+/J mutants. All experiments were performed with a minimum of 3 pairs of littermates. Data are shown as means ± SD (n = 3 mice/group). Scale bars, 50 μm.

Article Snippet: Mouse Vcan cDNA (V2) was obtained from Origene (MR225930, Rockville, MD) and transfected (1 μg per well of 12-well plate) to mouse primary lung fibroblasts as described for siRNA.

Techniques: Western Blot, Control, Knock-Out, Isolation, Staining

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: GORAB promotes embryonic lung maturation through antagonizing AKT phosphorylation, versican expression and mesenchymal cell migration

doi: 10.1096/fj.201902075R

Figure Lengend Snippet: (A) H&E staining of distal regions of the lungs of wild type (Gorab+/+;PDGFRα+/+), homozygous Gorab mutant (Gorab−/−;PDGFRα+/+), and homozygous Gorab mutant deficient for PDGF receptor α (Gorab−/−;PDGFRα+/EGFP). (B) Quantifications of the area of alveolar sac (left), the length of alveolar sac chord (middle), and the thickness of alveolar septum (right) (n = 3 pairs of littermates). (C) Western blotting of p-AKT and VCAN in lungs described in (A). (D, E) In situ hybridization (D) and immunofluorescence labeling (E) of VCAN in lungs described in (A). Nuclei are stained with hematoxylin (D) or DAPI (E). All experiments were performed with a minimum of 3 pairs of littermates. Data are shown as means ± SD (n = 3 mice/group). Bonferroni’s multiple comparison test, one-way ANOVA. Scale bars, 50 μm (A), 25 μm (D, E).

Article Snippet: Mouse Vcan cDNA (V2) was obtained from Origene (MR225930, Rockville, MD) and transfected (1 μg per well of 12-well plate) to mouse primary lung fibroblasts as described for siRNA.

Techniques: Staining, Mutagenesis, Western Blot, In Situ Hybridization, Immunofluorescence, Labeling, Comparison

Journal: eLife

Article Title: O-GlcNAc on NOTCH1 EGF repeats regulates ligand-induced Notch signaling and vascular development in mammals

doi: 10.7554/elife.24419

Figure Lengend Snippet: Figure 7. Reduced vessel integrity in the Eogt/ retina. (A) Immunostaining with fibrinogen (green) and a-SMA (magenta) antibodies in P15 wild-type, Eogt/, Tek-Cre, Tek-Cre:EogtF/F, Notch1+/, and Rbpj+/ retinas. Arrows indicate fibrinogen staining outside vessels stained by IB4 (white). Three- dimensional images were constructed from confocal images by maximum intensity projection. (B) Higher magnification three-dimensional images of Eogt/ retina constructed from confocal images using the Alpha-blend method. Below, single channel images showing fibrinogen (green) and IB4 (white) staining. (C) Sulfo-NHS-LC-biotin was perfused into P15 wild-type and Eogt/ mice and extravasation determined immediately after perfusion by staining with CF488A-conjugated streptavidin (green) and Dylight594-conjugated IB4 (white). Three-dimensional reconstructions were created by maximum intensity projection. Enlarged images of boxed area are shown (right). (D) Sulfo-NHS-LC-biotin was perfused into P15 wild-type, Eogt/, Notch1+/, Eogt/Notch1+/ mice as in (C). Quantification of the number of extravasation sites in 210 210 mm squares (n = 6 per retina per mouse) is shown. Note that sulfo-NHS-LC-biotin extravasation in Eogt/ retina is augmented in compound mutant mice. Data represent mean ± standard error; p values determined by Welch’s t test. ***p0.001. (E) Whole-mount images of wild-type or Eogt/ P15 retinas stained with IB4 (cyan) and anti-a SMA (magenta) antibody. (F) Whole-mount staining of wild-type and Eogt/ P15 retinas using IB4 (white) together with anti-fibrinogen (green) and anti-NG2 (magenta) antibodies. DOI: 10.7554/eLife.24419.018 The following source data and figure supplement are available for figure 7:

Article Snippet: Antibodies used in microscopy, flow cytometry and Western blot experiments: biotinylated isolectin B4 (IB4; Vector [B-1105]), Cy3-conjugated anti–a-smooth muscle actin (aSMA) antibody (clone 1A4; Sigma [C6198] or fluorescein isothiocyanate(FITC)-conjugated anti-aSMA antibody (clone 1A4; Sigma [F3777]), rabbit anti-human EOGT antibody (Sigma [HPA019460]), mouse anti-O-GlcNAc antibody (CTD110.6; Thermo Scientific [24565] or Sigma [07764]) (Comer et al., 2001), hamster antimouse NOTCH1 ECD antibody (8G10, Santa Cruz [sc-32756]), sheep anti-hamster NOTCH1 ECD antibody (R and D Systems, AF5267), rabbit anti-human NOTCH1 ECD antibody (H-131, Santa Cruz [sc-9170]), rabbit anti-human NOTCH1 ICD antibody (D6F11, Cell signaling), rabbit anti-mouse activated NOTCH1 (Val1744, Cell Signaling Technology [4147]) (Huppert et al., 2000), sheep anti-BiP antibody (BD Biosciences [51–9001980]), rabbit anti-NG2 chondroitin sulfate antibody (Millipore [AB5320]), rat anti-NG2 chondroitin sulfate antibody (R and D Systems [MAB6689]), mouse anti-Myc antibody (4A6, Upstate [05-724]), hamster anti-mouse JAG1 antibody (HMJ1-29; Biolegend [130902]), goat anti-mouse DLL4 antibody (R and D Systems [AF1389]), rabbit anti-fibrinogen antibody (Dako [A0080]), FITC-conjugated anti-hamster IgG antibody (Cappel [55400]), CF488A-conjugated anti-goat IgG antibody (Sigma [SAB4600032]), Dylight488–conjugated anti-rabbit IgG (Vector Labs [DI-1488]), CF488A-conjugated streptavidin (Biotium [29034]), CF640R-conjugated anti-rat IgG (Sigma [SAB4600156]), Rhodamine Red-X-conjugated donkey anti-sheep IgG (Jackson ImmunoResearch), Dylight649-conjugated streptavidin (Vector Labs [SA5649]), horseradish peroxidase (HRP)conjugated goat anti-mouse IgM (Thermo Scientific [31444]), HRP-conjugated goat anti-rabbit IgG (Invitrogen [65–6120]), HRP-conjugated goat anti-Armenian hamster IgG (Santa Cruz [sc-2443]), HRPconjugated horse anti-mouse IgG antibody (Cell signaling [7076S]), alkaline phosphatase (AP)-conjugated anti-FITC antibody (Roche [11-426-338-910]), FITC-conjugated IB4 (Vector Labs [FL-1201]), and Dylight594-conjugated IB4 (Vector Labs [DL-1178]).

Techniques: Immunostaining, Staining, Construct, Mutagenesis

Journal: Stem Cell Research & Therapy

Article Title: New advances of NG2-expressing cell subset in marrow mesenchymal stem cells as novel therapeutic tools for liver fibrosis/cirrhosis

doi: 10.1186/s13287-024-03817-x

Figure Lengend Snippet: Antibodies (abs), reagents, and kits

Article Snippet: Antibodies for FCM , Alexa Fluor 488-Mouse-NG2 , 562,413 , , , , , Flow Cytometry (FCM) , BD Biosciences , California , USA.

Techniques: Immunofluorescence, Western Blot, Flow Cytometry, Modification, Cell Culture, Cell Tracking Assay, TUNEL Assay, Apoptosis Assay, Fluorescence, Bicinchoninic Acid Protein Assay, Proliferation Assay, CCK-8 Assay, Staining

Journal: Stem Cell Research & Therapy

Article Title: New advances of NG2-expressing cell subset in marrow mesenchymal stem cells as novel therapeutic tools for liver fibrosis/cirrhosis

doi: 10.1186/s13287-024-03817-x

Figure Lengend Snippet: Comparison of the characteristics of ex vivo-expanded NG2/ BM MSCs and parental BM MSCs. (Aa-c) Comparison of the sizes of m-NG2/ BM MSCs (a) and parental m- BM MSCs (b) and quantification (c , n = 20). (B) FCM analyses and IF staining (red) of NG-2 + cells (arrow) in mouse marrow MSC (m- BM MSC) cultures; n = 6/type of experiment. (C) Measurement of the purity of the isolated and ex vivo-expanded NG2 + cells using FCM (left panel) and IF staining (green, arrow); n = 3/method. (D) NG2 + cells (boxes) within m- BM MSC cultures generated smaller spindle-shaped BM MSCs (thin arrows, S1 and S2 represent individual cultures. (Ea, b) IF staining for SSEA-3 in m-NG2/ BM MSCs (a , red) and m- BM MSCs (b , green) and quantification from a and b. (F , boxes), n = 3. The means ± SDs of duplicate preparations from three independent experiments are shown. Scale bars = 200 μm for all the images except for those in A and D; scale bars = 100 μm. ** ## p < 0.001 compared with m- BM MSCs

Article Snippet: Antibodies for FCM , Alexa Fluor 488-Mouse-NG2 , 562,413 , , , , , Flow Cytometry (FCM) , BD Biosciences , California , USA.

Techniques: Comparison, Ex Vivo, Staining, Isolation, Generated

Journal: Stem Cell Research & Therapy

Article Title: New advances of NG2-expressing cell subset in marrow mesenchymal stem cells as novel therapeutic tools for liver fibrosis/cirrhosis

doi: 10.1186/s13287-024-03817-x

Figure Lengend Snippet: Proliferation potential of the ex vivo-expanded m-NG2/ BM MSCs in normal cultures. ( A, B ) A CCK-8 assay for comparison of the two types of cells in growth rates from normal cultures at 24, 48, and 72 h. (Ca, b) Comparison of growth rates at 5 and 6 days (a) and quantification (b , boxes); n = 3/time point. (Da, b) Double IF staining to identify m-NG2/ BM MSCs (a , NG2, red) costained with Ki-67 (green, a , arrows), and the same procedure was used for parental BM MSCs (b , CD9, red, arrows); n = 6. (E) Quantification of the percentage of Ki-67-positive cells in D (merged, boxes) after 72 h in cultures; n = 6. (F) Fold changes in the expression of Ki-67 in m-NG2/ BM MSCs over that in BM MSCs were analyzed as described in (E). The means ± SDs of duplicate preparations from three independent experiments are shown. Scale bars: Ca = 100 μm, others = 200 μm. * p < 0.05 and ** p < 0.001 compared with m- BM MSCs

Article Snippet: Antibodies for FCM , Alexa Fluor 488-Mouse-NG2 , 562,413 , , , , , Flow Cytometry (FCM) , BD Biosciences , California , USA.

Techniques: Ex Vivo, CCK-8 Assay, Comparison, Staining, Expressing

Journal: Stem Cell Research & Therapy

Article Title: New advances of NG2-expressing cell subset in marrow mesenchymal stem cells as novel therapeutic tools for liver fibrosis/cirrhosis

doi: 10.1186/s13287-024-03817-x

Figure Lengend Snippet: Advantages of m-NG2/ BM MSCs over parental m- BM MSCs in promoting endogenous bile duct repair and improving functions in a DEN-induced mouse model four weeks after cell transplantation. ( Aa-e ) IF staining for CK7 (green) and CK19 (red) in naive (a) and DEN-treated (b) livers and quantification of CK7 (c) and CK19 (d) expression. RT‒qPCR analysis of the mRNA expression showed a similar trend (e) ; n = 6/group. (Ba-c) IF staining f or CK7 (green) and CK19 (red) in DEN-treated (+ PBS) and m- BM MSC-treated livers (a) and was quantified (b, c , boxes), n = 6/group. (Ca-c) The same staining procedure was used for comparisons of m- BM MSC- and m-NG2/ BM MSC-treated livers (a) , and the results were quantified (b, c , boxes); n = 6/group. (D) RT‒qPCR analysis of the expression of the ck7 and ck19 genes in the subgroups; n = 3. (Ea-f) IF staining for ɑ-SMA expression (red) in the liver according to subgroups (a-c) and analyses at both the protein (d, e) and mRNA (f) levels; n = 6/per group. (F) The serum levels of TBIL, ALP, ALT and AST in the subgroups were measured with a Beckman Coulter Chemistry Analyzer ( n = 3/set). The data are presented as the means ± SDs from several independent experiments. Scale bar = 200 μm. A, *# p < 0.05 compared with naive; B, # p < 0.05 compared with DEN (+ PBS); C, * p < 0.05 compared with m- BM MSCs; D, #* p < 0.05 compared with DEN or m- BM MSCs; E, #* p < 0.05 compared with DEN or m- BM MSCs; F, #* p < 0.05 compared with DEN or m- BM MSCs

Article Snippet: Antibodies for FCM , Alexa Fluor 488-Mouse-NG2 , 562,413 , , , , , Flow Cytometry (FCM) , BD Biosciences , California , USA.

Techniques: Transplantation Assay, Staining, Expressing

Journal: Stem Cell Research & Therapy

Article Title: New advances of NG2-expressing cell subset in marrow mesenchymal stem cells as novel therapeutic tools for liver fibrosis/cirrhosis

doi: 10.1186/s13287-024-03817-x

Figure Lengend Snippet: m-NG2/ BM MSCs homed to lesions, differentiated into BDCs and promoted host BDC-mediated hepatocyte regeneration as a core component of m- BM MSC functions. ( A ) CFSE-labeled m-NG2/ BM MSCs and m- BM MSCs migrated into injured areas of the liver 72 h after transplantation. (B) Quantitative analysis of the data in A (boxes, n = 6). (Ca-c) IF staining of CK19 + cells (red) differentiated from CFSE-labeled m-NG2/ BM MSCs (a) and m- BM MSCs (b) and quantification (c , merged, n = 6, the. The boxes represent CK19 + cells that formed vessel-like structures). (Da-c) Double IF staining of host CK19 + cells (green, arrows) after 72 h covered by ALB + cells (red) in mice (livers) treated with m-NG2/ BM MSCs (a) or m- BM MSCs (b) and quantification of the merged cells (arrows, c , n = 6). (Ea-c) A nalysis of CK7 by IF staining (red) (a, b) and mRNA levels using RT‒qPCR (c) ; n = 6. (Fa-c) Similar analysis to E for CK19, n = 6. The data are presented as the means ± SDs from several independent experiments. Scale bar = 200 μm. ** P < 0.001 compared with m- BM MSCs; ns represents no significant difference

Article Snippet: Antibodies for FCM , Alexa Fluor 488-Mouse-NG2 , 562,413 , , , , , Flow Cytometry (FCM) , BD Biosciences , California , USA.

Techniques: Labeling, Transplantation Assay, Staining

Journal: Stem Cell Research & Therapy

Article Title: New advances of NG2-expressing cell subset in marrow mesenchymal stem cells as novel therapeutic tools for liver fibrosis/cirrhosis

doi: 10.1186/s13287-024-03817-x

Figure Lengend Snippet: Direct differentiation of BDCs from m-NG2/ BM MSCs in response to DEN-induced liver injury cues. ( Aa-b ) The morphology of m-NG2/ BM MSCs in control medium (Ctrl-CM, a) and in the DEN CM (b) , bold arrows indicate assumed NG2 + cells, red arrows indicate as bile duct-like cells. Little change was observed in parental m- BM MSCs during this period (b , right panels) compared to that in Ctrl-CM (a , right panel): S1, S2 in b represent individuals, scale bar = 100 μm; remaining panels: scale bar = 200 μm. (Ba, b) Double IF staining for NG2 + cells (red) or CD9 + cells (green) covered by CK19 + cells (green/red) of m-NG2/ BM MSCs (a) and m- BM MSCs (b) cultured in DEN CM for 18–24 h. (C) Quantification (Ba, b) of the merged cells. (Da-d) IF staining (red) for host Lyve-1 + cell expression (boxes) in subgroups of liver sections. (Ea, b) Quantificative analysis of Lyve-1 expression in subgroups from boxes in Da-d (a) and the number of vessel-like structures formed from the groups in Dc, d (b , n = 6). (Fa-d) Direct differentiation of Lyve-1 + cells from donor m-NG2/ BM MSCs (a) and parental m- BM MSCs (b) in DEN CM and comparative quantification of total numbers per quarter area (boxes, c) and the number of vessel-like formations (red arrows, d , n = 6). At least three independent experiments were performed, and the data are presented as the means ± SDs. Scale bar = 200 μm. # p < 0.05 compared with naive; ** p < 0.001 compared with m- BM MSCs; ns: no significance compared with m- BM MSCs

Article Snippet: Antibodies for FCM , Alexa Fluor 488-Mouse-NG2 , 562,413 , , , , , Flow Cytometry (FCM) , BD Biosciences , California , USA.

Techniques: Control, Staining, Cell Culture, Expressing

Journal: Stem Cell Research & Therapy

Article Title: New advances of NG2-expressing cell subset in marrow mesenchymal stem cells as novel therapeutic tools for liver fibrosis/cirrhosis

doi: 10.1186/s13287-024-03817-x

Figure Lengend Snippet: Characterization of NG2 + cells isolated from human marrow MSCs (h- BM MSCs) via the PPWP. (Aa-c) h- BM MSC-sourced NG2 + cells (h-NG2/ BM MSCs) (a) isolated from h- BM MSCs (b) and comparative analysis in size (c) . The bold arrow indicates NG2 + cells, and the thin arrow indicates spindle-shaped BM MSCs. (Ba, b) IF staining for Ki-67 + cells (red, arrows) in the two types of cells when their in normal cultures (a) and quantification (b) ; n = 3. (Ca-c) In normal BM MSC cultures, larger flaky cells (a bold arrow, assumed to indicate NG2 + cells) appeared to produce smaller spindle-shaped cells (a thin arrow) (a , S1 and S2 represent individual cultures). IF staining for SSEA-3 (arrows, b) and comparative quantification of the two types of cells are shown (c) ; the box shows a cell clone. (Da-f) IF staining (red) of host CK19 + and Alb + cells in the liver subgroups four weeks after cell transplantation (a, d) , quantification ( b , arrows; e , boxes) n = 6, and x-fold changes in host CK19 + cells stimulated by h-NG2/ BM MSCs compared with those stimulated by parental h- BM MSCs (c, f) . (Ea-f) IF staining (red) of host ɑ-SMA + cells in DEN-induced mouse livers 4 weeks after transplantation of the two types of donor cells (b, c) compared to DEN (a) and quantification of protein ( d , boxes, n = 6) and mRNA levels using RT‒qPCR (e , n = 6); blood functional hepatic parameters were also compared in these subgroups (f , n = 6 ) . (Fa-f) IF staining (red) for host Lyve-1 + cells in subgroups of naive (a), DEN (b), two types of donor cells (c, d) four weeks after cell transplantation, and quantification of the number of Lyve-1 + cells per quarter area (boxes) of the staining (e , n = 6 ); mRNA levels in livers from the same subgroups were determined using RT‒qPCR (f , n = 6). Scale bar = 200 μm in all images; at least three independent experiments were performed, and the data are presented as the means ± SDs. *# p < 0.05 compared with naive or DEN or h- BM MSCs; ** p < 0.001 h- BM MSCs; ns: indicate no significance

Article Snippet: Antibodies for FCM , Alexa Fluor 488-Mouse-NG2 , 562,413 , , , , , Flow Cytometry (FCM) , BD Biosciences , California , USA.

Techniques: Isolation, Staining, Transplantation Assay, Functional Assay

Journal: Stem Cell Research & Therapy

Article Title: New advances of NG2-expressing cell subset in marrow mesenchymal stem cells as novel therapeutic tools for liver fibrosis/cirrhosis

doi: 10.1186/s13287-024-03817-x

Figure Lengend Snippet: Potential of h-NG2/ BM MSCs to develop BDCs and their unique ability to directly differentiate into sinusoidal cells and structures in response to DEN-induced liver injury cues. (Aa-c) During 1–4 h of culture in DEN CM, BDC-like morphological changes were observed in culturing h-NG2/ BM MSC cells that differentiated from h-NG2/ BM MSCs (a , red arrows, box), but few similar changes were observed in cells that differentiated from parental h- BM MSCs (b , box), and the number of changed cells per image field was analyzed (c , n = 10). (Ba, b) Double IF staining of CK19 + cells (a , red, second panels) covered with h-NG2/ BM MSCs (NG2, green, top panel) or h- BM MSCs (CD90, green, top panel) and analysis of the percentage of developed CK19 + cells/image half-field from merged cells (b , brown, boxes) during 18–24 h of culture in DEN CM, n = 6. (Ca-c) Double IF staining revealed that during the 18–24 h period in DEN CM cultures, the CK19 + cells developmed from h-NG2/ BM MSCs formed obvious vessel-like structures ( a , merged, arrows) that were not observed in the parental h- BM MSCs (b) , and quantification was performed (c , n = 6). (Da-c) Double IF staining of h-NG2/ BM MSCs (NG2, green, a ) and h- BM MSCs (CD90, green, b ) covered with Lyve-1 + cells (red) after 4–6 h culture periods in DEN CM and quantification of the merged cells in Da, b (c , boxes), n = 6. (Ea, b) In the DEN CM cultures, Lyve-1 + cell (red)-generated from h-NG2/ BM MSC cells (green) formed vessel-like structures ( a , arrows) and this phenomena was not appeared in the h- BM MSCs (b) . (F) Quantification of the data in E ( n = 6). Scale bar = 200 μm for all images. At least three independent experiments were performed, and the data are presented as the means ± SDs; ** p < 0.001 compared with h- BM MSCs

Article Snippet: Antibodies for FCM , Alexa Fluor 488-Mouse-NG2 , 562,413 , , , , , Flow Cytometry (FCM) , BD Biosciences , California , USA.

Techniques: Staining, Generated