Journal: Frontiers in Physiology

Article Title: Velvet Antler Mobilizes Endothelial Progenitor Cells to Promote Angiogenesis and Repair Vascular Endothelial Injury in Rats Following Myocardial Infarction

doi: 10.3389/fphys.2018.01940

Figure Lengend Snippet: CD133 and MVD in the border zone of MI. (A) Immunofluorescence on days 7 after MI. (B) Expression of CD133 stained by immunofluorescence (per/field). (C) MVD stained by CD31 (per/field). CD133 present star like and distribute on vessels marked by CD31, suggesting that angiogenesis might be promoted by EPCs marked by CD133. ∗∗ P < 0.01 compared with the sham group; # P < 0.05 and ## P < 0.01 compared with the model group; Δ P < 0.05 compared with the VA group. N = 8.

Article Snippet: PE-conjugated anti-CD34 (Bioss, China), PE-CY7 conjugated anti-CD133 (Bioss), and FITC conjugated anti- VEGFR2 antibody (Bioss) were used.

Techniques: Immunofluorescence, Expressing, Staining

Journal: Aging (Albany NY)

Article Title: Spindle pole body component 25 regulates stemness of prostate cancer cells

doi: 10.18632/aging.101631

Figure Lengend Snippet: Differentiation of SPC25+ from SPC25- PrC cells with genetic manipulation. ( A ) The DU145 and LNCap cell lines were transduced with 2 AAVs. The first AAV carries a luciferase and a mCherry fluorescent reporter under the control of a cytomegalovirus (CMV) promotor. The luciferase and mCherry reporters are connected by a p2A sequence to allow co-expression of 2 genes by one promoter with similar efficiency. Transduction of the cells with this AAV makes the cells red fluorescent to be sortable by flow cytometry and traceable in vivo by bioluminescence assay. The second AAV carries a nuclear green fluorescent protein (nGFP) reporter under the control of a SPC25 promoter. Transduction of the cells with this AAV makes the SPC25+ cells green fluorescent in the nuclei to be sortable by flow cytometry. Co-transduction of the cells with these 2 AAVs resulted in two populations of interest. Population 1, red fluorescent (expressing mCherry but not nGFP) cells represent SPC25- cells. Population 2, yellow fluorescent (expressing both mCherry and nGFP) cells represent SPC25+ cells. ( B ) The flow cytometry analysis on infected DU145 and LNCap cells. ( C ) The purified transduced cells were examined for fluorescence in culture. ( D ) RT-qPCR for SPC25 in different cell fractions. ( E ) Flow cytometry for CD133 in SPC25+ and SPC25- fractions. *p<0.05. N=5. Scale bars were 20 µm.

Article Snippet: For CD133, a PE-cy7 anti-human CD133 antibody (Becton-Dickinson Biosciences, San Jose, CA, USA) was used.

Techniques: Transduction, Luciferase, Control, Sequencing, Expressing, Flow Cytometry, In Vivo, ATP Bioluminescent Assay, Infection, Purification, Fluorescence, Quantitative RT-PCR

Journal: Molecular Therapy

Article Title: Adaptive Immune Response Impairs the Efficacy of Autologous Transplantation of Engineered Stem Cells in Dystrophic Dogs

doi: 10.1038/mt.2016.163

Figure Lengend Snippet: Characterization of 133+musSCs. (a) In vivo characterization of 133+musSCs (green) around untreated Golden Retriever muscular dystrophy (GRMD) muscle fibers (laminin in violet). (b) Fluorescence-activated cell sorting (FACS) analysis of muscle derived cells from homogenized tibialis cranialis showed CD133 and CD34 coexpression (c,d) FACS characterization of freshly isolated CD133+musSCs: median purity value (95%); CD133 and CD34 coexpression (>50%), and CXCR4 antigen expression (2.3%). No expression of CD45 antigen. (e) 133+musSCs 24 hours after cell sorting, and (f) in proliferation medium for 7 days. (g) Vascular structures of dystrophic 133+musSCs in endothelial differentiation medium for 21 days. (h) Pax7 (green) and desmin (red) coexpression of CD133+musSCs in muscle differentiation medium. (i,j) Desmin positive (green) fused myoblasts, desmin (green), and Myosin Heavy Chain (red) positive completely differentiated myotubes derived from 133+musSCs. (k) 133+muscSC proliferation rate during 4 weeks of culture. (l) Viability and 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) (MTT) assay showed cell viability as a percentage of viable cells on total cells (n = 3 replicates). (m) FACS immunophenotyping of LVex6-8133+musSCs and (n-) in vitro culture optical images. (o) Optical images of LVex6-8133+musSCs in colture in differentiation medium. (p) LVex6-8133+musSCs myotube desmin expression (green). Nuclei were costained with 4′,6-diamidino-2-phenylindole (DAPI) (blue) for all the confocal images. All images were captured with a confocal microscope Leica TCS SP2 (Leica, Germany): 20× magnification. (q,r) Proliferation rate during 4 weeks of culture and (r) viability assessed by MTT assay of LVex6-8133+musSCs demonstrating no change of cell behaviour after lentiviral transduction. (s) Fluorescent in situ hybridization (FISH) analysis of LVex6-8133+musSCs for the expression of U7 probe (red), showing the presence of vector in nuclei (DAPI) of transduced cells (t) and no signal could be observed in not-infected cells.

Article Snippet: 1.5 × 10 5 CD133+musSCs were incubated with FACS antibodies for five colors-flow cytometry characterizations: anti-CD133-APC, anti-CD34-PE, anti-CD45-FITC (BD Biosciences, Pharmingen), CXCR4-PE-Cy7 (CXC chemokine) (BD Biosciences, Pharmingen).

Techniques: In Vivo, Fluorescence, FACS, Derivative Assay, Isolation, Expressing, MTT Assay, In Vitro, Microscopy, Transduction, In Situ Hybridization, Plasmid Preparation, Infection

Journal: Molecular Therapy

Article Title: Adaptive Immune Response Impairs the Efficacy of Autologous Transplantation of Engineered Stem Cells in Dystrophic Dogs

doi: 10.1038/mt.2016.163

Figure Lengend Snippet: Characterization of 133+musSCs. (a) In vivo characterization of 133+musSCs (green) around untreated Golden Retriever muscular dystrophy (GRMD) muscle fibers (laminin in violet). (b) Fluorescence-activated cell sorting (FACS) analysis of muscle derived cells from homogenized tibialis cranialis showed CD133 and CD34 coexpression (c,d) FACS characterization of freshly isolated CD133+musSCs: median purity value (95%); CD133 and CD34 coexpression (>50%), and CXCR4 antigen expression (2.3%). No expression of CD45 antigen. (e) 133+musSCs 24 hours after cell sorting, and (f) in proliferation medium for 7 days. (g) Vascular structures of dystrophic 133+musSCs in endothelial differentiation medium for 21 days. (h) Pax7 (green) and desmin (red) coexpression of CD133+musSCs in muscle differentiation medium. (i,j) Desmin positive (green) fused myoblasts, desmin (green), and Myosin Heavy Chain (red) positive completely differentiated myotubes derived from 133+musSCs. (k) 133+muscSC proliferation rate during 4 weeks of culture. (l) Viability and 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) (MTT) assay showed cell viability as a percentage of viable cells on total cells (n = 3 replicates). (m) FACS immunophenotyping of LVex6-8133+musSCs and (n-) in vitro culture optical images. (o) Optical images of LVex6-8133+musSCs in colture in differentiation medium. (p) LVex6-8133+musSCs myotube desmin expression (green). Nuclei were costained with 4′,6-diamidino-2-phenylindole (DAPI) (blue) for all the confocal images. All images were captured with a confocal microscope Leica TCS SP2 (Leica, Germany): 20× magnification. (q,r) Proliferation rate during 4 weeks of culture and (r) viability assessed by MTT assay of LVex6-8133+musSCs demonstrating no change of cell behaviour after lentiviral transduction. (s) Fluorescent in situ hybridization (FISH) analysis of LVex6-8133+musSCs for the expression of U7 probe (red), showing the presence of vector in nuclei (DAPI) of transduced cells (t) and no signal could be observed in not-infected cells.

Article Snippet: 1.5 × 10 5 CD133+musSCs were incubated with FACS antibodies for five colors-flow cytometry characterizations: anti-CD133-APC, anti-CD34-PE, anti-CD45-FITC (BD Biosciences, Pharmingen), CXCR4-PE-Cy7 (CXC chemokine) (BD Biosciences, Pharmingen).

Techniques: In Vivo, Fluorescence, FACS, Derivative Assay, Isolation, Expressing, MTT Assay, In Vitro, Microscopy, Transduction, In Situ Hybridization, Plasmid Preparation, Infection

Journal: Molecular Therapy

Article Title: Adaptive Immune Response Impairs the Efficacy of Autologous Transplantation of Engineered Stem Cells in Dystrophic Dogs

doi: 10.1038/mt.2016.163

Figure Lengend Snippet: Efficacy characterization of 133+musSCs. (a) Fluorescent in situ hybridization (FISH) analysis of 5′TEX615 probe (red) demonstrated the presence of muscle fibers with peripheral U7 positive nuclei (arrows) on muscles isolated from LVex6-8 cell treated injected dogs. (b,c) Serial sections of dystrophin (green) positive muscle fibers expressing positive U7 nuclei (arrows; red) in LVex6-8 cell treated GRMD dogs muscles. Magnification 40X for all the images. (d) WB analysis of cell-treated GRMD dogs muscle biopsies showed no detectable dystrophin expression through the time points. (e) WB evaluation of skipped dystrophin expression on muscular biopsies from LVex6-8cell-treated GRMD mild and severe phenotype dogs: no dystrophin expression at T0; detection of skipped dystrophin in all dogs at T12; mild phenotype dog dystrophin expression at TS. Each muscular biopsy was loaded with 70 µg of proteins and a muscle from a control dog was used as positive control (each gel was loaded with serial dilution of control dog; Lane 1:70 µg, Lane 2:35 μg, Lane 3:17.5 μg, and Lane 4:8.75 μg to allow dystrophin quantification). (f) RT-PCR analysis and sequencing of exon skipping product in dystrophin-positive fibers (laser-dissected) from muscular biopsies of LVex6-8cell-treated dogs at T12 and (g) at TS. M: molecular weight marker; T01 and T02: mild phenotype dogs; NT: not treated GRMD dog; T03, T04, and T05: severe clinical phenotype dogs. PCR product sequencing confirmed ligation between the exons after the skipping: (i) no skipping (*), (ii) exon 5 and 9 in frame skipping (**), and (iii) exon 5 and 10 in frame skipping (***). GRMD, Golden Retriever muscular dystrophy; RT-PCR, reverse transcription-polymerase chain reaction; SCs, stem cells.

Article Snippet: 1.5 × 10 5 CD133+musSCs were incubated with FACS antibodies for five colors-flow cytometry characterizations: anti-CD133-APC, anti-CD34-PE, anti-CD45-FITC (BD Biosciences, Pharmingen), CXCR4-PE-Cy7 (CXC chemokine) (BD Biosciences, Pharmingen).

Techniques: In Situ Hybridization, Isolation, Injection, Expressing, Positive Control, Serial Dilution, Reverse Transcription Polymerase Chain Reaction, Sequencing, Molecular Weight, Marker, Ligation