Journal: Frontiers in Physiology

Article Title: CD13 promotes mesenchymal stem cell-mediated regeneration of ischemic muscle

doi: 10.3389/fphys.2013.00402

Figure Lengend Snippet: Mesenchymal stem cell culture and characterization. (A) Phase contrast image of BM derived MSCs at Day 0 and Day 21(Bar = 100 μm). (B) CD13 expression in WT-MSCs by fluorescence immunostaining (Bar = 20 μm) and protein expression of CD13 in WT-MSC. (C) RT PCR analysis of stem cell expression profiles. (D) Flow cytometric analysis of MSCs. Cells were characteristically positive for CD29, CD49e and negative for CD19, CD31, and CD34. Unstained;WT-MSC; KO-MSC–. (E) Both WT-MSC and KO-MSC expressed transcription factor OCT3/4 (Bar = 20 μm). (F) Confluent MSCs were transferred to adipogenic and osteogenic medium for 3 weeks. Adipocytes were detected by oil red O staining and osteoblasts by alizarin red staining (Bar = 200 μm). (G) 1 × 10 5 cells were seeded on Matrigel coated 6-well plates and incubated for 12 h. cells isolated from CD13 KO mice are unable to form capillary networks and form fewer branches (Bar = 200 μm).

Article Snippet: Equal amount of protein from each group were separated by SDS-PAGE and transferred on to PVDF membrane and incubated with respective primary antibodies; CD13 monoclonal antibody for mouse CD13 (SL-13, custom made by ProMab Biotechnologies, Inc. Richmond, CA); 452 for human CD13 (Dr. Meenhard Herlyn, Philadelphia, PA); pFAK 397, pFAK925, and tFAK (cell signaling); β-Actin (Sigma); followed by incubation with horseradish peroxidase-conjugated secondary antibodies.

Techniques: Stem Cell Culture, Derivative Assay, Expressing, Fluorescence, Immunostaining, Reverse Transcription Polymerase Chain Reaction, Staining, Incubation, Isolation

Journal: Frontiers in Physiology

Article Title: CD13 promotes mesenchymal stem cell-mediated regeneration of ischemic muscle

doi: 10.3389/fphys.2013.00402

Figure Lengend Snippet: Lack of CD13 impairs MSC adhesion, proliferation, migration, and invasion. (A) Adhesion assay: Cells (1 × 10 4 ) were seeded in 96 well plates coated separately with fibronectin, Matrigel, or gelatin and allowed to adhere for 1 h at 37°C. After PBS wash, adherent cells were detected by MTT assay. n = 6, ** P < 0.01. (B,C) Proliferation assay: Cells (0.5 × 10 4 ) were seeded in 96 well plate and cell proliferation detected by MTT assay at the indicated time points. n = 6, * P < 0.05, ** P < 0.01. (D) Migration assay: 1 × 10 4 cells were seeded in FluoroBlok chambers. After 4 h. incubation the cells were stained with DAPI and counted. n = 4, ** P < 0.01. (E) Invasion assay: 1 × 10 5 cells were seeded on Matrigel (1:5 dilution) coated FluoroBlok chambers. After 6 h. incubation the cells were counted. n = 4, ** P < 0.01.

Article Snippet: Equal amount of protein from each group were separated by SDS-PAGE and transferred on to PVDF membrane and incubated with respective primary antibodies; CD13 monoclonal antibody for mouse CD13 (SL-13, custom made by ProMab Biotechnologies, Inc. Richmond, CA); 452 for human CD13 (Dr. Meenhard Herlyn, Philadelphia, PA); pFAK 397, pFAK925, and tFAK (cell signaling); β-Actin (Sigma); followed by incubation with horseradish peroxidase-conjugated secondary antibodies.

Techniques: Migration, Cell Adhesion Assay, MTT Assay, Proliferation Assay, Incubation, Staining, Invasion Assay

Journal: Frontiers in Physiology

Article Title: CD13 promotes mesenchymal stem cell-mediated regeneration of ischemic muscle

doi: 10.3389/fphys.2013.00402

Figure Lengend Snippet: Mesenchymal stem cell defects in CD13 KO mice are cell intrinsic. (A) phalloidin-stained MSC cells isolated from injured muscles of CD13 KO mice showed remarkable cytoskeletal disruption compared to cells from WT mice; Objective 40X (Bar = 100 μm). (B,C) Immunofluorescent detection of FAK phosphorylation at tyrosine residues 397 (B) and 925 (C) . Protein lysates of MSC were probed for phospho-FAK (Y397) and phospho-FAK (Y925) with β-actin as the loading control. CD13 KO MSCs expressed lower levels of phospho-FAK protein (Bar = 20 μm).

Article Snippet: Equal amount of protein from each group were separated by SDS-PAGE and transferred on to PVDF membrane and incubated with respective primary antibodies; CD13 monoclonal antibody for mouse CD13 (SL-13, custom made by ProMab Biotechnologies, Inc. Richmond, CA); 452 for human CD13 (Dr. Meenhard Herlyn, Philadelphia, PA); pFAK 397, pFAK925, and tFAK (cell signaling); β-Actin (Sigma); followed by incubation with horseradish peroxidase-conjugated secondary antibodies.

Techniques: Staining, Isolation, Muscles, Disruption, Phospho-proteomics, Control

Journal: Frontiers in Physiology

Article Title: CD13 promotes mesenchymal stem cell-mediated regeneration of ischemic muscle

doi: 10.3389/fphys.2013.00402

Figure Lengend Snippet: CD13 activation increases monolayer adhesion and FAK phosphorylation in human MSCs. (A) Human mesenchymal stem cells also express CD13 by immunofluorescence (green); Objective 63X (Bar = 20 μm) and immunoblot of human cell lysates. (B) Colorimetric quantification of adhesion of human MSCs treated with the CD13 activating mAb 452 to HUVEC monolayers. Data represents the mean ± s.e.m. n = 3 from two independent experiments ( ** P < 0.01). (C) CD13 crosslinking with activating mAb 452 temporally induces FAK tyrosine phosphorylation in human MSCs.

Article Snippet: Equal amount of protein from each group were separated by SDS-PAGE and transferred on to PVDF membrane and incubated with respective primary antibodies; CD13 monoclonal antibody for mouse CD13 (SL-13, custom made by ProMab Biotechnologies, Inc. Richmond, CA); 452 for human CD13 (Dr. Meenhard Herlyn, Philadelphia, PA); pFAK 397, pFAK925, and tFAK (cell signaling); β-Actin (Sigma); followed by incubation with horseradish peroxidase-conjugated secondary antibodies.

Techniques: Activation Assay, Phospho-proteomics, Immunofluorescence, Western Blot

Journal: Frontiers in Physiology

Article Title: CD13 promotes mesenchymal stem cell-mediated regeneration of ischemic muscle

doi: 10.3389/fphys.2013.00402

Figure Lengend Snippet: Lack of CD13 on exogenously administered MSCs impairs perfusion recovery in an in vivo hindlimb ischemia model. (A) Laser Doppler flow imaging of perfusion in mice at the indicated time points. Representative color-coded images of three groups (PBS, WT-MSC, and KO-MSC) of mice on day 0, 3, 7, 14, and 21 after surgery and cell transplantation assessed by laser Doppler imaging. Red indicates highest perfusion velocity, green intermediate, and blue, lowest velocity. (B) Cumulative results for PBS ( n = 7), WT-MSC ( n = 6), and KO-MSC ( n = 6) injected mice are shown graphically as ratios of blood flow in ischemic limb (I) to that in the non-ischemic limb (NI) at each time point. Functional assessment of ischemic muscles. Cumulative results are shown graphically as (C) the ambulatory impairment score; (D) ischemic tissue damage score as described in Methods. (E) Images of Non-ischemic and PBS, WT-MSC, and KO-MSC injected ischemic paw. Black nail indicates necrosis. # P < 0.05 compare to PBS and * P < 0.05 compare to KO-MSC; (score assessment criteria in Methods).

Article Snippet: Equal amount of protein from each group were separated by SDS-PAGE and transferred on to PVDF membrane and incubated with respective primary antibodies; CD13 monoclonal antibody for mouse CD13 (SL-13, custom made by ProMab Biotechnologies, Inc. Richmond, CA); 452 for human CD13 (Dr. Meenhard Herlyn, Philadelphia, PA); pFAK 397, pFAK925, and tFAK (cell signaling); β-Actin (Sigma); followed by incubation with horseradish peroxidase-conjugated secondary antibodies.

Techniques: In Vivo, Imaging, Transplantation Assay, Injection, Functional Assay, Muscles

Journal: Frontiers in Physiology

Article Title: CD13 promotes mesenchymal stem cell-mediated regeneration of ischemic muscle

doi: 10.3389/fphys.2013.00402

Figure Lengend Snippet: Muscle regeneration and capillary formation are impaired in mice injected with CD13 KO MSC following ischemic injury. (A) Hematoxylin and eosin (H&E) staining of gastrocnemius muscle regeneration was confirmed by the presence of multiple, centrally located myocyte nuclei, 20X objective; Bar = 100 μm. (B) Capillaries were visualized by immunofluorescent staining with CD31 (red) and nuclei with DAPI (blue); Objective, 40X objective; Bar = 50 μm. (C) The area of the injured tissue was compared among PBS, WT-MSC, and KO-MSC groups. A significant increase in muscle regeneration (average) was observed in WT-MSC group compared with PBS group at day 21. (D) The ratio of capillary density per fiber was measured in ischemic gastrocnemius muscles. Capillary density was significantly increased in WT-MSC compared with other groups, PBS and KO-MSC. All data were quantified by ImagePro Plus. Values are shown as mean ± s.e.m. ( * P < 0.05) (PBS n = 7; WT-MSC n = 6; KO-MSC n = 6).

Article Snippet: Equal amount of protein from each group were separated by SDS-PAGE and transferred on to PVDF membrane and incubated with respective primary antibodies; CD13 monoclonal antibody for mouse CD13 (SL-13, custom made by ProMab Biotechnologies, Inc. Richmond, CA); 452 for human CD13 (Dr. Meenhard Herlyn, Philadelphia, PA); pFAK 397, pFAK925, and tFAK (cell signaling); β-Actin (Sigma); followed by incubation with horseradish peroxidase-conjugated secondary antibodies.

Techniques: Injection, Staining, Muscles

Journal: Virulence

Article Title: Establishment of an oral infection model resembling the periodontal pocket in a perfusion bioreactor system

doi: 10.4161/21505594.2014.978721

Figure Lengend Snippet: Masson's trichrome staining used for histological analysis of the collagen sponges, in which the 3D gingival tissue had been reconstructed in vitro. The tissue was co-cultured for 24 h with hydroxyapatite discs without ( a ) or with the biofilm ( b ).

Article Snippet: Briefly, 200 μl of bacterial cell suspensions containing equal densities (OD 550 = 1.0) of each strain were mixed with 1.6 ml of growth medium consisting 60% saliva, 10% human serum, 30%mFUM, and 0.5% hemin to initiate biofilm formation on hydroxyapatite discs (diameter 13 mm) (Clarkson Chromatography Products, custom made), pre-coated for 4 h with saliva, which was diluted 1:2 in a mixture of 0.9% NaCl and distilled water.

Techniques: Staining, In Vitro, Cell Culture

Journal: Virulence

Article Title: Establishment of an oral infection model resembling the periodontal pocket in a perfusion bioreactor system

doi: 10.4161/21505594.2014.978721

Figure Lengend Snippet: Quantification of cytokine secretion. The 3D in vitro gingival tissue were cultured either in the presence of only pellicle coated hydroxyapatite discs (cell) or co-cultured with biofilm-grown hydroxyapatite discs (biofilm + cell) for 24 h. These values represent mean values of triplicate experiments ± standard deviation (SD). The results are presented on a logarithmic scale. Asterisk (*) represents the significance of differences ( P ≤ 0.05) between the 2 groups.

Article Snippet: Briefly, 200 μl of bacterial cell suspensions containing equal densities (OD 550 = 1.0) of each strain were mixed with 1.6 ml of growth medium consisting 60% saliva, 10% human serum, 30%mFUM, and 0.5% hemin to initiate biofilm formation on hydroxyapatite discs (diameter 13 mm) (Clarkson Chromatography Products, custom made), pre-coated for 4 h with saliva, which was diluted 1:2 in a mixture of 0.9% NaCl and distilled water.

Techniques: In Vitro, Cell Culture, Standard Deviation