mscs conditioned media  (Dawley Inc)

Journal: Stem Cells Translational Medicine

Article Title: Spontaneous In Vivo Chondrogenesis of Bone Marrow-Derived Mesenchymal Progenitor Cells by Blocking Vascular Endothelial Growth Factor Signaling

doi: 10.5966/sctm.2015-0321

Figure Lengend Snippet: Inhibitory effect of sFlk-1 expressed by bone marrow-derived mesenchymal stromal/stem cells (MSCs) on in vitro endothelial organization and proliferation. Human umbilical vein endothelial cell proliferation assay performed by using supernatants collected by naïve (control) or sFlk-1-expressing MSCs. (A): Cell metabolic activity was measured on the basis of the colorimetric MTS assay and represented by using optical density units. (B): Immunofluorescence staining for CD31 in green showing the capillary-like structure formation in the direct coculture of either control or sFlk-1-expressing MSCs with human dermal microvascular endothelial cells. (C): Representative images of tubular structure formation of endothelial cells (stained with calcein assay medium in green after 48 hours) cultured in medium conditioned by control or sFlk-1 expressing MSCs supplemented with 0, 10, or 50 ng/ml of VEGF. Scale bars = 300 µm. (D): Total tube number, length, branching points, and loops were quantified by image analysis. Ten images per sample were analyzed. Data are presented as mean ± SD (n = 4 samples/group from 2 independent experiments). ∗, p < .05; ∗∗, p < .01. Abbreviations: OD, optical density; VEGF, vascular endothelial growth factor.

Article Snippet: In vitro tube formation assay was performed with human dermal microvascular endothelial cells (HDMECs) as previously described [ 29 ], either by using control or sFlk-1 MSC conditioned media (Endothelial Cell Basal Medium MV [PromoCell, Heidelberg, Germany, http://www.promocell.com/ ] with 5% FBS and 1% penicillin-streptomycin solution) extracted after 48 hours or by direct coculture of control or sFlk-1 MSC.

Techniques: Derivative Assay, In Vitro, Proliferation Assay, Expressing, Activity Assay, MTS Assay, Immunofluorescence, Staining, Cell Culture

Journal: Stem Cells Translational Medicine

Article Title: Spontaneous In Vivo Chondrogenesis of Bone Marrow-Derived Mesenchymal Progenitor Cells by Blocking Vascular Endothelial Growth Factor Signaling

doi: 10.5966/sctm.2015-0321

Figure Lengend Snippet: In vivo blocking of angiogenesis. (A): Representative macroscopic picture of the explants of engineered tissue generated by naïve (left) or sFlk-1-expressing (right) MSCs after 12 weeks in vivo. (B): Vessel density quantified within the total area of the implant generated by control (naïve) or sFlk-1 MSCs at different time points by histomorphometric analysis (n = 3–4). ∗, p < .05. (C): Immunohistochemistry for mouse CD31. Representative images at ×20 magnification include the central implant area generated by naive (top row) or sFlk-1 (bottom row) MSCs at 1, 4, 8, or 12 weeks. Black arrowheads indicate the blood vessels. Scale bar = 100 µm. Abbreviation: MSC, bone marrow-derived mesenchymal stromal/stem cell.

Article Snippet: In vitro tube formation assay was performed with human dermal microvascular endothelial cells (HDMECs) as previously described [ 29 ], either by using control or sFlk-1 MSC conditioned media (Endothelial Cell Basal Medium MV [PromoCell, Heidelberg, Germany, http://www.promocell.com/ ] with 5% FBS and 1% penicillin-streptomycin solution) extracted after 48 hours or by direct coculture of control or sFlk-1 MSC.

Techniques: In Vivo, Blocking Assay, Generated, Expressing, Immunohistochemistry, Derivative Assay

Journal: Stem Cells Translational Medicine

Article Title: Spontaneous In Vivo Chondrogenesis of Bone Marrow-Derived Mesenchymal Progenitor Cells by Blocking Vascular Endothelial Growth Factor Signaling

doi: 10.5966/sctm.2015-0321

Figure Lengend Snippet: In vivo chondrogenesis. Histological staining with Safranin-O for glycosaminoglycans and immunohistochemistry for type II collagen of engineered tissue generated by naïve (control) or sFlk-1 MSCs after 4 (A) or 12 (B) weeks in vivo. Fluorescence staining with DAPI (in blue) and a specific anti-human nuclei antibody (in red) of constructs generated by control or sFlk-1 MSCs after 4 (A) or 12 (B) weeks in vivo. Scale bar = 100 µm. Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; MSC, bone marrow-derived mesenchymal stromal/stem cell.

Article Snippet: In vitro tube formation assay was performed with human dermal microvascular endothelial cells (HDMECs) as previously described [ 29 ], either by using control or sFlk-1 MSC conditioned media (Endothelial Cell Basal Medium MV [PromoCell, Heidelberg, Germany, http://www.promocell.com/ ] with 5% FBS and 1% penicillin-streptomycin solution) extracted after 48 hours or by direct coculture of control or sFlk-1 MSC.

Techniques: In Vivo, Staining, Immunohistochemistry, Generated, Fluorescence, Construct, Derivative Assay

Journal: Toxicology and applied pharmacology

Article Title: Protective role of mesenchymal stem cells and mesenchymal stem cell-derived exosomes in cigarette smoke-induced mitochondrial dysfunction in mice

doi: 10.1016/j.taap.2019.114788

Figure Lengend Snippet: Characterization of mMSC-derived exosomes and the effect of their combination MSC+EXO on various pro-infammatory mediators in BAL fluid of acute 10days CS exposed mice. A) Isolation and characterization of mMSC-derived EXO using TEM, qNano and Western blotting. B) Intraperitoneal injection of MSC and EXO combination decreased lung cell infiltrations in mice exposed to acute CS estimated in the BAL fluid. C) Effect of MSC and EXO on different pro-inflammatory mediators in response to acute CS. Data were shown as mean ± SEM (n=6/group). ***P < 0.001 vs. air; # P < 0.05, ### P < 0.001 vs. CS.

Article Snippet: Exosome isolation and characterization Exosomes from mouse MSC conditioned media was isolated as per the manufacturer’s instructions (Norgen Biotek kit for cell culture media-exosomes-midi preparation # 60500).

Techniques: Derivative Assay, Isolation, Western Blot, Injection

Journal: Stem Cells Translational Medicine

Article Title: Mesenchymal Stem Cells Modulate the Functional Properties of Microglia via TGF-β Secretion

doi: 10.5966/sctm.2015-0217

Figure Lengend Snippet: MSC-CM suppressed the inflammatory reaction and enhanced phagocytosis in LPS-stimulated microglia. (A): To determine the effect of MSC-CM on the level of cytokines in LPS-stimulated microglia, we applied MSC-CM for 24, 48, and 72 hours to LPS-stimulated microglia. LPS increased mRNA expression of TNF-α, IL-1β, IL-6, and iNOS, and nitrate secretion in LPS-stimulated microglia, whereas MSC-CM inhibited the inflammatory reaction. (B): Immunostaining shows that MSC-CM significantly suppressed iNOS expression in LPS-stimulated microglia. The microglia and nuclei were stained with Iba-1 and DAPI, respectively. (C): LPS-stimulated microglia ingested more latex beads than did untreated controls, and the phagocytic activity of microglia was significantly enhanced when the MSC-CM was applied to LPS-stimulated microglia following LPS treatment. The data are means ± SEM of three independent experiments. ∗, p < .05; ∗∗, p < .01; ∗∗∗, p < .001; in comparison with control. §, p < .05; §§, p < .05; §§§, p < .001; in comparison with LPS. Abbreviations: CON, control; DAPI, 4′,6-diamidino-2-phenylindole; hr, hour; iNOS, inducible nitric oxide; IL, interleukin; LPS, lipopolysaccharide; MSC-CM, mesenchymal stromal cell-conditioned media; RQ, relative quantity; TNF, tumor necrosis factor.

Article Snippet: Rat MSC Conditioned Media and Drug Treatment in Primary Cultured Microglia Rat (Sprague Dawley) MSCs were purchased from Gibco (#S1601-100; Thermo Fisher Scientific).

Techniques: Expressing, Immunostaining, Staining, Activity Assay, Comparison, Control

Journal: Stem Cells Translational Medicine

Article Title: Mesenchymal Stem Cells Modulate the Functional Properties of Microglia via TGF-β Secretion

doi: 10.5966/sctm.2015-0217

Figure Lengend Snippet: MSC-CM restored CX3CR1, CD206, and CD200R expression in LPS-stimulated microglia. To investigate the effect of MSC-CM on the expression of CD86, CX3CR1, CD206, and CD200R in LPS-stimulated microglia, we performed flow cytometry. (A): Microglia were treated with LPS for 24 hours and MSC-CM was incubated for 72 hours. The isotype control was indicated as gray color. Control group (red), LPS group (green), and LPS + MSC-CM (blue) were indicated, respectively. The table box beside the diagram indicates the percentage of positive cells in each group. (B): Rather than divide this microglia population further by imposing strict boundaries on a continuous expression pattern, we then determined MFI of the entire remaining population for each marker, and we calculated the M2:M1 MFI ratio (CX3CR1, CD206, or CD200R/CD86). MSC-CM restored CX3CR1, CD206, and CD200R MFI, whereas it inhibited the CD86 in LPS-stimulated microglia, showing the normalization of M2:M1 MFI ratio. (C): Immunofluorescence results showed a similar pattern with flow cytometry analysis. Microglia was stained with Iba-1 and DAPI, respectively. (D): MSC-CM restored mRNA expression of CX3CR1 and CD206 in LPS-stimulated microglia following to incubation time. The data are means ± SEM of three independent experiments. §, p < .001 in comparison with control (CON); §§, p < .01; §§§, p < .001 in comparison with LPS. Abbreviations: CON, control; DAPI, 4′,6-diamidino-2-phenylindole; hr, hour; LPS, lipopolysaccharide; MFI, mean fluorescence intensity; MSC-CM, mesenchymal stromal cell-conditioned media; RQ, relative quantity.

Article Snippet: Rat MSC Conditioned Media and Drug Treatment in Primary Cultured Microglia Rat (Sprague Dawley) MSCs were purchased from Gibco (#S1601-100; Thermo Fisher Scientific).

Techniques: Expressing, Flow Cytometry, Incubation, Control, Marker, Immunofluorescence, Staining, Comparison, Fluorescence

Journal: Stem Cells Translational Medicine

Article Title: Mesenchymal Stem Cells Modulate the Functional Properties of Microglia via TGF-β Secretion

doi: 10.5966/sctm.2015-0217

Figure Lengend Snippet: TGF-β mediates the effect of MSC-CM on LPS-stimulated microglia. To determine which soluble factor was involved in the effect of MSC-CM on LPS-stimulated microglia, we treated LPS-stimulated microglia with MSC-CM + a TGF-βR inhibitor (A), a CX3CL1 antibody (B), or siMSC-CM (siMSC-CM indicates conditioned medium that was obtained from TGF-β siRNA-transfected MSCs) (C). LPS increased TNF-α, IL-1β, IL-6, and iNOS mRNA levels, and MSC-CM inhibited the increased cytokines and led to CX3CR1 restoration. TGF-β inhibition abolished the effect of MSC-CM on LPS-stimulated microglia, whereas CX3CL1 antibody did not. siMSC-CM did not affect LPS-stimulated microglia. The data are means ± SEM of three independent experiments. ∗, p < .01; ∗∗, p < .001; in comparison with control (CON). §, p < .05; §§, p < .01; §§§, p < .001; in comparison with LPS. †, p < .05; ††, p < .01; †††, p < .001; in comparison with two groups (LPS + MSC-CM vs. LPS + MSC-CM + antibody/inhibitor or siMSC-CM). Abbreviations: CON, control; iNOS, inducible nitric oxide; IL, interleukin; LPS, lipopolysaccharide; MSC-CM, mesenchymal stromal cell-conditioned media; n.s., not significant; RQ, relative quantity; TGF, transforming growth factor; TNF, tumor necrosis factor.

Article Snippet: Rat MSC Conditioned Media and Drug Treatment in Primary Cultured Microglia Rat (Sprague Dawley) MSCs were purchased from Gibco (#S1601-100; Thermo Fisher Scientific).

Techniques: Transfection, Inhibition, Comparison, Control

Journal: Stem Cells Translational Medicine

Article Title: Mesenchymal Stem Cells Modulate the Functional Properties of Microglia via TGF-β Secretion

doi: 10.5966/sctm.2015-0217

Figure Lengend Snippet: TGF-βR inhibition alone can abolish the effect of MSC-CM in LPS-stimulated microglia. To investigate whether CX3CL1 plays a role in the anti-inflammatory effect of TGF-β in MSC-CM, we added a CX3CL1 antibody, a TGF-βR inhibitor, and both together to MSC-CM. LPS reduced CX3CR1 (A) and increased TNF-α mRNA expression (B), IL-1β mRNA expression (C), IL-6 mRNA expression (D), and iNOS mRNA expression (E), and. MSC-CM rescued these changes. The TGF-βR inhibitor alone showed the same effect on LPS-stimulated microglia, regardless of the presence of the CX3CL1 antibody. When the TGF-βR inhibitor was added to the MSC-CM, the effect of the MSC-CM on TNF-α, IL-1β, IL-6, iNOS, and CX3CR1 was abolished. The data are means ± SEM of three independent experiments. ∗, p < .05; ∗∗, p < .01; ∗∗∗, p < .001; in comparison with control. §, p < .05; §§, p < .01; §§§, p < .001; in comparison with LPS. †, p < .05; †††, p < .001; compared between two groups. Abbreviations: iNOS, inducible nitric oxide; IL, interleukin; LPS, lipopolysaccharide; MSC-CM, mesenchymal stromal cell-conditioned media; RQ, relative quantity; TGF, transforming growth factor; TNF, tumor necrosis factor.

Article Snippet: Rat MSC Conditioned Media and Drug Treatment in Primary Cultured Microglia Rat (Sprague Dawley) MSCs were purchased from Gibco (#S1601-100; Thermo Fisher Scientific).

Techniques: Inhibition, Expressing, Comparison, Control

Journal: Stem Cells Translational Medicine

Article Title: Mesenchymal Stem Cells Modulate the Functional Properties of Microglia via TGF-β Secretion

doi: 10.5966/sctm.2015-0217

Figure Lengend Snippet: Recombinant TGF-β showed an effect similar to that of MSC-CM on LPS-stimulated microglia. Recombinant TGF-β (rTGF-β, 10 ng/ml) induced similar effects as did MSC-CM. On the basis of a previous study, recombinant CX3CL1 (rCX3CL1, 10 ng/ml) was also tested. The rTGF-β and rCX3CL1 were applied for 24 hours. rTGF-β rescued the reduced CX3CR1 expression (A) and inhibited the increased gene expression of TNF-α (B), IL-1β (C), IL-6 (D), and iNOS (E), similar to MSC-CM in LPS-stimulated microglia. rCX3CL1 (10 ng/ml) also inhibited IL-1β and iNOS expression and restored CX3CR1 expression but did not affect TNF-α and IL-6 expression. The data are means ± SEM of three independent experiments. ∗, p < .05; ∗∗∗, p < .001; in comparison with control. §, p < .05; §§§, p < .001; in comparison with LPS. ††, p < .05; †††, p < .001; compared between two groups. Abbreviations: iNOS, inducible nitric oxide; IL, interleukin; LPS, lipopolysaccharide; MSC-CM, mesenchymal stromal cell-conditioned media; RQ, relative quantity; TGF, transforming growth factor; TNF, tumor necrosis factor.

Article Snippet: Rat MSC Conditioned Media and Drug Treatment in Primary Cultured Microglia Rat (Sprague Dawley) MSCs were purchased from Gibco (#S1601-100; Thermo Fisher Scientific).

Techniques: Recombinant, Expressing, Gene Expression, Comparison, Control

Journal: Stem Cells Translational Medicine

Article Title: Mesenchymal Stem Cells Modulate the Functional Properties of Microglia via TGF-β Secretion

doi: 10.5966/sctm.2015-0217

Figure Lengend Snippet: MSC-CM inhibits the NF-κB pathway and rescues CX3CR1 expression via the TGF-β signaling pathway in LPS-stimulated microglia. (A): LPS increased p-IκB expression and reduced CX3CR1 expression and Smad2/3 phosphorylation by Western blot. MSC-CM restored these changes in LPS-stimulated microglia. However, the effect of MSC-CM was abolished when TGF-βR inhibitor was applied with MSC-CM. TGF-βR inhibitor alone did not affect CX3CR1 expression, IκB, or Smad2/3 phosphorylation in primary cultured microglia. The p-IκB (B), p-smad2 (C), p-smad3 (D), and CX3CR1 (E) expression levels were measured using densitometry. The data are means ± SEM of three independent experiments. ∗, p < .05, compared with control. §, p < .05; §§, p < .01; §§§, p < .001; in comparison with LPS. †, p < .05; ††, p < .001; compared between two groups. Abbreviations: CON, control; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; LPS, lipopolysaccharide; MSC-CM, mesenchymal stromal cell-conditioned media; TGF, transforming growth factor.

Article Snippet: Rat MSC Conditioned Media and Drug Treatment in Primary Cultured Microglia Rat (Sprague Dawley) MSCs were purchased from Gibco (#S1601-100; Thermo Fisher Scientific).

Techniques: Expressing, Phospho-proteomics, Western Blot, Cell Culture, Control, Comparison