Journal: Bioengineering & Translational Medicine

Article Title: Mesenchymal stem cell extracellular vesicle vascularization bioactivity and production yield are responsive to cell culture substrate stiffness

doi: 10.1002/btm2.10743

Figure Lengend Snippet: Substrate stiffness influences BM‐MSC EV production and bioactivity. (a) EV production as quantified by EVs per cell from BM‐MSCs seeded on Sylgard 184 PDMS substrates with different base‐to‐crosslinker ratios. EVs used for this data were from 1 day of collection and isolated and counted separately from the conditioned media from the other 2 days. After media collection, cells were trypsinized and counted ( n = 3). (b) After a scratch was induced, HUVECs were treated with BM‐MSC EVs from the different substrates or growth or basal media, and percent gap closure after 20 h was evaluated via microscopy ( n = 3). (c) HUVECs were resuspended in EV treatments or growth or basal endothelial media, seeded in Matrigel‐coated wells, and tube formation after 3–6 h was quantified by the number of loops that had formed ( n = 3). All values expressed as mean ± SD. All data are representative of at least three independent experiments ( n = 3). Statistical significance was determined by ANOVA; *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Article Snippet: To measure in vitro angiogenesis, 48‐well plates were coated with 60 μL of growth factor reduced Matrigel (Corning; 356230) and incubated at 37°C for 30 min. P4 HUVECs were then seeded at 35,000 cells/well with either endothelial growth media (PromoCell; C‐22121) with 1% penicillin–streptomycin (positive control), endothelial basal media (negative control), or endothelial basal media (PromoCell; C‐22221) with 0.1% FBS and 1% penicillin–streptomycin with 5E9 EVs/mL.

Techniques: Isolation, Microscopy

Journal: Bioengineering & Translational Medicine

Article Title: Mesenchymal stem cell extracellular vesicle vascularization bioactivity and production yield are responsive to cell culture substrate stiffness

doi: 10.1002/btm2.10743

Figure Lengend Snippet: Softer 184:527 PDMS substrates improve the angiogenic bioactivity of BM‐MSC EVs. (a) EV production quantified as EV per cell from BM‐MSCs seeded on each substrate made with different ratios of Sylgard 184 and Sylgard 527 ( n = 2). EVs used for this data were from 1 day of collection and isolated and counted separately from the conditioned media from the other 2 days. After media collection, cells were trypsinized and counted. (b) After a scratch was induced, HUVECs were treated with BM‐MSC EVs from the different substrates or growth or basal media, and percent gap closure after 20 h was evaluated via microscopy ( n = 3). (c) HUVECs were resuspended in the different EV treatments or growth or basal endothelial basal media, and tube formation after 3–6 h was quantified by the number of loops that had formed ( n = 3). All values expressed as mean ± SD. Statistical significance was determined by ANOVA; * p < 0.05, ** p < 0.01, and **** p < 0.0001.

Article Snippet: To measure in vitro angiogenesis, 48‐well plates were coated with 60 μL of growth factor reduced Matrigel (Corning; 356230) and incubated at 37°C for 30 min. P4 HUVECs were then seeded at 35,000 cells/well with either endothelial growth media (PromoCell; C‐22121) with 1% penicillin–streptomycin (positive control), endothelial basal media (negative control), or endothelial basal media (PromoCell; C‐22221) with 0.1% FBS and 1% penicillin–streptomycin with 5E9 EVs/mL.

Techniques: Isolation, Microscopy

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