Journal: Stem Cells International

Article Title: VEGF Contributes to Mesenchymal Stem Cell-Mediated Reversion of Nor1-Dependent Hypertrophy in iPS Cell-Derived Cardiomyocytes

doi: 10.1155/2021/8888575

Figure Lengend Snippet: MSC-derived soluble mediators induce hypertrophy regression in iPS-CM. Bone marrow-derived MSCs were stimulated with 30 ng/ml IFN- γ and 3 ng/ml IL-1 β for 24 h. Then, preconditioned MSCs (acMSCS) were indirectly cocultured with hypertrophied iPS-CM. In some experiments, hypertrophied iPS-CM were cultured in medium supplemented with 20% MSC-conditioned medium (CM). (a) Cell area quantification in iPS-CM was performed by immunofluorescent staining. Representative images are displayed. Bar: 100 μ m. n = 6. (b) Nor1 protein expression in iPS-CM. n = 5. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001.

Article Snippet: For experiments, MSCs were stimulated with 30 ng/ml recombinant murine IFN- γ (PeproTech) and 3 ng/ml recombinant murine IL-1 β (PeproTech) in MSC culture medium (Pan-Biotech) supplemented with 2.5 ng/ml human basic fibroblast growth factor FGF (FGF-b, PeproTech), 100 U/ml penicillin, and 10 μ g/ml streptomycin (Sigma-Aldrich) for 24 h. Preconditioned MSCs possess anti-inflammatory and immunomodulatory capacities [ ] and were used for coculture experiments.

Techniques: Derivative Assay, Cell Culture, Staining, Expressing

Journal: Stem Cells International

Article Title: VEGF Contributes to Mesenchymal Stem Cell-Mediated Reversion of Nor1-Dependent Hypertrophy in iPS Cell-Derived Cardiomyocytes

doi: 10.1155/2021/8888575

Figure Lengend Snippet: MSC-derived VEGF is essential for hypertrophy regression in iPS-CM. (a) VEGF was quantified in culture supernatants of MSCs stimulated with 30 ng/ml IFN- γ and 3 ng/ml IL-1 β for 24 h and those cultured for additional 24 h in serum-free medium (48 h). In addition, VEGF levels in fractionated MSC-conditioned medium (concentrates and filtrates) were determined by ELISA. n = 5. (b) PE-treated hypertrophied iPS-CM were cultured in the presence of 20% MSC-conditioned medium (CM) for 24 h. As a control, cells were cultured in medium supplemented with >30 kDa concentrates. For VEGF neutralization, 50 ng/ml or 150 ng/ml neutralizing anti-VEGF antibodies was added to the culture medium. The cell surface area was quantified by F-actin staining. Representative images are displayed. Bar: 100 μ m. n = 5. (c) Nor1 gene and protein expression was quantified in iPS-CM cultured in the presence of MSC-CM supplemented with VEGF-neutralizing antibodies. n = 4. (d) Intracellular VEGF levels were quantified in hypertrophied iPS-CM cultured in the presence of 20% CM for 24 h. MSCs with and without preconditioning served as a control. n = 4. (e) VEGF levels were quantified in culture supernatants of PE-treated iPS-CM cultured in medium supplemented with 20% CM for 24 h. n = 6. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001.

Article Snippet: For experiments, MSCs were stimulated with 30 ng/ml recombinant murine IFN- γ (PeproTech) and 3 ng/ml recombinant murine IL-1 β (PeproTech) in MSC culture medium (Pan-Biotech) supplemented with 2.5 ng/ml human basic fibroblast growth factor FGF (FGF-b, PeproTech), 100 U/ml penicillin, and 10 μ g/ml streptomycin (Sigma-Aldrich) for 24 h. Preconditioned MSCs possess anti-inflammatory and immunomodulatory capacities [ ] and were used for coculture experiments.

Techniques: Derivative Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Neutralization, Staining, Expressing

Journal: Basic Research in Cardiology

Article Title: Small extracellular vesicles obtained from hypoxic mesenchymal stromal cells have unique characteristics that promote cerebral angiogenesis, brain remodeling and neurological recovery after focal cerebral ischemia in mice

doi: 10.1007/s00395-021-00881-9

Figure Lengend Snippet: Small extracellular vesicles (sEVs) obtained from mesenchymal stromal cells (MSCs) cultured under hypoxic conditions increase cerebral microvascular endothelial cell proliferation. Relative number of human microvascular endothelial cells (hCMEC/D3) after exposure to different concentrations of A sEVs obtained from MSC culture media that contain platelet lysates (sEV platelet ), B sEVs obtained from MSCs ( source 41.5) cultured under regular ‘normoxic’ conditions (21% O 2 ; sEV normoxic ) or C sEVs obtained from MSCs (source 41.5) cultured under hypoxic conditions (1% O 2 ; sEV hypoxic ). In D , representative microphotographs for hCMEC/D3 cells exposed to control conditions or sEVs at a concentration of 50 µg/mL are shown. Data are mean ± SD values ( n = 9 independent experiments). * p < 0.05, ** p < 0.01, *** p < 0.001 compared with control. Scale bar: 400 µm in D

Article Snippet: Samples of 3 × 10 5 hCMEC/D3 cells were seeded in 6-well plates containing EBM-2 medium complemented with 1.25% FBS (other supplements as above). sEVs at various concentrations (0–250 μg/mL) obtained from (a) MSC culture medium (DMEM low, Lonza) supplemented with 10% platelet lysate (sEV platelet ), (b) supernatants of MSCs cultured under regular ‘normoxic’ conditions (21% O 2 ; sEV normoxic ), or (c) supernatants of MSCs cultured under hypoxic conditions (1% O 2 ; sEV hypoxic ) were added.

Techniques: Cell Culture, Control, Concentration Assay

Journal: Basic Research in Cardiology

Article Title: Small extracellular vesicles obtained from hypoxic mesenchymal stromal cells have unique characteristics that promote cerebral angiogenesis, brain remodeling and neurological recovery after focal cerebral ischemia in mice

doi: 10.1007/s00395-021-00881-9

Figure Lengend Snippet: sEVs from hypoxic MSCs increase cerebral microvascular endothelial cell migration. Relative number of migrating hCMEC/D3 cells, determined in a modified Boyden chamber transwell migration assay, after exposure to different concentrations of A sEVs obtained from MSC culture media that contain platelet lysates (sEV platelet ), B sEVs obtained from MSCs ( source 41.5) cultured under ‘normoxic’ conditions (21% O 2 ; sEV normoxic ) or C sEVs obtained from MSCs (source 41.5) cultured under hypoxic conditions (1% O 2 ; sEV hypoxic ). In D , representative microphotographs for hCMEC/D3 cells exposed to control conditions or sEVs at a 50 µg/mL concentration are shown. Data are mean ± SD values ( n = 9 independent experiments). * p < 0.05, ** p < 0.01, *** p < 0.001 compared with control. Scale bars: 125 µm in D

Article Snippet: Samples of 3 × 10 5 hCMEC/D3 cells were seeded in 6-well plates containing EBM-2 medium complemented with 1.25% FBS (other supplements as above). sEVs at various concentrations (0–250 μg/mL) obtained from (a) MSC culture medium (DMEM low, Lonza) supplemented with 10% platelet lysate (sEV platelet ), (b) supernatants of MSCs cultured under regular ‘normoxic’ conditions (21% O 2 ; sEV normoxic ), or (c) supernatants of MSCs cultured under hypoxic conditions (1% O 2 ; sEV hypoxic ) were added.

Techniques: Migration, Modification, Transwell Migration Assay, Cell Culture, Control, Concentration Assay

Journal: Basic Research in Cardiology

Article Title: Small extracellular vesicles obtained from hypoxic mesenchymal stromal cells have unique characteristics that promote cerebral angiogenesis, brain remodeling and neurological recovery after focal cerebral ischemia in mice

doi: 10.1007/s00395-021-00881-9

Figure Lengend Snippet: sEVs from hypoxic MSCs increase the tube formation of cerebral microvascular endothelial cells. Relative tube number, evaluated in a Matrigel-based tube formation assay, of hCMEC/D3 cells exposed to different concentrations of A sEVs obtained from MSC culture media that contain platelet lysates (sEV platelet ), B sEVs obtained from MSCs ( source 41.5) cultured under regular ‘normoxic’ conditions (21% O 2 ; sEV normoxic ) or C sEVs obtained from MSCs (source 41.5) cultured under hypoxic conditions (1% O 2 ; sEV hypoxic ). D Microvascular tube length density, E microvascular branching point density and F mean branch length between two branching points of hCMEC/D3 cells exposed to control conditions or sEV platelet , sEV normoxic (MSC source 41.5) or sEV hypoxic (MSC source 41.5) at a concentration of 50 µg/mL ( n = 3–9 independent experiments). In G , representative microphotographs for hCMEC/D3 cells exposed to control conditions or 50 µg/mL sEVs are shown. Data are mean ± SD values ( n = 9 independent experiments [in A – C ], 3–9 independent experiments [in D – F ]). * p < 0.05, *** p < 0.001 compared with control/ † p < 0.05, ††† p < 0.001 compared with sEV platelet / ‡ p < 0.05, ‡‡‡ p < 0.001 compared with sEV normoxic . Scale bars: 500 µm

Article Snippet: Samples of 3 × 10 5 hCMEC/D3 cells were seeded in 6-well plates containing EBM-2 medium complemented with 1.25% FBS (other supplements as above). sEVs at various concentrations (0–250 μg/mL) obtained from (a) MSC culture medium (DMEM low, Lonza) supplemented with 10% platelet lysate (sEV platelet ), (b) supernatants of MSCs cultured under regular ‘normoxic’ conditions (21% O 2 ; sEV normoxic ), or (c) supernatants of MSCs cultured under hypoxic conditions (1% O 2 ; sEV hypoxic ) were added.

Techniques: Tube Formation Assay, Cell Culture, Control, Concentration Assay

Journal: Basic Research in Cardiology

Article Title: Small extracellular vesicles obtained from hypoxic mesenchymal stromal cells have unique characteristics that promote cerebral angiogenesis, brain remodeling and neurological recovery after focal cerebral ischemia in mice

doi: 10.1007/s00395-021-00881-9

Figure Lengend Snippet: sEVs from hypoxic MSCs increase the survival of cerebral microvascular endothelial cells exposed to oxygen–glucose deprivation (OGD), but do not influence the viability of cells cultured under regular ‘normoxic’ conditions. Relative absorbance of hCMEC/D3 cells cultured under ( A–C ) regular ‘normoxic’ conditions (21% O 2 ) or ( D–F ) 24 h OGD (1% O 2 , glucose deprivation) followed by 6 h reoxygenation (21% O 2 )/glucose recultivation, determined in a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay after exposure to different concentrations of A , D sEVs obtained from MSC culture media that contain platelet lysates (sEV platelet ), B , E sEVs obtained from MSCs ( source 41.5) cultured under regular ‘normoxic’ conditions (21% O 2 ; sEV normoxic ) or C , F sEVs obtained from MSCs (source 41.5) cultured under hypoxic conditions (1% O 2 ; sEV hypoxic ). Data are mean ± SD values ( n = 3 independent experiments [in A – E ], 8 independent experiments [in F ]). ** p < 0.01, *** p < 0.001 compared with control

Article Snippet: Samples of 3 × 10 5 hCMEC/D3 cells were seeded in 6-well plates containing EBM-2 medium complemented with 1.25% FBS (other supplements as above). sEVs at various concentrations (0–250 μg/mL) obtained from (a) MSC culture medium (DMEM low, Lonza) supplemented with 10% platelet lysate (sEV platelet ), (b) supernatants of MSCs cultured under regular ‘normoxic’ conditions (21% O 2 ; sEV normoxic ), or (c) supernatants of MSCs cultured under hypoxic conditions (1% O 2 ; sEV hypoxic ) were added.

Techniques: Cell Culture, MTT Assay, Control

Journal: Basic Research in Cardiology

Article Title: Small extracellular vesicles obtained from hypoxic mesenchymal stromal cells have unique characteristics that promote cerebral angiogenesis, brain remodeling and neurological recovery after focal cerebral ischemia in mice

doi: 10.1007/s00395-021-00881-9

Figure Lengend Snippet: Cerebral microvascular endothelial cells exposed to sEVs obtained from hypoxic MSCs exhibit a distinct microRNA signature associated with angiogenesis. Total counts of A miR-126-3p, B miR-140-5p, C let-7c-5p, D miR-186-5p, E miR-370-3p and F miR-409-3p, evaluated by NanoString analysis, in hCMEC/D3 cells exposed to control conditions, sEVs obtained from MSC culture media that contain platelet lysates (50 µg/mL; sEV platelet ), sEVs obtained from MSCs ( source 41.5) cultured under regular ‘normoxic’ conditions (21% O 2 ; 50 µg/mL; sEV normoxic ) or sEVs obtained from MSCs (source 41.5) cultured under hypoxic conditions (1% O 2 ; 50 µg/mL; sEV hypoxic ). Data are box plots with medians (lines inside boxes)/means (crosses inside boxes) ± interquartile ranges (IQR; boxes) with minimum/maximum values as whiskers ( n = 4–6 samples per group). * p < 0.05, ** p < 0.01, *** p < 0.001 compared with control/ †† p < 0.01, ††† p < 0.001 compared with sEV platelet / ‡ p < 0.05, ‡‡ p < 0.01 compared with sEV normoxic

Article Snippet: Samples of 3 × 10 5 hCMEC/D3 cells were seeded in 6-well plates containing EBM-2 medium complemented with 1.25% FBS (other supplements as above). sEVs at various concentrations (0–250 μg/mL) obtained from (a) MSC culture medium (DMEM low, Lonza) supplemented with 10% platelet lysate (sEV platelet ), (b) supernatants of MSCs cultured under regular ‘normoxic’ conditions (21% O 2 ; sEV normoxic ), or (c) supernatants of MSCs cultured under hypoxic conditions (1% O 2 ; sEV hypoxic ) were added.

Techniques: Control, Cell Culture

Journal: Basic Research in Cardiology

Article Title: Small extracellular vesicles obtained from hypoxic mesenchymal stromal cells have unique characteristics that promote cerebral angiogenesis, brain remodeling and neurological recovery after focal cerebral ischemia in mice

doi: 10.1007/s00395-021-00881-9

Figure Lengend Snippet: sEVs obtained from hypoxic MSCs induce post-ischemic angiogenesis, brain remodeling and neurological recovery in a mouse model of ischemic stroke. A Density of CD31 + cerebral microvessels in the previously ischemic striatum, B number of NeuN + surviving neurons in the previously ischemic striatum and C area of GFAP + astrocytic scar in the brain infarct at the rostrocaudal level of the bregma, which is the core of the middle cerebral artery territory, as well as D striatum volume, E whole-brain volume and F neurological deficits evaluated using the Clark score of mice exposed to 40 min middle cerebral artery occlusion (MCAO), which were intravenously treated after 24 h, 72 h and 120 h with vehicle (normal saline), sEVs obtained from MSC culture media that contain platelet lysate (sEV platelet ), sEVs released by MSCs ( source 41.5) cultured under regular ‘normoxic’ conditions (21% O 2 ; sEV normoxic ; equivalent released by 2 × 10 6 cells) or sEVs released by MSCs (source 41.5) cultured under hypoxic conditions (1% O 2 ; sEV hypoxic ; equivalent released by 2 × 10 6 cells) followed by animal sacrifice after 56 days. Representative microphotographs are also shown. Data are box plots with medians (lines inside boxes)/means (crosses inside boxes) ± IQR (boxes) with minimum/maximum values as whiskers (in A – E ) or mean ± SD values (in F ) ( n = 10 animals vehicle, 6 animals sEV platelet , 9 animals sEV normoxic , 9 animals sEV hypoxic ). * p < 0.05, ** p < 0.01 compared with control/ † p < 0.05, †† p < 0.01 compared with sEV platelet / ‡ p < 0.05 compared with sEV normoxic . Scale bars: 50 µm (in A – C )/1 mm (in D , E )

Article Snippet: Samples of 3 × 10 5 hCMEC/D3 cells were seeded in 6-well plates containing EBM-2 medium complemented with 1.25% FBS (other supplements as above). sEVs at various concentrations (0–250 μg/mL) obtained from (a) MSC culture medium (DMEM low, Lonza) supplemented with 10% platelet lysate (sEV platelet ), (b) supernatants of MSCs cultured under regular ‘normoxic’ conditions (21% O 2 ; sEV normoxic ), or (c) supernatants of MSCs cultured under hypoxic conditions (1% O 2 ; sEV hypoxic ) were added.

Techniques: Saline, Cell Culture, Control