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You searched for: EV200171 (EV-TRACK ID)

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Results list Study Tree
Experiment number
  • If needed, multiple experiments were identified in a single publication based on differing sample types, separation protocols and/or vesicle types of interest.
Species
  • Species of origin of the EVs.
Separation protocol
  • Gives a short, non-chronological overview of the different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
Details EV-TRACK ID Experiment nr. Species Sample type Separation protocol First author Year EV-METRIC
EV200171 1/8 Homo sapiens primary adipose tissue-derived mesenchymal stromal cells (d)(U)C Gorgun, Cansu 2020 56%

Study summary

Full title
Dissecting the effects of preconditioning with inflammatory cytokines and hypoxia on the angiogenic potential of mesenchymal stromal cell (MSC)-derived soluble proteins and extracellular vesicles (EVs)
All authors
Cansu Gorgun, Davide Ceresa, Raphaelle Lesage, Federico Villa, Daniele Reverberi, Carolina Balbi, Sara Santamaria, Katia Cortese, Paolo Malatesta, Liesbet Geris, Rodolfo Quarto, Roberta Tasso
Journal
Biomaterials
Abstract
Mesenchymal stromal cells (MSCs) are characterized by a regulatory phenotype and respond promptly to (show more...)Mesenchymal stromal cells (MSCs) are characterized by a regulatory phenotype and respond promptly to the environmental signals modulating their secretory activity. An appropriate preconditioning may induce MSCs to release secretomes with an enhanced regenerative potential. However, it fails to take into account that secretomes are composed by both soluble factors and extracellular vesicles (EVs), whose functions could be altered differently by the preconditioning approach. Here we demonstrate that the MSC secretome is strongly modulated by the simultaneous stimulation with hypoxia and pro-inflammatory cytokines, used to mimic the harsh environment present at the site of injury. We observed that the environmental variations strongly influenced the angiogenic potential of the different secretome fractions. Upon inflammation, the pro-angiogenic capacity of the soluble component of the MSC secretome was strongly inhibited, regardless of the oxygen level, while the EV-encapsulated component was not significantly affected by the inflammatory stimuli. These effects were accompanied by the modulation of the secreted proteins. On one hand, inflammation-activated MSCs release proteins mainly involved in the interaction with innate immune cells and in tissue remodeling/repair; on the other hand, when MSCs are not exposed to an inflamed environment, they respond to the different oxygen levels modulating the expression of proteins involved in the angiogenic process. The cargo content (in terms of miRNAs) of the corresponding EV fractions was less sensitive to the influence of the external stimuli. Our findings suggest that the therapeutic efficacy of MSC-based therapies could be enhanced by selecting the appropriate preconditioning approach and carefully discriminating its effects on the different secretome components. (hide)
EV-METRIC
56% (90th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Protein markers
EV: CD81/ Mitofilin/ CD63/ CD9/ Syntenin-1
non-EV: GRP94/ Lamin A
Proteomics
no
Show all info
Study aim
Function/Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
primary adipose tissue-derived mesenchymal stromal cells
EV-harvesting Medium
Serum free medium
Cell viability (%)
70
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 10,000 g and 50,000 g
Between 50,000 g and 100,000 g
Pelleting performed
Yes
Pelleting: time(min)
40
Pelleting: rotor type
SW 28
Pelleting: speed (g)
10000
Wash: volume per pellet (ml)
5
Wash: time (min)
40
Wash: Rotor Type
SW 55 Ti
Wash: speed (g)
10000
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
Syntenin-1
Not detected EV-associated proteins
Mitofilin
Not detected contaminants
GRP94/ Lamin A
Flow cytometry
Type of Flow cytometry
BD FACSAria II
Hardware adaptation to ~100nm EV's
Gorgun, Cansu, et al. ""Isolation and Flow Cytometry Characterization of ExtracellularVesicle Subpopulations Derived from Human Mesenchymal Stromal Cells."" Current protocols in stem cell biology 48.1 (2019): e76.
Calibration bead size
100,160,200,240,300,500,900
Antibody details provided?
No
Detected EV-associated proteins
CD63/ CD9/ CD81
Characterization: RNA analysis
RNA analysis
Type
RNA sequencing
Database
No
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
232
EV concentration
Yes
Particle yield
Yes, as number of particles per milliliter of starting sample 2.19E+07
Particle analysis: flow cytometry
Flow cytometer type
BD FACSAria II
Hardware adjustment
Gorgun, Cansu, et al. ""Isolation and Flow Cytometry Characterization of ExtracellularVesicle Subpopulations Derived from Human Mesenchymal Stromal Cells."" Current protocols in stem cell biology 48.1 (2019): e76.
Calibration bead size
100,160,200,240,300,500,900
EM
EM-type
Transmission-EM
Image type
Wide-field
EV200171 2/8 Homo sapiens primary adipose tissue-derived mesenchymal stromal cells (d)(U)C Gorgun, Cansu 2020 56%

Study summary

Full title
Dissecting the effects of preconditioning with inflammatory cytokines and hypoxia on the angiogenic potential of mesenchymal stromal cell (MSC)-derived soluble proteins and extracellular vesicles (EVs)
All authors
Cansu Gorgun, Davide Ceresa, Raphaelle Lesage, Federico Villa, Daniele Reverberi, Carolina Balbi, Sara Santamaria, Katia Cortese, Paolo Malatesta, Liesbet Geris, Rodolfo Quarto, Roberta Tasso
Journal
Biomaterials
Abstract
Mesenchymal stromal cells (MSCs) are characterized by a regulatory phenotype and respond promptly to (show more...)Mesenchymal stromal cells (MSCs) are characterized by a regulatory phenotype and respond promptly to the environmental signals modulating their secretory activity. An appropriate preconditioning may induce MSCs to release secretomes with an enhanced regenerative potential. However, it fails to take into account that secretomes are composed by both soluble factors and extracellular vesicles (EVs), whose functions could be altered differently by the preconditioning approach. Here we demonstrate that the MSC secretome is strongly modulated by the simultaneous stimulation with hypoxia and pro-inflammatory cytokines, used to mimic the harsh environment present at the site of injury. We observed that the environmental variations strongly influenced the angiogenic potential of the different secretome fractions. Upon inflammation, the pro-angiogenic capacity of the soluble component of the MSC secretome was strongly inhibited, regardless of the oxygen level, while the EV-encapsulated component was not significantly affected by the inflammatory stimuli. These effects were accompanied by the modulation of the secreted proteins. On one hand, inflammation-activated MSCs release proteins mainly involved in the interaction with innate immune cells and in tissue remodeling/repair; on the other hand, when MSCs are not exposed to an inflamed environment, they respond to the different oxygen levels modulating the expression of proteins involved in the angiogenic process. The cargo content (in terms of miRNAs) of the corresponding EV fractions was less sensitive to the influence of the external stimuli. Our findings suggest that the therapeutic efficacy of MSC-based therapies could be enhanced by selecting the appropriate preconditioning approach and carefully discriminating its effects on the different secretome components. (hide)
EV-METRIC
56% (90th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Protein markers
EV: CD81/ Mitofilin/ CD63/ CD9/ Syntenin-1
non-EV: GRP94/ Lamin A
Proteomics
no
Show all info
Study aim
Function/Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
primary adipose tissue-derived mesenchymal stromal cells
EV-harvesting Medium
Serum free medium
Cell viability (%)
70
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 10,000 g and 50,000 g
Between 50,000 g and 100,000 g
Pelleting performed
Yes
Pelleting: time(min)
120
Pelleting: rotor type
SW 28
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
5
Wash: time (min)
120
Wash: Rotor Type
SW 55 Ti
Wash: speed (g)
100000
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
Syntenin-1
Not detected EV-associated proteins
Mitofilin
Not detected contaminants
GRP94/ Lamin A
Flow cytometry
Type of Flow cytometry
BD FACSAria II
Hardware adaptation to ~100nm EV's
Gorgun, Cansu, et al. ""Isolation and Flow Cytometry Characterization of ExtracellularVesicle Subpopulations Derived from Human Mesenchymal Stromal Cells."" Current protocols in stem cell biology 48.1 (2019): e76.
Calibration bead size
100,160,200,240,300,500,900
Antibody details provided?
No
Detected EV-associated proteins
CD63/ CD9/ CD81
Characterization: RNA analysis
RNA analysis
Type
RNAsequencing
Database
No
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
164
EV concentration
Yes
Particle yield
Yes, as number of particles per milliliter of starting sample 2.39E+07
Particle analysis: flow cytometry
Flow cytometer type
BD FACSAria II
Hardware adjustment
Gorgun, Cansu, et al. ""Isolation and Flow Cytometry Characterization of ExtracellularVesicle Subpopulations Derived from Human Mesenchymal Stromal Cells."" Current protocols in stem cell biology 48.1 (2019): e76.
Calibration bead size
100,160,200,240,300,500,900
Report type
Size range/distribution
Reported size (nm)
Smaller than 160 nm
EM
EM-type
Transmission-EM
Image type
Wide-field
EV200171 3/8 Homo sapiens primary adipose tissue-derived mesenchymal stromal cells (d)(U)C
 Gorgun, Cansu 2020 56%

Study summary

Full title
Dissecting the effects of preconditioning with inflammatory cytokines and hypoxia on the angiogenic potential of mesenchymal stromal cell (MSC)-derived soluble proteins and extracellular vesicles (EVs)
All authors
Cansu Gorgun, Davide Ceresa, Raphaelle Lesage, Federico Villa, Daniele Reverberi, Carolina Balbi, Sara Santamaria, Katia Cortese, Paolo Malatesta, Liesbet Geris, Rodolfo Quarto, Roberta Tasso
Journal
Biomaterials
Abstract
Mesenchymal stromal cells (MSCs) are characterized by a regulatory phenotype and respond promptly to (show more...)Mesenchymal stromal cells (MSCs) are characterized by a regulatory phenotype and respond promptly to the environmental signals modulating their secretory activity. An appropriate preconditioning may induce MSCs to release secretomes with an enhanced regenerative potential. However, it fails to take into account that secretomes are composed by both soluble factors and extracellular vesicles (EVs), whose functions could be altered differently by the preconditioning approach. Here we demonstrate that the MSC secretome is strongly modulated by the simultaneous stimulation with hypoxia and pro-inflammatory cytokines, used to mimic the harsh environment present at the site of injury. We observed that the environmental variations strongly influenced the angiogenic potential of the different secretome fractions. Upon inflammation, the pro-angiogenic capacity of the soluble component of the MSC secretome was strongly inhibited, regardless of the oxygen level, while the EV-encapsulated component was not significantly affected by the inflammatory stimuli. These effects were accompanied by the modulation of the secreted proteins. On one hand, inflammation-activated MSCs release proteins mainly involved in the interaction with innate immune cells and in tissue remodeling/repair; on the other hand, when MSCs are not exposed to an inflamed environment, they respond to the different oxygen levels modulating the expression of proteins involved in the angiogenic process. The cargo content (in terms of miRNAs) of the corresponding EV fractions was less sensitive to the influence of the external stimuli. Our findings suggest that the therapeutic efficacy of MSC-based therapies could be enhanced by selecting the appropriate preconditioning approach and carefully discriminating its effects on the different secretome components. (hide)
EV-METRIC
56% (90th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Hypoxia
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
No extra separation steps
Protein markers
EV: CD81/ Mitofilin/ CD63/ CD9/ Syntenin-1
non-EV: GRP94/ Lamin A
Proteomics
no
Show all info
Study aim
Function/Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
primary adipose tissue-derived mesenchymal stromal cells
EV-harvesting Medium
Serum free medium
Cell viability (%)
70
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 10,000 g and 50,000 g
Between 50,000 g and 100,000 g
Pelleting performed
Yes
Pelleting: time(min)
120
Pelleting: rotor type
SW 28
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
5
Wash: time (min)
120
Wash: Rotor Type
SW 55 Ti
Wash: speed (g)
100000
Other
Name other separation method
No extra separation steps
Characterization: Protein analysis
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
Syntenin-1
Not detected EV-associated proteins
Mitofilin
Detected contaminants
Lamin A
Not detected contaminants
GRP94
Flow cytometry
Hardware adaptation to ~100nm EV's
Gorgun, Cansu, et al. ""Isolation and Flow Cytometry Characterization of ExtracellularVesicle Subpopulations Derived from Human Mesenchymal Stromal Cells."" Current protocols in stem cell biology 48.1 (2019): e76.
Calibration bead size
100,160,200,240,300,500,900
Antibody details provided?
No
Detected EV-associated proteins
CD63/ CD9/ CD81
Characterization: RNA analysis
RNA analysis
Type
RNAsequencing
Database
No
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
164
EV concentration
Yes
Particle yield
Yes, as number of particles per milliliter of starting sample 7.06E+07
Particle analysis: flow cytometry
Flow cytometer type
BD FACSAria II
Hardware adjustment
Gorgun, Cansu, et al. ""Isolation and Flow Cytometry Characterization of ExtracellularVesicle Subpopulations Derived from Human Mesenchymal Stromal Cells."" Current protocols in stem cell biology 48.1 (2019): e76.
Calibration bead size
100,160,200,240,300,500,900
Report type
Size range/distribution
Reported size (nm)
Smaller than 160 nm
EM
EM-type
Transmission-EM
Image type
Wide-field
EV200171 4/8 Homo sapiens primary adipose tissue-derived mesenchymal stromal cells (d)(U)C Gorgun, Cansu 2020 56%

Study summary

Full title
Dissecting the effects of preconditioning with inflammatory cytokines and hypoxia on the angiogenic potential of mesenchymal stromal cell (MSC)-derived soluble proteins and extracellular vesicles (EVs)
All authors
Cansu Gorgun, Davide Ceresa, Raphaelle Lesage, Federico Villa, Daniele Reverberi, Carolina Balbi, Sara Santamaria, Katia Cortese, Paolo Malatesta, Liesbet Geris, Rodolfo Quarto, Roberta Tasso
Journal
Biomaterials
Abstract
Mesenchymal stromal cells (MSCs) are characterized by a regulatory phenotype and respond promptly to (show more...)Mesenchymal stromal cells (MSCs) are characterized by a regulatory phenotype and respond promptly to the environmental signals modulating their secretory activity. An appropriate preconditioning may induce MSCs to release secretomes with an enhanced regenerative potential. However, it fails to take into account that secretomes are composed by both soluble factors and extracellular vesicles (EVs), whose functions could be altered differently by the preconditioning approach. Here we demonstrate that the MSC secretome is strongly modulated by the simultaneous stimulation with hypoxia and pro-inflammatory cytokines, used to mimic the harsh environment present at the site of injury. We observed that the environmental variations strongly influenced the angiogenic potential of the different secretome fractions. Upon inflammation, the pro-angiogenic capacity of the soluble component of the MSC secretome was strongly inhibited, regardless of the oxygen level, while the EV-encapsulated component was not significantly affected by the inflammatory stimuli. These effects were accompanied by the modulation of the secreted proteins. On one hand, inflammation-activated MSCs release proteins mainly involved in the interaction with innate immune cells and in tissue remodeling/repair; on the other hand, when MSCs are not exposed to an inflamed environment, they respond to the different oxygen levels modulating the expression of proteins involved in the angiogenic process. The cargo content (in terms of miRNAs) of the corresponding EV fractions was less sensitive to the influence of the external stimuli. Our findings suggest that the therapeutic efficacy of MSC-based therapies could be enhanced by selecting the appropriate preconditioning approach and carefully discriminating its effects on the different secretome components. (hide)
EV-METRIC
56% (90th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Hypoxia
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Protein markers
EV: CD81/ Mitofilin/ CD63/ CD9/ Syntenin-1
non-EV: GRP94/ Lamin A
Proteomics
no
Show all info
Study aim
Function/Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
primary adipose tissue-derived mesenchymal stromal cells
EV-harvesting Medium
Serum free medium
Cell viability (%)
70
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 10,000 g and 50,000 g
Between 50,000 g and 100,000 g
Pelleting performed
Yes
Pelleting: time(min)
40
Pelleting: rotor type
SW 28
Pelleting: speed (g)
10000
Wash: volume per pellet (ml)
5
Wash: time (min)
40
Wash: Rotor Type
SW 55 Ti
Wash: speed (g)
10000
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
Syntenin-1
Not detected EV-associated proteins
Mitofilin
Not detected contaminants
GRP94/ Lamin A
Flow cytometry
Type of Flow cytometry
BD FACSAria II
Hardware adaptation to ~100nm EV's
Gorgun, Cansu, et al. ""Isolation and Flow Cytometry Characterization of ExtracellularVesicle Subpopulations Derived from Human Mesenchymal Stromal Cells."" Current protocols in stem cell biology 48.1 (2019): e76.
Calibration bead size
100,160,200,240,300,500,900
Antibody details provided?
No
Detected EV-associated proteins
CD63/ CD9/ CD81
Characterization: RNA analysis
RNA analysis
Type
RNAsequencing
Database
No
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
164
EV concentration
Yes
Particle yield
Yes, as number of particles per milliliter of starting sample 4.54E+07
Particle analysis: flow cytometry
Flow cytometer type
BD FACSAria II
Hardware adjustment
Gorgun, Cansu, et al. ""Isolation and Flow Cytometry Characterization of ExtracellularVesicle Subpopulations Derived from Human Mesenchymal Stromal Cells."" Current protocols in stem cell biology 48.1 (2019): e76.
Calibration bead size
100,160,200,240,300,500,900
Report type
Size range/distribution
Reported size (nm)
Smaller than 160 nm
EM
EM-type
Transmission-EM
Image type
Wide-field
EV200171 5/8 Homo sapiens primary adipose tissue-derived mesenchymal stromal cells (d)(U)C Gorgun, Cansu 2020 56%

Study summary

Full title
Dissecting the effects of preconditioning with inflammatory cytokines and hypoxia on the angiogenic potential of mesenchymal stromal cell (MSC)-derived soluble proteins and extracellular vesicles (EVs)
All authors
Cansu Gorgun, Davide Ceresa, Raphaelle Lesage, Federico Villa, Daniele Reverberi, Carolina Balbi, Sara Santamaria, Katia Cortese, Paolo Malatesta, Liesbet Geris, Rodolfo Quarto, Roberta Tasso
Journal
Biomaterials
Abstract
Mesenchymal stromal cells (MSCs) are characterized by a regulatory phenotype and respond promptly to (show more...)Mesenchymal stromal cells (MSCs) are characterized by a regulatory phenotype and respond promptly to the environmental signals modulating their secretory activity. An appropriate preconditioning may induce MSCs to release secretomes with an enhanced regenerative potential. However, it fails to take into account that secretomes are composed by both soluble factors and extracellular vesicles (EVs), whose functions could be altered differently by the preconditioning approach. Here we demonstrate that the MSC secretome is strongly modulated by the simultaneous stimulation with hypoxia and pro-inflammatory cytokines, used to mimic the harsh environment present at the site of injury. We observed that the environmental variations strongly influenced the angiogenic potential of the different secretome fractions. Upon inflammation, the pro-angiogenic capacity of the soluble component of the MSC secretome was strongly inhibited, regardless of the oxygen level, while the EV-encapsulated component was not significantly affected by the inflammatory stimuli. These effects were accompanied by the modulation of the secreted proteins. On one hand, inflammation-activated MSCs release proteins mainly involved in the interaction with innate immune cells and in tissue remodeling/repair; on the other hand, when MSCs are not exposed to an inflamed environment, they respond to the different oxygen levels modulating the expression of proteins involved in the angiogenic process. The cargo content (in terms of miRNAs) of the corresponding EV fractions was less sensitive to the influence of the external stimuli. Our findings suggest that the therapeutic efficacy of MSC-based therapies could be enhanced by selecting the appropriate preconditioning approach and carefully discriminating its effects on the different secretome components. (hide)
EV-METRIC
56% (90th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Normoxia + IL1Alpha + TNFAlpha
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Protein markers
EV: CD81/ Mitofilin/ CD63/ CD9/ Syntenin-1
non-EV: GRP94/ Lamin A
Proteomics
no
Show all info
Study aim
Function/Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
primary adipose tissue-derived mesenchymal stromal cells
EV-harvesting Medium
Serum free medium
Cell viability (%)
70
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 10,000 g and 50,000 g
Between 50,000 g and 100,000 g
Pelleting performed
Yes
Pelleting: time(min)
120
Pelleting: rotor type
SW 28
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
5
Wash: time (min)
120
Wash: Rotor Type
SW 55 Ti
Wash: speed (g)
100000
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
Syntenin-1
Not detected EV-associated proteins
Mitofilin
Not detected contaminants
GRP94/ Lamin A
Flow cytometry
Type of Flow cytometry
BD FACSAria II
Hardware adaptation to ~100nm EV's
Gorgun, Cansu, et al. ""Isolation and Flow Cytometry Characterization of ExtracellularVesicle Subpopulations Derived from Human Mesenchymal Stromal Cells."" Current protocols in stem cell biology 48.1 (2019): e76.
Calibration bead size
100,160,200,240,300,500,900
Antibody details provided?
No
Detected EV-associated proteins
CD63/ CD9/ CD81
Characterization: RNA analysis
RNA analysis
Type
RNAsequencing
Database
No
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
232
EV concentration
Yes
Particle yield
Yes, as number of particles per milliliter of starting sample 2.57E+07
Particle analysis: flow cytometry
Flow cytometer type
BD FACSAria II
Hardware adjustment
Gorgun, Cansu, et al. ""Isolation and Flow Cytometry Characterization of ExtracellularVesicle Subpopulations Derived from Human Mesenchymal Stromal Cells."" Current protocols in stem cell biology 48.1 (2019): e76.
Calibration bead size
100,160,200,240,300,500,900
Report type
Size range/distribution
Reported size (nm)
Bigger than 160 nm
EM
EM-type
Transmission-EM
Image type
Wide-field
EV200171 7/8 Homo sapiens primary adipose tissue-derived mesenchymal stromal cells (d)(U)C Gorgun, Cansu 2020 56%

Study summary

Full title
Dissecting the effects of preconditioning with inflammatory cytokines and hypoxia on the angiogenic potential of mesenchymal stromal cell (MSC)-derived soluble proteins and extracellular vesicles (EVs)
All authors
Cansu Gorgun, Davide Ceresa, Raphaelle Lesage, Federico Villa, Daniele Reverberi, Carolina Balbi, Sara Santamaria, Katia Cortese, Paolo Malatesta, Liesbet Geris, Rodolfo Quarto, Roberta Tasso
Journal
Biomaterials
Abstract
Mesenchymal stromal cells (MSCs) are characterized by a regulatory phenotype and respond promptly to (show more...)Mesenchymal stromal cells (MSCs) are characterized by a regulatory phenotype and respond promptly to the environmental signals modulating their secretory activity. An appropriate preconditioning may induce MSCs to release secretomes with an enhanced regenerative potential. However, it fails to take into account that secretomes are composed by both soluble factors and extracellular vesicles (EVs), whose functions could be altered differently by the preconditioning approach. Here we demonstrate that the MSC secretome is strongly modulated by the simultaneous stimulation with hypoxia and pro-inflammatory cytokines, used to mimic the harsh environment present at the site of injury. We observed that the environmental variations strongly influenced the angiogenic potential of the different secretome fractions. Upon inflammation, the pro-angiogenic capacity of the soluble component of the MSC secretome was strongly inhibited, regardless of the oxygen level, while the EV-encapsulated component was not significantly affected by the inflammatory stimuli. These effects were accompanied by the modulation of the secreted proteins. On one hand, inflammation-activated MSCs release proteins mainly involved in the interaction with innate immune cells and in tissue remodeling/repair; on the other hand, when MSCs are not exposed to an inflamed environment, they respond to the different oxygen levels modulating the expression of proteins involved in the angiogenic process. The cargo content (in terms of miRNAs) of the corresponding EV fractions was less sensitive to the influence of the external stimuli. Our findings suggest that the therapeutic efficacy of MSC-based therapies could be enhanced by selecting the appropriate preconditioning approach and carefully discriminating its effects on the different secretome components. (hide)
EV-METRIC
56% (90th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Hypoxia + IL1Alpha + TNFAlpha
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Protein markers
EV: CD81/ Mitofilin/ CD63/ CD9/ Syntenin-1
non-EV: GRP94/ Lamin A
Proteomics
no
Show all info
Study aim
Function/Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
primary adipose tissue-derived mesenchymal stromal cells
EV-harvesting Medium
Serum free medium
Cell viability (%)
70
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 10,000 g and 50,000 g
Between 50,000 g and 100,000 g
Pelleting performed
Yes
Pelleting: time(min)
120
Pelleting: rotor type
SW 28
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
5
Wash: time (min)
120
Wash: Rotor Type
SW 55 Ti
Wash: speed (g)
100000
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
Syntenin-1
Not detected EV-associated proteins
Mitofilin
Not detected contaminants
GRP94/ Lamin A
Flow cytometry
Type of Flow cytometry
BD FACSAria II
Hardware adaptation to ~100nm EV's
Gorgun, Cansu, et al. ""Isolation and Flow Cytometry Characterization of ExtracellularVesicle Subpopulations Derived from Human Mesenchymal Stromal Cells."" Current protocols in stem cell biology 48.1 (2019): e76.
Calibration bead size
100,160,200,240,300,500,900
Antibody details provided?
No
Detected EV-associated proteins
CD63/ CD9/ CD81
Characterization: RNA analysis
RNA analysis
Type
RNAsequencing
Database
No
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
164
EV concentration
Yes
Particle yield
Yes, as number of particles per milliliter of starting sample 6.23E+07
Particle analysis: flow cytometry
Flow cytometer type
BD FACSAria II
Hardware adjustment
Gorgun, Cansu, et al. ""Isolation and Flow Cytometry Characterization of ExtracellularVesicle Subpopulations Derived from Human Mesenchymal Stromal Cells."" Current protocols in stem cell biology 48.1 (2019): e76.
Calibration bead size
100,160,200,240,300,500,900
Report type
Size range/distribution
Reported size (nm)
Smaller than 160 nm
EM
EM-type
Transmission-EM
Image type
Wide-field
EV200171 8/8 Homo sapiens primary adipose tissue-derived mesenchymal stromal cells (d)(U)C Gorgun, Cansu 2020 56%

Study summary

Full title
Dissecting the effects of preconditioning with inflammatory cytokines and hypoxia on the angiogenic potential of mesenchymal stromal cell (MSC)-derived soluble proteins and extracellular vesicles (EVs)
All authors
Cansu Gorgun, Davide Ceresa, Raphaelle Lesage, Federico Villa, Daniele Reverberi, Carolina Balbi, Sara Santamaria, Katia Cortese, Paolo Malatesta, Liesbet Geris, Rodolfo Quarto, Roberta Tasso
Journal
Biomaterials
Abstract
Mesenchymal stromal cells (MSCs) are characterized by a regulatory phenotype and respond promptly to (show more...)Mesenchymal stromal cells (MSCs) are characterized by a regulatory phenotype and respond promptly to the environmental signals modulating their secretory activity. An appropriate preconditioning may induce MSCs to release secretomes with an enhanced regenerative potential. However, it fails to take into account that secretomes are composed by both soluble factors and extracellular vesicles (EVs), whose functions could be altered differently by the preconditioning approach. Here we demonstrate that the MSC secretome is strongly modulated by the simultaneous stimulation with hypoxia and pro-inflammatory cytokines, used to mimic the harsh environment present at the site of injury. We observed that the environmental variations strongly influenced the angiogenic potential of the different secretome fractions. Upon inflammation, the pro-angiogenic capacity of the soluble component of the MSC secretome was strongly inhibited, regardless of the oxygen level, while the EV-encapsulated component was not significantly affected by the inflammatory stimuli. These effects were accompanied by the modulation of the secreted proteins. On one hand, inflammation-activated MSCs release proteins mainly involved in the interaction with innate immune cells and in tissue remodeling/repair; on the other hand, when MSCs are not exposed to an inflamed environment, they respond to the different oxygen levels modulating the expression of proteins involved in the angiogenic process. The cargo content (in terms of miRNAs) of the corresponding EV fractions was less sensitive to the influence of the external stimuli. Our findings suggest that the therapeutic efficacy of MSC-based therapies could be enhanced by selecting the appropriate preconditioning approach and carefully discriminating its effects on the different secretome components. (hide)
EV-METRIC
56% (90th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Hypoxia + IL1Alpha + TNFAlpha
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Protein markers
EV: CD81/ Mitofilin/ CD63/ CD9/ Syntenin-1
non-EV: GRP94/ Lamin A
Proteomics
no
Show all info
Study aim
Function/Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
primary adipose tissue-derived mesenchymal stromal cells
EV-harvesting Medium
Serum free medium
Cell viability (%)
70
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 10,000 g and 50,000 g
Between 50,000 g and 100,000 g
Pelleting performed
Yes
Pelleting: time(min)
40
Pelleting: rotor type
SW 28
Pelleting: speed (g)
10000
Wash: volume per pellet (ml)
5
Wash: time (min)
40
Wash: Rotor Type
SW 55 Ti
Wash: speed (g)
10000
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
Syntenin-1
Not detected EV-associated proteins
Mitofilin
Not detected contaminants
GRP94/ Lamin A
Flow cytometry
Type of Flow cytometry
BD FACSAria II
Hardware adaptation to ~100nm EV's
Gorgun, Cansu, et al. ""Isolation and Flow Cytometry Characterization of ExtracellularVesicle Subpopulations Derived from Human Mesenchymal Stromal Cells."" Current protocols in stem cell biology 48.1 (2019): e76.
Calibration bead size
100,160,200,240,300,500,900
Antibody details provided?
No
Detected EV-associated proteins
CD63/ CD9/ CD81
Characterization: RNA analysis
RNA analysis
Type
RNAsequencing
Database
No
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
232
EV concentration
Yes
Particle yield
Yes, as number of particles per milliliter of starting sample 2.91E+07
Particle analysis: flow cytometry
Flow cytometer type
BD FACSAria II
Hardware adjustment
Gorgun, Cansu, et al. ""Isolation and Flow Cytometry Characterization of ExtracellularVesicle Subpopulations Derived from Human Mesenchymal Stromal Cells."" Current protocols in stem cell biology 48.1 (2019): e76.
Calibration bead size
100,160,200,240,300,500,900
Report type
Size range/distribution
Reported size (nm)
Bigger than 160 nm
EM
EM-type
Transmission-EM
Image type
Wide-field
EV200171 6/8 Homo sapiens primary adipose tissue-derived mesenchymal stromal cells (d)(U)C Gorgun, Cansu 2020 14%

Study summary

Full title
Dissecting the effects of preconditioning with inflammatory cytokines and hypoxia on the angiogenic potential of mesenchymal stromal cell (MSC)-derived soluble proteins and extracellular vesicles (EVs)
All authors
Cansu Gorgun, Davide Ceresa, Raphaelle Lesage, Federico Villa, Daniele Reverberi, Carolina Balbi, Sara Santamaria, Katia Cortese, Paolo Malatesta, Liesbet Geris, Rodolfo Quarto, Roberta Tasso
Journal
Biomaterials
Abstract
Mesenchymal stromal cells (MSCs) are characterized by a regulatory phenotype and respond promptly to (show more...)Mesenchymal stromal cells (MSCs) are characterized by a regulatory phenotype and respond promptly to the environmental signals modulating their secretory activity. An appropriate preconditioning may induce MSCs to release secretomes with an enhanced regenerative potential. However, it fails to take into account that secretomes are composed by both soluble factors and extracellular vesicles (EVs), whose functions could be altered differently by the preconditioning approach. Here we demonstrate that the MSC secretome is strongly modulated by the simultaneous stimulation with hypoxia and pro-inflammatory cytokines, used to mimic the harsh environment present at the site of injury. We observed that the environmental variations strongly influenced the angiogenic potential of the different secretome fractions. Upon inflammation, the pro-angiogenic capacity of the soluble component of the MSC secretome was strongly inhibited, regardless of the oxygen level, while the EV-encapsulated component was not significantly affected by the inflammatory stimuli. These effects were accompanied by the modulation of the secreted proteins. On one hand, inflammation-activated MSCs release proteins mainly involved in the interaction with innate immune cells and in tissue remodeling/repair; on the other hand, when MSCs are not exposed to an inflamed environment, they respond to the different oxygen levels modulating the expression of proteins involved in the angiogenic process. The cargo content (in terms of miRNAs) of the corresponding EV fractions was less sensitive to the influence of the external stimuli. Our findings suggest that the therapeutic efficacy of MSC-based therapies could be enhanced by selecting the appropriate preconditioning approach and carefully discriminating its effects on the different secretome components. (hide)
EV-METRIC
14% (44th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Normoxia + IL1Alpha + TNFAlpha
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Protein markers
EV: None
non-EV: None
Proteomics
no
Show all info
Study aim
Function/Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
primary adipose tissue-derived mesenchymal stromal cells
EV-harvesting Medium
Serum free medium
Cell viability (%)
70
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 10,000 g and 50,000 g
Between 50,000 g and 100,000 g
Pelleting performed
Yes
Pelleting: time(min)
40
Pelleting: rotor type
SW 28
Pelleting: speed (g)
10000
Wash: volume per pellet (ml)
5
Wash: time (min)
40
Wash: Rotor Type
SW 55 Ti
Wash: speed (g)
10000
Characterization: Protein analysis
None
Protein Concentration Method
Not determined
Characterization: Lipid analysis
No
Characterization: Particle analysis
None
1 - 8 of 8
  • CM = Commercial method
  • dUC = differential ultracentrifugation
  • DG = density gradient
  • UF = ultrafiltration
  • SEC = size-exclusion chromatography
EV-TRACK ID
EV200171
species
Homo
sapiens
sample type
Cell
culture
cell type
primary
adipose
tissue-derived
mesenchymal
stromal
cells
condition
Control
condition
Control
condition
Hypoxia
Hypoxia
Normoxia
IL1Alpha
TNFAlpha
Hypoxia
IL1Alpha
TNFAlpha
Hypoxia
IL1Alpha
TNFAlpha
Normoxia
IL1Alpha
TNFAlpha
separation protocol
dUC
dUC
dUC
No
extra
separation
steps
dUC
dUC
dUC
dUC
dUC
Exp. nr.
1
2
3
4
5
7
8
6
EV-METRIC %
56
56
56
56
56
56
56
14