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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV190081	1/3	Homo sapiens	hTERT-immortalized adipose tissue derived mesenchymal stromal cells	(d)(U)C Filtration	Haghighitalab, Azadeh	2021	33%
Study summary Full title Investigating the effects of IDO1, PTGS2, and TGF-β1 overexpression on immunomodulatory properties of hTERT-MSCs and their extracellular vesicles All authors Azadeh Haghighitalab, Maryam M Matin, Ahmad Amin, Shima Minaee, Hamid Reza Bidkhori, Thorsten R Doeppner, Ahmad Reza Bahrami Journal Sci Rep Abstract The therapeutic potential of mesenchymal stem cells (MSCs) is out of the question. Yet, recent drawb (show more...)The therapeutic potential of mesenchymal stem cells (MSCs) is out of the question. Yet, recent drawbacks have resulted in a strategic shift towards the application of MSC-derived cell-free products such as extracellular vesicles (EVs). Recent reports revealed that functional properties of MSCs, including EV secretion patterns, correlate with microenvironmental cues. These findings highlight the urgent need for defining the optimal circumstances for EV preparation. Considering the limitations of primary cells, we employed immortalized cells as an alternative source to prepare therapeutically sufficient EV numbers. Herein, the effects of different conditional environments are explored on human TERT-immortalized MSCs (hTERT-MSCs). The latter were transduced to overexpress IDO1, PTGS2, and TGF-β1 transgenes either alone or in combination, and their immunomodulatory properties were analyzed thereafter. Likewise, EVs derived from these various MSCs were extensively characterized. hTERT-MSCs-IDO1 exerted superior inhibitory effects on lymphocytes, significantly more than hTERT-MSCs-IFN-γ. As such, IDO1 overexpression promoted the immunomodulatory properties of such enriched EVs. Considering the limitations of cell therapy like tumor formation and possible immune responses in the host, the results presented herein might be considered as a feasible model for the induction of immunomodulation in off-the-shelf and cell-free therapeutics, especially for autoimmune diseases. (hide) EV-METRIC 33% (75th 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 (d)(U)C Filtration Protein markers EV: TSG101/ CD63 non-EV: Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells hTERT-immortalized adipose tissue derived mesenchymal stromal cells EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS Cell viability (%) 80 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 50,000 g and 100,000 g Pelleting performed Yes Pelleting: time(min) 60 Pelleting: rotor type An-50Ti Pelleting: speed (g) 70 Filtration steps 0.45µm > x > 0.22µm, 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins CD63/ TSG101 Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Size range/distribution Reported size (nm) 40-400 NTA Report type Mean Reported size (nm) 176 EV concentration Yes EM EM-type Atomic force-EM/ Transmission-EM Image type Wide-field

	EV190081	2/3	Homo sapiens	hTERT-immortalized adipose tissue derived mesenchymal stromal cells	(d)(U)C Filtration	Haghighitalab, Azadeh	2021	33%
Study summary Full title Investigating the effects of IDO1, PTGS2, and TGF-β1 overexpression on immunomodulatory properties of hTERT-MSCs and their extracellular vesicles All authors Azadeh Haghighitalab, Maryam M Matin, Ahmad Amin, Shima Minaee, Hamid Reza Bidkhori, Thorsten R Doeppner, Ahmad Reza Bahrami Journal Sci Rep Abstract The therapeutic potential of mesenchymal stem cells (MSCs) is out of the question. Yet, recent drawb (show more...)The therapeutic potential of mesenchymal stem cells (MSCs) is out of the question. Yet, recent drawbacks have resulted in a strategic shift towards the application of MSC-derived cell-free products such as extracellular vesicles (EVs). Recent reports revealed that functional properties of MSCs, including EV secretion patterns, correlate with microenvironmental cues. These findings highlight the urgent need for defining the optimal circumstances for EV preparation. Considering the limitations of primary cells, we employed immortalized cells as an alternative source to prepare therapeutically sufficient EV numbers. Herein, the effects of different conditional environments are explored on human TERT-immortalized MSCs (hTERT-MSCs). The latter were transduced to overexpress IDO1, PTGS2, and TGF-β1 transgenes either alone or in combination, and their immunomodulatory properties were analyzed thereafter. Likewise, EVs derived from these various MSCs were extensively characterized. hTERT-MSCs-IDO1 exerted superior inhibitory effects on lymphocytes, significantly more than hTERT-MSCs-IFN-γ. As such, IDO1 overexpression promoted the immunomodulatory properties of such enriched EVs. Considering the limitations of cell therapy like tumor formation and possible immune responses in the host, the results presented herein might be considered as a feasible model for the induction of immunomodulation in off-the-shelf and cell-free therapeutics, especially for autoimmune diseases. (hide) EV-METRIC 33% (75th 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 Chemically primed 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 (d)(U)C Filtration Protein markers EV: TSG101/ CD63 non-EV: Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells hTERT-immortalized adipose tissue derived mesenchymal stromal cells EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS Cell viability (%) 80 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 50,000 g and 100,000 g Pelleting performed Yes Pelleting: time(min) 60 Pelleting: rotor type An-50Ti Pelleting: speed (g) 70 Filtration steps 0.45µm > x > 0.22µm, 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins CD63/ TSG101 Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Size range/distribution Reported size (nm) 40-400 NTA Report type Mean Reported size (nm) 176-180 EV concentration Yes EM EM-type Atomic force-EM Image type Wide-field

	EV190081	3/3	Homo sapiens	hTERT-immortalized adipose tissue derived mesenchymal stromal cells	(d)(U)C Filtration	Haghighitalab, Azadeh	2021	29%
Study summary Full title Investigating the effects of IDO1, PTGS2, and TGF-β1 overexpression on immunomodulatory properties of hTERT-MSCs and their extracellular vesicles All authors Azadeh Haghighitalab, Maryam M Matin, Ahmad Amin, Shima Minaee, Hamid Reza Bidkhori, Thorsten R Doeppner, Ahmad Reza Bahrami Journal Sci Rep Abstract The therapeutic potential of mesenchymal stem cells (MSCs) is out of the question. Yet, recent drawb (show more...)The therapeutic potential of mesenchymal stem cells (MSCs) is out of the question. Yet, recent drawbacks have resulted in a strategic shift towards the application of MSC-derived cell-free products such as extracellular vesicles (EVs). Recent reports revealed that functional properties of MSCs, including EV secretion patterns, correlate with microenvironmental cues. These findings highlight the urgent need for defining the optimal circumstances for EV preparation. Considering the limitations of primary cells, we employed immortalized cells as an alternative source to prepare therapeutically sufficient EV numbers. Herein, the effects of different conditional environments are explored on human TERT-immortalized MSCs (hTERT-MSCs). The latter were transduced to overexpress IDO1, PTGS2, and TGF-β1 transgenes either alone or in combination, and their immunomodulatory properties were analyzed thereafter. Likewise, EVs derived from these various MSCs were extensively characterized. hTERT-MSCs-IDO1 exerted superior inhibitory effects on lymphocytes, significantly more than hTERT-MSCs-IFN-γ. As such, IDO1 overexpression promoted the immunomodulatory properties of such enriched EVs. Considering the limitations of cell therapy like tumor formation and possible immune responses in the host, the results presented herein might be considered as a feasible model for the induction of immunomodulation in off-the-shelf and cell-free therapeutics, especially for autoimmune diseases. (hide) EV-METRIC 29% (68th 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 Genetically engineered 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 (d)(U)C Filtration Protein markers EV: non-EV: Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells hTERT-immortalized adipose tissue derived mesenchymal stromal cells EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS Cell viability (%) 80 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 50,000 g and 100,000 g Pelleting performed Yes Pelleting: time(min) 60 Pelleting: rotor type An-50Ti Pelleting: speed (g) 70 Filtration steps 0.45µm > x > 0.22µm, 0.22µm or 0.2µm Protein Concentration Method microBCA Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Size range/distribution Reported size (nm) 40-400 EM EM-type Atomic force-EM/ Transmission-EM Image type Wide-field

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EV-TRACK ID	EV190081
species	Homo sapiens
sample type	Cell culture
cell type	hTERT-immortalized adipose tissue derived mesenchymal stromal cells
condition	Control condition	Chemically primed	Genetically engineered
separation protocol	(d)(U)C Filtration	(d)(U)C Filtration	(d)(U)C Filtration
Exp. nr.	1	2	3
EV-METRIC %	33	33	29