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CD73 activity of mesenchymal stromal cell-derived extracellular vesicle preparations is detergent-resistant and does not correlate with immunomodulatory capabilities

Open AccessPublished:October 14, 2022DOI:https://doi.org/10.1016/j.jcyt.2022.09.006

      Abstract

      Background aims

      Extracellular vesicles (EVs) derived from human mesenchymal stromal cells (MSCs) show immunomodulatory activity in different assays both in vitro and in vivo. In previous work, the authors compared the immunomodulatory potential of independent MSC-EV preparations in a multi-donor mixed lymphocyte reaction (mdMLR) assay and an optimized steroid-refractory acute graft-versus-host disease (aGVHD) mouse model. The authors observed that only a proportion of the MSC-EV preparations showed immunomodulatory capabilities and demonstrated that only MSC-EV preparations with mdMLR immunomodulating activities were able to suppress aGVHD symptoms in vivo and vice versa. Since the mdMLR assay is complex and depends on primary human cells of different donors, the authors sought to establish an assay that is much easier to standardize and fulfills the requirements for becoming qualified as a potency assay.

      Methods

      The bona fide MSC antigen CD73 possesses ecto-5’-nucleotidase activity that cleaves pro-inflammatory extracellular adenosine monophosphate into anti-inflammatory adenosine and free phosphate. To test whether the ecto-5’-nucleotidase activity of the MSC-EV preparations reflected their immunomodulatory potential, the authors adopted an enzymatic assay that monitors the ecto-5’-nucleotidase activity of CD73 in a quantitative manner and compared the activity of well-characterized MSC-EV preparations containing or lacking mdMLR immunomodulatory activity.

      Results

      The authors showed that the ecto-5’-nucleotidase activity of the MSC-EV preparations did not correlate with their ability to modulate T-cell responses in the mdMLR assay and thus with their potency in improving disease symptomatology in the optimized mouse aGVHD model. Furthermore, the ecto-5’-nucleotidase activity was resistant to EV-destroying detergent treatment.

      Conclusions

      Ecto-5’-nucleotidase activity neither reflects the potency of the authors’ MSC-EV preparations nor provides any information about the integrity of the respective EVs. Thus, ecto-5’-nucleotidase enzyme activity is not indicative for the immunomodulatory potency of the authors’ MSC-EV products. The development of appropriate potency assays for MSC-EV products remains challenging.

      Graphical abstract

      Key Words

      Introduction

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      ,
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      Extracellular metabolism of ATP and other nucleotides.
      ,
      • Kerkela E.
      Adenosinergic Immunosuppression by Human Mesenchymal Stromal Cells Requires Co-Operation with T cells.
      ]. Since deficiency of CD73 enhances the severity of clinical aGVHD symptoms in mice [
      • Tsukamoto H.
      • Chernogorova P.
      • Ayata K.
      • Gerlach U.V.
      • Rughani A.
      • Ritchey J.W.
      • Ganesan J.
      • Follo M.
      • Zeiser R.
      • Thompson L.F.
      • Idzko M.
      Deficiency of CD73/ecto-5′-nucleotidase in mice enhances acute graft-versus-host disease.
      ], the authors decided to explore the utility of ecto-5’-nucleotidase activity in distinguishing MSC-EV preparations revealing or lacking immunomodulatory activity in the mdMLR assay. Accordingly, in this study, the authors adopted an enzymatic ecto-5’-nucleotidase activity assay quantifying free phosphate concentration and determined this activity in independent MSC-EV preparations.

      Methods

      Cultivation of MSCs

      Human MSCs were cultivated from the bone marrow of healthy donors after obtaining informed consent according to the Declaration of Helsinki as described previously [
      • Kordelas L.
      • Rebmann V.
      • Ludwig A.K.
      • Radtke S.
      • Ruesing J.
      • Doeppner T.R.
      • Epple M.
      • Horn P.A.
      • Beelen D.W.
      • Giebel B.
      MSC-derived exosomes: a novel tool to treat therapy-refractory graft-versus-host disease.
      ,
      • Radtke S.
      • Gorgens A.
      • Liu B.
      • Horn P.A.
      • Giebel B.
      Human mesenchymal and murine stromal cells support human lympho-myeloid progenitor expansion but not maintenance of multipotent haematopoietic stem and progenitor cells.
      ]. Briefly, MSCs were expanded in low-glucose Dulbecco's Modified Eagle's Medium (Lonza, Basel, Switzerland) supplemented with 10% human platelet lysate (hPL), 100 U/mL penicillin–streptomycin–L-glutamine (Thermo Fisher Scientific, Waltham, MA, USA) and 5 IU/mL heparin (Ratiopharm, Ulm, Germany). Medium exchange was performed when MSCs reached a confluency of 50%, after which media were collected every 48 h and centrifuged at 2000 × g for 15 min to remove cellular debris until MSC culture reached a confluency of about 80%. CMs were stored at –20°C until use. All processed CMs tested negative for mycoplasma. Cultivated MSCs were characterized by flow cytometry, and their osteogenic and adipogenic differentiation potential was confirmed in vitro in full accordance with the MSC identity criteria recently recommended by MSC-EV experts [
      • Witwer K.W.
      • Van Balkom B.W.M.
      • Bruno S.
      • Choo A.
      • Dominici M.
      • Gimona M.
      • Hill A.F.
      • De Kleijn D.
      • Koh M.
      • Lai R.C.
      • Mitsialis S.A.
      • Ortiz L.A.
      • Rohde E.
      • Asada T.
      • Toh W.S.
      • Weiss D.J.
      • Zheng L.
      • Giebel B.
      • Lim S.K.
      Defining mesenchymal stromal cell (MSC)-derived small extracellular vesicles for therapeutic applications.
      ].

      MSC-EV preparation with polyethylene glycol from MSC-CM

      EVs were prepared from the CM of MSCs by applying a polyethylene glycol (PEG) ultracentrifugation-based purification strategy as described previously [
      • Ludwig A.K.
      • De Miroschedji K.
      • Doeppner T.R.
      • Borger V.
      • Ruesing J.
      • Rebmann V.
      • Durst S.
      • Jansen S.
      • Bremer M.
      • Behrmann E.
      • Singer B.B.
      • Jastrow H.
      • Kuhlmann J.D.
      • El Magraoui F.
      • Meyer H.E.
      • Hermann D.M.
      • Opalka B.
      • Raunser S.
      • Epple M.
      • Horn P.A.
      • Giebel B.
      Precipitation with polyethylene glycol followed by washing and pelleting by ultracentrifugation enriches extracellular vesicles from tissue culture supernatants in small and large scales.
      ,
      • Borger V.
      • Staubach S.
      • Dittrich R.
      • Stambouli O.
      • Giebel B.
      Scaled Isolation of Mesenchymal Stem/Stromal Cell-Derived Extracellular Vesicles.
      ]. Briefly, MSC-CMs were thawed and centrifuged at 6800 × g for 45 min at 4°C in an Avanti J-26 XP centrifuge using a JS-5.3 swing-out rotor with a k-factor of 7728 (Beckman Coulter, Krefeld, Germany). This was followed by a 0.22-µm filtration step using a Nalgene bottle-top filter (Thermo Fisher Scientific). PEG 6000 (Sigma-Aldrich, Deisenhofen, Germany) and sodium chloride (NaCl) were added to clarified MSC-CM to a final concentration of 10% PEG 6000 (v/v) and 75 mM NaCl. Following incubation overnight at 4°C, EVs were pelleted in a JS-5.3 swing-out rotor in an Avanti J-26 XP centrifuge at 1500 × g for 30 min at 4°C (k-factor, 7728). Subsequently, pellets were resuspended, washed in 65 mL 0.9% NaCl and transferred into 70-mL polycarbonate centrifuge tubes (Beckman Coulter). EVs were reprecipitated at 110 000 × g for 130 min at 4°C in an XPN-80 ultracentrifuge using a Ti45 tight-angle rotor with a k-factor of 133 (Beckman Coulter). Obtained pellets were resuspended in 0.9% NaCl and 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES;Thermo Fisher Scientific) at 4 × 107 cell equivalents/mL, aliquoted into micro-centrifugation low-retention tubes (Kisker Biotech GmbH & Co KG, Steinfurt, Germany) and stored at –80°C until use.

      Nanoparticle tracking analysis

      MSC-EV preparations were characterized according to the updated Minimal Information for Studies of Extracellular Vesicle guidelines [
      • Thery C.
      • Witwer K.W.
      • Aikawa E.
      • Alcaraz M.J.
      • Anderson J.D.
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      • Antoniou A.
      • Arab T.
      • Archer F.
      • Atkin-Smith G.K.
      • Ayre D.C.
      • Bach J.M.
      • Bachurski D.
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      • Balaj L.
      • Baldacchino S.
      • Bauer N.N.
      • Baxter A.A.
      • Bebawy M.
      • Beckham C.
      • Zavec A.Bedina
      • Benmoussa A.
      • Berardi A.C.
      • Bergese P.
      • Bielska E.
      • Blenkiron C.
      • Bobis-Wozowicz S.
      • Boilard E.
      • Boireau W.
      • Bongiovanni A.
      • Borras F.E.
      • Bosch S.
      • Boulanger C.M.
      • Breakefield X.
      • Breglio A.M.
      • Brennan M.A.
      • Brigstock D.R.
      • Brisson A.
      • Broekman M.L.
      • Bromberg J.F.
      • Bryl-Gorecka P.
      • Buch S.
      • Buck A.H.
      • Burger D.
      • Busatto S.
      • Buschmann D.
      • Bussolati B.
      • Buzas E.I.
      • Byrd J.B.
      • Camussi G.
      • Carter D.R.
      • Caruso S.
      • Chamley L.W.
      • Chang Y.T.
      • Chen C.
      • Chen S.
      • Cheng L.
      • Chin A.R.
      • Clayton A.
      • Clerici S.P.
      • Cocks A.
      • Cocucci E.
      • Coffey R.J.
      • Cordeiro-da-Silva A.
      • Couch Y.
      • Coumans F.A.
      • Coyle B.
      • Crescitelli R.
      • Criado M.F.
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      • Das S.
      • Chaudhuri A.Datta
      • de Candia P.
      • De Santana E.F.
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      • Demaret T.
      • Deville S.
      • Devitt A.
      • Dhondt B.
      • Vizio D.Di
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      • Dolo V.
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      • Dominici M.
      • Dourado M.R.
      • Driedonks T.A.
      • Duarte F.V.
      • Duncan H.M.
      • Eichenberger R.M.
      • Ekstrom K.
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      • Erdbrugger U.
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      • Fatima F.
      • Fish J.E.
      • Flores-Bellver M.
      • Forsonits A.
      • Frelet-Barrand A.
      • Fricke F.
      • Fuhrmann G.
      • Gabrielsson S.
      • Gamez-Valero A.
      • Gardiner C.
      • Gartner K.
      • Gaudin R.
      • Gho Y.S.
      • Giebel B.
      • Gilbert C.
      • Gimona M.
      • Giusti I.
      • Goberdhan D.C.
      • Gorgens A.
      • Gorski S.M.
      • Greening D.W.
      • Gross J.C.
      • Gualerzi A.
      • Gupta G.N.
      • Gustafson D.
      • Handberg A.
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      • Harrison P.
      • Hegyesi H.
      • Hendrix A.
      • Hill A.F.
      • Hochberg F.H.
      • Hoffmann K.F.
      • Holder B.
      • Holthofer H.
      • Hosseinkhani B.
      • Hu G.
      • Huang Y.
      • Huber V.
      • Hunt S.
      • Ibrahim A.G.
      • Ikezu T.
      • Inal J.M.
      • Isin M.
      • Ivanova A.
      • Jackson H.K.
      • Jacobsen S.
      • Jay S.M.
      • Jayachandran M.
      • Jenster G.
      • Jiang L.
      • Johnson S.M.
      • Jones J.C.
      • Jong A.
      • Jovanovic-Talisman T.
      • Jung S.
      • Kalluri R.
      • Kano S.I.
      • Kaur S.
      • Kawamura Y.
      • Keller E.T.
      • Khamari D.
      • Khomyakova E.
      • Khvorova A.
      • Kierulf P.
      • Kim K.P.
      • Kislinger T.
      • Klingeborn M.
      • Klinke 2nd, D.J.
      • Kornek M.
      • Kosanovic M.M.
      • Kovacs A.F.
      • Kramer-Albers E.M.
      • Krasemann S.
      • Krause M.
      • Kurochkin I.V.
      • Kusuma G.D.
      • Kuypers S.
      • Laitinen S.
      • Langevin S.M.
      • Languino L.R.
      • Lannigan J.
      • Lasser C.
      • Laurent L.C.
      • Lavieu G.
      • Lazaro-Ibanez E.
      • Lay S.Le
      • Lee M.S.
      • Lee Y.X.F.
      • Lemos D.S.
      • Lenassi M.
      • Leszczynska A.
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      • Liao K.
      • Libregts S.F.
      • Ligeti E.
      • Lim R.
      • Lim S.K.
      • Line A.
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      • Llorente A.
      • Lombard C.A.
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      • Lorincz A.M.
      • Lotvall J.
      • Lovett J.
      • Lowry M.C.
      • Loyer X.
      • Lu Q.
      • Lukomska B.
      • Lunavat T.R.
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      • Marcilla A.
      • Mariani J.
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      • Martens-Uzunova E.S.
      • Martin-Jaular L.
      • Martinez M.C.
      • Martins V.R.
      • Mathieu M.
      • Mathivanan S.
      • Maugeri M.
      • McGinnis L.K.
      • McVey M.J.
      • Meckes Jr., D.G.
      • Meehan K.L.
      • Mertens I.
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      • Moller A.
      • Jorgensen M.Moller
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      • Morhayim J.
      • Mullier F.
      • Muraca M.
      • Musante L.
      • Mussack V.
      • Muth D.C.
      • Myburgh K.H.
      • Najrana T.
      • Nawaz M.
      • Nazarenko I.
      • Nejsum P.
      • Neri C.
      • Neri T.
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      • Nimrichter L.
      • Nolan J.P.
      • Nolte-'t Hoen E.N.
      • Hooten N.Noren
      • O'Driscoll L.
      • O'Grady T.
      • O'Loghlen A.
      • Ochiya T.
      • Olivier M.
      • Ortiz A.
      • Ortiz L.A.
      • Osteikoetxea X.
      • Ostergaard O.
      • Ostrowski M.
      • Park J.
      • Pegtel D.M.
      • Peinado H.
      • Perut F.
      • Pfaffl M.W.
      • Phinney D.G.
      • Pieters B.C.
      • Pink R.C.
      • Pisetsky D.S.
      • von Strandmann E.Pogge
      • Polakovicova I.
      • Poon I.K.
      • Powell B.H.
      • Prada I.
      • Pulliam L.
      • Quesenberry P.
      • Radeghieri A.
      • Raffai R.L.
      • Raimondo S.
      • Rak J.
      • Ramirez M.I.
      • Raposo G.
      • Rayyan M.S.
      • Regev-Rudzki N.
      • Ricklefs F.L.
      • Robbins P.D.
      • Roberts D.D.
      • Rodrigues S.C.
      • Rohde E.
      • Rome S.
      • Rouschop K.M.
      • Rughetti A.
      • Russell A.E.
      • Saa P.
      • Sahoo S.
      • Salas-Huenuleo E.
      • Sanchez C.
      • Saugstad J.A.
      • Saul M.J.
      • Schiffelers R.M.
      • Schneider R.
      • Schoyen T.H.
      • Scott A.
      • Shahaj E.
      • Sharma S.
      • Shatnyeva O.
      • Shekari F.
      • Shelke G.V.
      • Shetty A.K.
      • Shiba K.
      • Siljander P.R.
      • Silva A.M.
      • Skowronek A.
      • Snyder 2nd, O.L.
      • Soares R.P.
      • Sodar B.W.
      • Soekmadji C.
      • Sotillo J.
      • Stahl P.D.
      • Stoorvogel W.
      • Stott S.L.
      • Strasser E.F.
      • Swift S.
      • Tahara H.
      • Tewari M.
      • Timms K.
      • Tiwari S.
      • Tixeira R.
      • Tkach M.
      • Toh W.S.
      • Tomasini R.
      • Torrecilhas A.C.
      • Tosar J.P.
      • Toxavidis V.
      • Urbanelli L.
      • Vader P.
      • van Balkom B.W.
      • van der Grein S.G.
      • Van Deun J.
      • van Herwijnen M.J.
      • Van Keuren-Jensen K.
      • van Niel G.
      • van Royen M.E.
      • van Wijnen A.J.
      • Vasconcelos M.H.
      • Vechetti Jr., I.J.
      • Veit T.D.
      • Vella L.J.
      • Velot E.
      • Verweij F.J.
      • Vestad B.
      • Vinas J.L.
      • Visnovitz T.
      • Vukman K.V.
      • Wahlgren J.
      • Watson D.C.
      • Wauben M.H.
      • Weaver A.
      • Webber J.P.
      • Weber V.
      • Wehman A.M.
      • Weiss D.J.
      • Welsh J.A.
      • Wendt S.
      • Wheelock A.M.
      • Wiener Z.
      • Witte L.
      • Wolfram J.
      • Xagorari A.
      • Xander P.
      • Xu J.
      • Yan X.
      • Yanez-Mo M.
      • Yin H.
      • Yuana Y.
      • Zappulli V.
      • Zarubova J.
      • Zekas V.
      • Zhang J.Y.
      • Zhao Z.
      • Zheng L.
      • Zheutlin A.R.
      • Zickler A.M.
      • Zimmermann P.
      • Zivkovic A.M.
      • Zocco D.
      • Zuba-Surma E.K.
      Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines.
      ]. Nanoparticle tracking analyses were performed on a ZetaView (Particle Metrix GmbH, Meerbusch, Germany) for particle quantification and average size estimation. Polystyrene beads measuring 100 nm (Thermo Fisher Scientific) were used for calibration. Videos were recorded at all 11 positions with five repetitions. Positions two and 10 were excluded for the final result. The machine was set up as follows: sensitivity 75, shutter 75, minimum brightness 20, minimum size 5 and maximum size 200 (see supplementary Table 1).

      Protein content of MSC-EV preparations

      The protein content of the MSC-EV preparations was determined using a bicinchoninic acid protein assay kit (Thermo Fisher Scientific) according to the manufacturer's instructions (see supplementary Table 1).

      Western blot

      Western blot was performed as described previously [
      • Ludwig A.K.
      • De Miroschedji K.
      • Doeppner T.R.
      • Borger V.
      • Ruesing J.
      • Rebmann V.
      • Durst S.
      • Jansen S.
      • Bremer M.
      • Behrmann E.
      • Singer B.B.
      • Jastrow H.
      • Kuhlmann J.D.
      • El Magraoui F.
      • Meyer H.E.
      • Hermann D.M.
      • Opalka B.
      • Raunser S.
      • Epple M.
      • Horn P.A.
      • Giebel B.
      Precipitation with polyethylene glycol followed by washing and pelleting by ultracentrifugation enriches extracellular vesicles from tissue culture supernatants in small and large scales.
      ]. Briefly, MSC-EV preparations (25 µg) were solubilized and denatured using Laemmli sample buffer (4×) containing dithiothreitol (AppliChem GmbH, Darmstadt, Germany) and separated by 12% one-dimensional sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Proteins were plotted on polyvinylidene fluoride membranes (MilliporeSigma, Darmstadt, Germany), and membranes were blocked with Tris-buffered saline and 0.1% Tween 20 containing 5% (w/v) skim milk powder (Sigma-Aldrich). For the detection of exosomal marker proteins, the following antibodies were used: anti-Syntenin (clone EPR8102; Abcam, Cambridge, UK), anti-CD81 (clone JS-81; BD Biosciences, San Jose, CA, USA), anti-CD9 (clone VJ1/20.3.1; kindly provided by Francisco Sánchez, Madrid, Spain) and anti-CD63 (clone H5C6; BioLegend, San Diego, CA, USA). Anti-calnexin (clone ab10286; Abcam) antibodies were used for the detection of contaminants. Bound antibodies were counter labeled with horseradish peroxidase-conjugated secondary antibodies (Dianova, Hamburg, Germany). SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, Darmstadt, Germany) was used for signal detection. Protein bands were visualized using a Fusion FX7 chemiluminescence detection system (Vilber Lourmat Deutschland GmbH, Eberhardzell, Germany). EM-MENU 4.09.83 image acquisition software was used to take 16-bit images. Image post-processing was carried out using ImageJ 1.52b software (National Institutes of Health, Bethesda, MD, USA).

      Imaging flow cytometry

      Unless otherwise mentioned, staining of MSC-EV preparations was performed as described previously [
      • Tertel T.
      • Gorgens A.
      • Giebel B.
      Analysis of individual extracellular vesicles by imaging flow cytometry.
      ,
      • Tertel T.
      • Bremer M.
      • Maire C.
      • Lamszus K.
      • Peine S.
      • Jawad R.
      • Andaloussi S.E.L.
      • Giebel B.
      • Ricklefs F.L.
      • Gorgens A.
      High-Resolution Imaging Flow Cytometry Reveals Impact of Incubation Temperature on Labeling of Extracellular Vesicles with Antibodies.
      ]. Briefly, 5 µL PEG-prepared MSC-EV samples were labeled with 0.5 µL allophycocyanin (APC)-conjugated mouse anti-human CD63 (clone MEM-259; EXBIO Praha, a.s., Vestec, Czech Republic) or mouse IgG1 isotype (clone MOPC-21; BioLegend) for the detergent experiments. Additionally, for further characterization, 5 µL PEG-prepared MSC-EV samples were labeled with phycoerythrin (PE)-conjugated mouse anti-human CD9 (clone MEM-61; EXBIO Praha, a.s.) or fluorescein isothiocyanate (FITC)-conjugated mouse anti-human CD81 (clone JS-84; Beckman Coulter). Unstained samples and buffer controls without MSC-EVs but with antibodies were used as controls. All samples were incubated for 1 h in the dark at room temperature and diluted 100-fold for anti-CD9 and 40-fold for anti-CD63 or anti-CD81 with pH 7.4 phosphate-buffered saline (PBS, Thermo Fisher Scientific) before analysis.
      Next, without further washing, samples were analyzed with an Amnis ImageStreamXMk II instrument (Luminex Corporation, Seattle, WA, USA). Different concentrations of non-ionic detergents Tween 20 and NP-40 (0.1%, 0.25%, 0.5% and 1%) were used for evaluation of the EV characteristics of recorded objects. All samples, which were applied in U-bottom 96-well Falcon plates (Corning GmbH, Kaiserslautern, Germany), were analyzed in triplicate with 5-min acquisition time per well. All data were acquired at 60× magnification at low flow rate and with the removed beads option deactivated. More details are provided in supplementary Tables 2 and 3. Data analysis was performed exactly as described before using IDEAS 6.2 software (Luminex Corporation) [
      • Tertel T.
      • Gorgens A.
      • Giebel B.
      Analysis of individual extracellular vesicles by imaging flow cytometry.
      ,
      • Tertel T.
      • Bremer M.
      • Maire C.
      • Lamszus K.
      • Peine S.
      • Jawad R.
      • Andaloussi S.E.L.
      • Giebel B.
      • Ricklefs F.L.
      • Gorgens A.
      High-Resolution Imaging Flow Cytometry Reveals Impact of Incubation Temperature on Labeling of Extracellular Vesicles with Antibodies.
      ]. All fluorescent events were plotted against the side scatter. A combined mask feature (MC and NMC) was used to improve the detection of fluorescent images. Images were analyzed for coincidences (swarm detection) using the spot counting feature; any event with multiple spots was excluded. All remaining events with low side scatter (<500) and a fluorescent intensity higher than 300 were used for calculation of concentrations. Data were visualized with Prism 8.4.2 software (GraphPad Software, San Diego, CA, USA).

      Multi-donor mixed lymphocyte reaction

      To evaluate in vitro the immunomodulatory activities of prepared MSC-EV samples, the authors performed mdMLR assays as described previously [
      • Madel R.J.
      • Börger V.
      • Dittrich R.
      • Bremer M.
      • Tertel T.
      • Ngo Thi Phuong N.
      • Babo H.A.
      • Kordelas L.
      • Buer J.
      • Horn P.A.
      • Westendorf A.M.
      • Brandau S.
      • Kirschning C.J.
      • Giebel B.
      Independent human mesenchymal stromal cell-derived extracellular vesicle preparations differentially affect symptoms in an advanced murine Graft-versus-Host-Disease model.
      ]. Briefly, peripheral blood MNCs (PBMCs) from 12 different donors were isolated by conventional Ficoll density gradient centrifugation and pooled using equivalent cell numbers from each donor and aliquoted. The aliquots of PBMC pools were stored in the vapor phase of liquid nitrogen until use. Upon thawing, PBMCs were cultured in Roswell Park Memorial Institute 1640 medium (Thermo Fisher Scientific) supplemented with 10% human AB serum (produced in-house), 100 U/mL penicillin and 100 µg/mL streptomycin (Thermo Fisher Scientific). A total of 6 × 105 cells in a final volume of 200 µL per well were cultured at 37°C and 5% carbon dioxide in the presence and absence of MSC-EV preparations in 96-well U-bottom Falcon plates. PBMCs cultured in the absence of MSC-EVs served as the stimulated control. For functional testing, 25 µg of given MSC-EV preparations was applied to the respective wells. After 5 days of culture, cells were harvested and stained with a cocktail of fluorescently labeled antibodies: anti-CD4-BV785 (clone RPA-T4, 300554; BioLegend), anti-CD25-PE (clone BC-96, 12-0259-42; Thermo Fischer Scientific) and anti-CD54-AF700 (clone 1H4, A7-429-T100; EXBIO Praha, a.s.). The authors used 7-aminoactinomycin D for the identification of dead cells. Data acquisition was performed on a CytoFLEX flow cytometer with CytExpert 2.3 software (Beckman Coulter). Obtained data were analyzed with Kaluza Analysis 2.1 software (Beckman Coulter). Flow cytometry data are shown as total cell counts.

      CD73 assay

      A Malachite Green colorimetric assay kit (Sigma-Aldrich) was applied to quantify the production of inorganic phosphate from 5’ AMP substrate according to the manufacturer's instructions. Briefly, MSC-EV preparations (25 µg) diluted in NaCl and 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid were added to 5′ AMP (50 µM) diluted in the same buffer. Following incubation for 1 h at 37°C, the colorimetric reagent was added. The absorbance was measured after 30 min at 620 nm on a PowerWave XS microplate reader (BioTek Instruments, Winooski, VT, USA). Processed AMP amounts were calculated by comparing the absorbance values with a pre-recorded standard curve. As a control, the CD73 inhibitor α,β-methyleneadenosine 5′-diphosphate (APCP) (Sigma-Aldrich) was added to selected samples to a final concentration of 10 µM. To study the requirements for EV integrity, the authors treated selected samples with different concentrations of non-ionic detergents Tween 20 and NP-40 (0.1%, 0.25%, 0.5% and 1%).

      Statistical analysis

      Data are presented as mean ± standard deviation. After testing for Gaussian distribution, continuous variables were compared using an unpaired t-test. Statistical significance was defined as P ≤ 0.05. Statistical analyses were performed using Prism 8.4.2 (GraphPad Software).

      Results

      Immunomodulatory properties differ among independent MSC-EV preparations

      With the aim of exploring the utility of enzymatic CD73 assays for the prediction of functional properties of independent MSC-EV preparations, the authors selected a collection of eight independent MSC-EV preparations that showed or lacked immunomodulatory activities in the mdMLR assay in the past. Since in addition to inter-donor variability the authors also observed, to various degrees, batch-to-batch activity variations for MSC-EV products produced from aliquots of the same donor MSC stocks, the authors selected different MSC-EV batches of the two most frequently used donor MSC stocks (i.e., MSC16.3 and MSC41.5). Notably, although most MSC16.3-EV preparations lack immunomodulatory in vitro activities, most MSC41.5-EVs contain them.
      The authors selected the two atypical MSC16.3-EV preparations that appeared active in mdMLR (16.3-EVa1, 16.3-EVa2) and the two atypical MSC41.5-EV preparations that appeared inactive (41.5-EVi1, 41.5-EVi2). A representative inactive MSC16.3-EV preparation (16.3-EVi) and a representative active MSC41.5-EV preparation (41.5-EVa) were also selected in addition to an active and inactive MSC-EV preparation from other donors (70.5-EVa, 84-EVi).
      To exclude the possibility that functional properties had changed during storage of the MSC-EV samples and to have appropriate references, the authors at first retested the immunomodulatory activity of all eight samples in the mdMLR assay. Compared with the stimulated MNCs that were cultured in the absence of MSC-EVs (control), four of the eight MSC-EV preparations did not modulate the content of activated CD4 T cells (i.e., CD54+CD25+CD4+ cells), whereas the other four MSC-EV preparations significantly reduced CD54+CD25+T-cell numbers. Confirming previous evaluations, the authors qualified the MSC-EVs 16.3-EVa1, 16.3-EVa2, 41.5-EVa and 70.5-EVa as MSC-EV preparations with mdMLR immunomodulatory activities (MSC-EVa) and the MSC-EV 16.3-EVi, 41.5-EVi1, 41.5-EVi2 and 84-EVi as MSC-EV preparations without mdMLR immunomodulatory activities (MSC-EVi) (Figure 1).
      Fig 1
      Fig. 1MSC-EV preparations differ regarding their immunomodulatory activity. The immunomodulatory activity of different MSC-EV preparations was evaluated in the mdMLR assay. Within a culture period of 5 days, in comparison with control cultures, MSC-EV preparations considered to be immunomodulatory active reduced the frequency of activated CD4+ T cells that were identified as CD25+CD54+ cells. By contrast, MSC-EV preparations judged as immunomodulatory inactive did not considerably influence the content of activated CD4+ T cells in comparison with the respective control cultures. (A) MNCs were gated according to their light scatter features. Among single, non-7-AAD-labeled cells, CD4 T cells were identified as CD4+ cells. (B) Frequencies of living activated CD4 T cells (CD25+CD54+) cultured for 5 days in the absence (Stim) or presence of MSC-EV preparations derived from different MSC cell stocks (16.3-EVa1, 16.3-EVa2, 16.3-EVi, 41.5-Eva, 41.5-EVi1, 41.5-EVi2, 70.5-EVa and 84-EVi). Each row represents mdMLR assays that were performed in parallel. FSC-A, forward scatter area; 7-AAD, 7-aminoactinomycin D; SSC-A, side scatter area; Stim, stimulated.

      Immunomodulatory activities of MSC-EV preparations are not reflected by metric EV data

      To compare the mdMLR data with metric features of the given MSC-EV preparations accurately, the authors again performed western blot and imaging flow cytometry analyses of all MSC-EV samples used in this study in parallel. 25-µg of given MSC-EV samples and as a control of the respective hPL-EV samples that had been prepared from fresh MSC media (10% pooled hPL-supplemented Dulbecco's Modified Eagle's Medium), the authors observed variabilities in the band intensities of all EV markers tested (CD9, CD63, CD81 and Syntenin) (Figure 2A). Notably, as before, the authors detected lower CD81 and higher CD9 levels in the hPL-EV control than the MSC-EV samples [
      • Madel R.J.
      • Börger V.
      • Dittrich R.
      • Bremer M.
      • Tertel T.
      • Ngo Thi Phuong N.
      • Babo H.A.
      • Kordelas L.
      • Buer J.
      • Horn P.A.
      • Westendorf A.M.
      • Brandau S.
      • Kirschning C.J.
      • Giebel B.
      Independent human mesenchymal stromal cell-derived extracellular vesicle preparations differentially affect symptoms in an advanced murine Graft-versus-Host-Disease model.
      ]. Calnexin, which was used as an impurity marker, was not detected in any of the EV samples but was detected in a control sample containing material of lysed MSCs (see supplementary Figure 1). For analysis, the authors quantified the intensity of the western blot bands and compared their intensities with the immunomodulating activities of MSC-EVs. However, the authors did not find any correlation (Figure 2B). Next, the authors analyzed the composition of CD9+, CD63+ and CD81+ objects in each of the given MSC-EV samples with imaging flow cytometry (Figure 2C; also see supplementary Figure 2). Consistent with the results of western blot, the abundance of CD9+, CD63+ and CD81+ object populations did not correlate with the mdMLR modulatory properties of the investigated MSC-EV preparations (Figure 2D). Thus, as in the authors’ previous study [
      • Madel R.J.
      • Börger V.
      • Dittrich R.
      • Bremer M.
      • Tertel T.
      • Ngo Thi Phuong N.
      • Babo H.A.
      • Kordelas L.
      • Buer J.
      • Horn P.A.
      • Westendorf A.M.
      • Brandau S.
      • Kirschning C.J.
      • Giebel B.
      Independent human mesenchymal stromal cell-derived extracellular vesicle preparations differentially affect symptoms in an advanced murine Graft-versus-Host-Disease model.
      ], obtained metric EV data did not correlate with the explored immunomodulatory activity of the given MSC-EV preparations.
      Fig 2
      Fig. 2Western blot and IFCM analyses reveal no correlation of EV marker abundancies with mdMLR results. Western blot and IFCM analyses of all samples were performed in parallel. For western blot, EV samples prepared from fresh hPL-supplemented cell culture media (hPL-EVs) and lysed MSCs were used as controls. (A) Western blot bands following sequential anti-CD81, anti-CD9 and anti-CD63 staining as well as anti-Syntenin staining (images of whole blots are presented in supplementary Figure 1). (B) Correlation analysis performed with unpaired t-test for western blot band intensities as calculated by ImageJ and immunomodulatory properties of active (MSC-EVa) and inactive (MSC-EVi) MSC-EV samples. (C) Abundance of CD9+, CD63+ and CD81+ objects in the different MSC-EV samples as quantified by IFCM. The applied gating strategy and examples for the analyses are provided in supplementary Figure 2. The concentration of recorded objects is provided as object/mL ± standard deviation. (D) Correlation analysis performed with unpaired t-test for obtained IFCM data and immunomodulatory properties of active (MSC-EVa) and inactive (MSC-EVi) MSC-EV samples. IFCM, imaging flow cytometry.
      The p values for the western blots are: CD81 p=0.551; CD9 p=0.976; CD63 p=0.964.
      The p values for the IFCM analyses in D are: CD9 p=0.383; CD63 p=0.944; CD81 P=0.884.

      MSC-EV preparations possess ecto-5’-nucleotidase activity

      The authors performed dose-finding experiments to explore ecto-5’-nucleotidase concentrations in mdMLR immunomodulatory and non-immunomodulatory MSC-EV preparations. To this end, the authors applied the MSC-EV preparations 16.3-EVa1, 16.3-EVi and 41.5-EVi2 in different concentrations to monitor CD73 enzyme activity. For simplicity, doses were defined according to the protein content of the MSC-EV preparations. In total, six different doses, ranging from 5 µg to 100 µg per sample, were applied in duplicate in three independent experiments. To monitor the impact of the buffer, 5′ AMP and the CD73 inhibitor APCP were applied as controls separately or together with the respective MSC-EV preparations. Both active and inactive MSC preparations showed a dose-dependent increase in free phosphate production that correlated with the amount of MSC-EV preparation that was applied (Figure 3A). As expected, in the presence of APCP, EV samples did not show any ecto-5’-nucleotidase activity.
      Fig 3
      Fig. 3Ecto-5’-nucleotidase activity is of MSC origin and can be analyzed quantitatively. For quantification of ecto-5’-nucleotidase activity within MSC-EV preparations, a Malachite Green colorimetric assay was used. This assay is based on the quantification of inorganic phosphate that is cleaved by the ecto-5’-nucleotidase activity of provided samples from a given AMP substrate. (A) MSC-EV preparation samples were applied according to their protein content (5–100 µg). A couple of controls were provided, containing different combinations of sample buffer (Buffer), 25 µg of given MSC-EV preparations, the ecto-5’-nucleotidase inhibitor APCP and/or the AMP substrate. (B) As additional controls, 25 µg of each of three independent hPL-EV preparations was tested for ecto-5’-nucleotidase activity. Data were obtained from three independent experiments performed in duplicates. Results are presented as mean ± standard deviation of duplicates.
      Since the MSC-EV preparations contained human platelet-derived EVs [
      • Tertel T.
      • Bremer M.
      • Maire C.
      • Lamszus K.
      • Peine S.
      • Jawad R.
      • Andaloussi S.E.L.
      • Giebel B.
      • Ricklefs F.L.
      • Gorgens A.
      High-Resolution Imaging Flow Cytometry Reveals Impact of Incubation Temperature on Labeling of Extracellular Vesicles with Antibodies.
      ,
      • Gorgens A.
      • Bremer M.
      • Ferrer-Tur R.
      • Murke F.
      • Tertel T.
      • Horn P.A.
      • Thalmann S.
      • Welsh J.A.
      • Probst C.
      • Guerin C.
      • Boulanger C.M.
      • Jones J.C.
      • Hanenberg H.
      • Erdbrugger U.
      • Lannigan J.
      • Ricklefs F.L.
      • El-Andaloussi S.
      • Giebel B.
      Optimisation of imaging flow cytometry for the analysis of single extracellular vesicles by using fluorescence-tagged vesicles as biological reference material.
      ,
      • Wiklander O.P.B.
      • Bostancioglu R.B.
      • Welsh J.A.
      • Zickler A.M.
      • Murke F.
      • Corso G.
      • Felldin U.
      • Hagey D.W.
      • Evertsson B.
      • Liang X.M.
      • Gustafsson M.O.
      • Mohammad D.K.
      • Wiek C.
      • Hanenberg H.
      • Bremer M.
      • Gupta D.
      • Bjornstedt M.
      • Giebel B.
      • Nordin J.Z.
      • Jones J.C.
      • El Andaloussi S.
      • Gorgens A.
      Systematic Methodological Evaluation of a Multiplex Bead-Based Flow Cytometry Assay for Detection of Extracellular Vesicle Surface Signatures.
      ], the authors also tested the ecto-5’-nucleotidase activity of EV preparations obtained from hPL-supplemented fresh MSC medium. However, no ecto-5’-nucleotidase activity was detected in any of the three independently prepared hPL-EV samples (hPLa, hPLb and hPLc) (Figure 3B). Thus, the results demonstrated that ecto-5’-nucleotidase activity is of MSC origin.

      Ecto-5’-nucleotidase activity of MSC-EV preparations is donor-related and does not correlate with recorded mdMLR immunomodulatory activity

      To evaluate whether ecto-5’-nucleotidase concentration was comparable among independent MSC-EV preparations, the authors analyzed the free phosphate production of all selected MSC-EV preparations in duplicates in parallel, as before, the authors again applied 25 µg of each MSC-EV preparation. Remarkably, independent of their immunomodulatory activities within the mdMLR assay, all 16.3 and all 41.5 MSC-EV samples displayed comparable ecto-5’-nucleotidase activities. Of note, the ecto-5’-nucleotidase activities within the 41.5 MSC-EV samples were significantly higher than those in the 16.3 MSC-EV samples (P = 0.001) (Figure 4A,B). Notably, there was no correlation between the mdMLR immunomodulatory activity and the recorded ecto-5’-nucleotidase activity (P = 0.696) (Figure 4C). Thus, the authors concluded that ecto-5’-nucleotidase activity was independent of the MSC-EV preparation immunomodulatory activity measured in the mdMLR assay.
      Fig 4
      Fig. 4Ecto-5’-nucleotidase activity is related to the donor material rather than the MSC-EV preparation's ability to modulate allogeneic immune reactions within the mdMLR assay. (A) For the ecto-5’-nucleotidase assay, 25 µg of each of the eight different MSC-EV preparations was applied in duplicate. The recorded ecto-5’-nucleotidase activity is depicted in relation to the (A) different samples, (B) MSC donor and (C) MSC-EV preparation's ability to modulate allogeneic immune responses within the mdMLR assay. (B,C) Each data point represents the mean of the duplicate. Data are presented as mean ± standard deviation. Unpaired t-test was used for statistical analysis. ns, not significant (P = 0.696).

      EV integrity is not required for ecto-5’-nucleotidase activity in MSC-EV preparations

      Next, the authors investigated whether ecto-5’-nucleotidase activity depended on the integrity of the EVs within the MSC-EV preparation samples. Since EVs are detergent-sensitive, 25 µg aliquots of the MSC-EV preparation 16.3i were treated with different concentrations of the non-ionic detergent Tween 20 or NP-40 (0.1%, 0.25%, 0.5% and 1%). The impact of the detergent on the EV samples was documented via anti-CD63 antibody labeling by imaging flow cytometry. Compared with untreated control samples, fewer CD63+ objects were recorded after treatment with 0.1% Tween 20 or 0.1% NP-40. After applying NP-40 at a final concentration of 0.25% or greater, almost no CD63+ objects were detected; however, a huge proportion of CD63+ objects were Tween 20-resistant (Figure 5A). These results demonstrated that CD63+ EVs in the authors’ MSC-EV preparations were NP-40-sensitive. In addition, the authors determined the ecto-5’-nucleotidase activity of detergent-treated samples. Independent of the detergent concentration, no significant reduction in recorded ecto-5’-nucleotidase activity was observed among detergent-treated and untreated MSC-EV samples (Figure 5B). Overall, in the authors’ MSC-EV preparations, the results demonstrated that EV integrity was not required for ecto-5’-nucleotidase activity.
      Fig 5
      Fig. 5Ecto-5’-nucleotidase activity in MSC-EV preparations is detergent-resistant. MSC-EV 16.3i was treated with different concentrations of the detergent Tween 20 or NP-40 (0.1%, 0.25%, 0.5% and 1%). (A) Ratio of detectable CD63+ objects in detergent-treated MSC-EV preparation samples compared with that of untreated control samples. (B) Recorded ecto-5’-nucleotidase activity of detergent-treated MSC-EV preparation samples. Data are presented as mean ± standard deviation of duplicates.

      Discussion

      Here the authors explored whether the ecto-5’-nucleotidase activity of given MSC-EV preparations reflected their immunomodulatory ability to suppress T-cell activation in an allogeneic setting, especially in the mdMLR assay. Independent of recorded immunomodulatory activity, the authors demonstrated that independent MSC-EV batches produced from CMs of MSCs cultivated from the same donor cell stocks contain comparable ecto-5’-nucleotidase activity and that this activity differs between MSC-EV preparations of different donors. Furthermore, the authors demonstrated that EV-destroying detergent treatment does not reduce the ecto-5’-nucleotidase activity of MSC-EV samples. Thus, ecto-5’-nucleotidase activity provides no information about the immunomodulatory activity of given MSC-EV samples, as measured by the mdMLR assay, or the quality of EVs, especially with regard to their integrity. Consequently, the authors conclude that although this assay is very precise and reproducible, it does not allow functional discrimination of MSC-EV samples containing or lacking immunomodulatory activity.
      Since the predictions obtained from the mdMLR assay correlate well with the ability of MSC-EV preparations to suppress aGVHD symptoms in aGVHD mice, the authors conclude that the CD73 activity assay is also not appropriate for discriminating MSC-EV samples containing or lacking the ability to suppress aGVHD symptoms.
      The fact that MSC-EV samples lacking the ability to suppress aGVHD symptoms contain ecto-5’-nucleotidase demonstrates that this activity is not sufficient for improving aGVHD. As discussed more recently, the authors hypothesize that MSC-EV preparations act in multifaceted ways and that, depending on disease-specific MoA combinations, attributes need to act together to suppress a certain disease [
      • Gimona M.
      • Brizzi M.F.
      • Choo A.B.H.
      • Dominici M.
      • Davidson S.M.
      • Grillari J.
      • Hermann D.M.
      • Hill A.F.
      • de Kleijn D.
      • Lai R.C.
      • Lai C.P.
      • Lim R.
      • Monguio-Tortajada M.
      • Muraca M.
      • Ochiya T.
      • Ortiz L.A.
      • Toh W.S.
      • Yi Y.W.
      • Witwer K.W.
      • Giebel B.
      • Lim S.K.
      Critical considerations for the development of potency tests for therapeutic applications of mesenchymal stromal cell-derived small extracellular vesicles.
      ]. Thus, even if the authors’ results clearly demonstrate that ecto-5’-nucleotidase itself is not sufficient to suppress aGVHD, it remains an open question as to whether CD73 activity contributes to MSC-EV anti-GVHD activity. The observation that CD73 deficiency enhances the severity of aGVHD symptoms in mice [
      • Tsukamoto H.
      • Chernogorova P.
      • Ayata K.
      • Gerlach U.V.
      • Rughani A.
      • Ritchey J.W.
      • Ganesan J.
      • Follo M.
      • Zeiser R.
      • Thompson L.F.
      • Idzko M.
      Deficiency of CD73/ecto-5′-nucleotidase in mice enhances acute graft-versus-host disease.
      ] argues for its involvement in controlling aGVHD. To understand whether CD73 contributes to the pre-clinical effect, CD73 activity needs to be specifically blocked or depleted in MSC-EVs. This could be achieved by, for example, CRISPR-Cas9-mediated knockout strategies as long as the loss of CD73 gene function does not significantly affect other biological properties of engineered MSCs. However, even though it will be interesting to learn about the role of CD73 activity in MSC-EV MoA, the authors’ MSC-EV preparations cannot be functionally discriminated by this assay. Therefore, there must be another component that determines whether the authors’ MSC-EV preparations can modulate the allogeneic immune reaction in vitro or in vivo. Although the authors compared the particle and EV abundance of selected EV markers in this study, it was impossible to identify any surrogate that allowed for discrimination of immunomodulatory active and inactive MSC-EV preparations. Notably, in the context of their aGVHD study [
      • Madel R.J.
      • Börger V.
      • Dittrich R.
      • Bremer M.
      • Tertel T.
      • Ngo Thi Phuong N.
      • Babo H.A.
      • Kordelas L.
      • Buer J.
      • Horn P.A.
      • Westendorf A.M.
      • Brandau S.
      • Kirschning C.J.
      • Giebel B.
      Independent human mesenchymal stromal cell-derived extracellular vesicle preparations differentially affect symptoms in an advanced murine Graft-versus-Host-Disease model.
      ], the authors compared potent and non-potent MSC-EV preparations at the proteomic and microRNA level and failed to identify any concrete surrogate marker. Thus, at the molecular level, immunomodulatory active and inactive MSC-EV samples are very similar, especially as presented by means of their ecto-5’-nucleotidase activity when they are derived from the same donor MSC stocks.

      Conclusions

      One of the challenges of the next few years is to identify differences among potent and non-potent MSC-EV samples. Thus, even if the authors cannot provide any explanation for the different activities of their different MSC-EV samples, the data underscore the need to set up reliable assays that can monitor the critical therapeutic activity of given MSC-EV products. Although the mdMLR assay may not become qualified as a potency assay as a result of its use of primary human cells, it is definitely a valuable surrogate for a number of animal experiments. In the future, the mdMLR assay may help to identify other assays that will provide comparable functional predictions and that, because of their characteristics, will be more easily qualified as potency assays.
      In the meantime, as exemplified here, appropriate potency testing remains one of the major hurdles in translating MSC-EV products to the clinic.

      Funding

      This study was supported by the LeitmarktAgentur.NRW and the European Union (European Regional Development Fund 2014-2020, EFRE-0800396), Stem Cell Network North Rhine-Westphalia and the European Union (ERA-NET EuroTransbio 11: EVTrust [031B0332B]).

      Author Contributions

      Conception and design of the study: FNB, TT and BG. Acquisition of data: FNB, TT, OS, CW, RD, and SS. Analysis and interpretation of data: all authors. Drafting or revising the manuscript: FNB and BG. All authors have approved the final article.

      Declaration of Competing Interest

      BG is a scientific advisory board member of Innovex Therapeutics SL and Mursla Ltd, a consultant of Fujifilm and a founding director of Exosla Ltd.

      Acknowledgments

      The authors thank the Westdeutsche Spender Zentrale for providing bone marrow samples of healthy donors. The authors are also grateful to all healthy blood donors whose cells were used in the MLR assay.

      Appendix. Supplementary materials

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