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MSC-derived exosomes: a novel tool to treat therapy-refractory graft-versus-host disease

Leukemia volume 28, pages 970–973 (2014)Cite this article

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Approximately 35–50% of the patients receiving matched related or unrelated allogeneic stem cell transplantation, develop severe forms of graft-versus-host disease (GvHD; Grade II–IV) that cannot be controlled with corticosteroids in up to 50% of the GvHD patients.1 Owing to the lack of confirmed treatment options Le Blanc et al.2 introduced mesenchymal stem cells (MSCs) as a strategy to treat severe therapy-refractory acute GvHD. Since then, a number of different studies have addressed the impact of MSC administration on GvHD with different outcomes.3 New data suggest that the beneficial effects of MSCs rather derive from secreted, immune response-modulating factors than from their tissue intercalation themselves.3 On the basis of a preclinical myocardial infarction model, evidence was provided that the immune-modulating factors of MSCs are also secreted ex vivo and reside in supernatant fractions that are highly enriched for small extracellular vesicles (EVs) such as exosomes4 and presumably small microvesicles (MVs) that bud from the plasma membrane. Exosomes are naturally occurring EVs (70–140 nm) that are released by exocytosis upon fusion of the so-called multivesicular bodies with the plasma membrane. Containing lipids, proteins and RNA, exosomes seem to participate in the intercellular communication processes. Together with the other EVs, exosomes are released from many cell types and can be enriched from virtually all body fluids including blood plasma, urine and saliva.5 Depending on their origin, some exosomes exert immune stimulatory or immune suppressive functions.5, 6 Considering MSC exosomes as the therapeutically active component of MSCs, the application of exosomes provides a number of advantages compared with the MSC application itself. Exosomes are non-self-replicating and owing to their small size can be sterilized by filtration. Thus, the regulatory items to produce exosome fractions for clinical treatment strategies should be less complicated than for any cellular therapeutic of in vitro expanded cells. Furthermore, it might be possible to harvest exosomes from supernatants of immortalized MSC cell lines, which otherwise could not be used for cellular therapies. Given the history of a therapy-refractory GvHD patient (Supplementary Figure S1), we decided to treat the patient in an individual treatment attempt with an exosome-enriched fraction processed from the collected MSC supernatants instead of administering the MSCs themselves.

According to reported difference in the outcome of MSC therapies in GvHD patients,3 we assumed that the exosome preparations from different MSCs might differ in their immune-modulating activities. Hence, MSCs of four different unrelated bone marrow donors were raised initially. The MSC nature of expanded cells was confirmed by flow cytometry and in differentiation assays according to the standards (data not shown). MSC-conditioned media were harvested every 48 h. To sterilize the supernatants and to remove larger EVs the conditioned media were passed through 0.22 μm filter membranes. As small EVs and viruses share a variety of features,7 and viruses can be purified by polyethylenglycol (PEG) precipitation,8, 9 we set up a PEG-based protocol to enrich for exosomes and potentially other EVs (Ludwig et al., in preparation). We used PEG6000 (10% final concentration) to enrich for the EV fractions of the sterilized supernatants. To remove residual polyethylene glycol and soluble proteins that might have been coprecipitated, pellets were washed with 0.9% NaCl and reprecipitated at 100.000 g for 2 h. Obtained pellets were dissolved in 0.9% NaCl to a final concentration of 1 unit EVs per 1 ml and stored as 1 ml aliquots at −80 °C until usage. The yield of an EV fraction prepared from supernatants of 4 × 107 MSCs that had been conditioned for 48 h was defined as 1 unit. The EV content of the four processed MSC supernatants was determined by nano-particle tracking analysis on the Zetaview (Particle Metrix, Diessen, Germany) and the NanoSight (Amesbury, UK) platforms.10 The particle and protein content of the four independent MSC supernatant fractions were all in same ranges (1.3–3.5 × 1010 particles/unit; 0.5–1.6 mg/unit). The average particle sizes calculated varied for the different preparations between 99–123 nm (ZetaView) and 133–138 nm (NanoSight). Western blot analyses demonstrated the presence of the exosomal marker proteins Tsg101 and CD8111, 12 in all fractions (Supplementary Figure S2A). These findings together with the vesicular appearances of obtained particles within transmission electron microscopy (Supplementary Figure S2C) indicated that the obtained samples were highly enriched for exosomes. However, owing to the general lack of methods to physically separate small MVs and exosomes from each other, we cannot exclude the fact that the MSC exosome-enriched fractions also contain proportions of other small EVs such MVs.13

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Acknowledgements

We thank Ferdi Grawe (Heinrich-Heine-University, Düsseldorf) for helping us to obtain exosome images and Andre Görgens for general discussion. We are grateful for the technical support of Sabine Schramm and Monika Collenburg and we thank Elisabeth H Weiss (Ludwig-Maximilians-University Munich) and Britta Eiz-Vesper (Hannover Medical School) for providing modified K562 cell line cells and Nahid Kordelas for proofreading.

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  1. L Kordelas and V Rebmann: Both authors share first authorship.

Authors and Affiliations

  1. Department of Bone Marrow Transplantation, University Hospital, University of Duisburg-Essen, Essen, Germany

    L Kordelas & D W Beelen

  2. German Cancer Consortium (DKTK), Germany

    L Kordelas, V Rebmann, S Radtke, P A Horn, D W Beelen & B Giebel

  3. Institute for Transfusion Medicine, University Hospital, University of Duisburg-Essen, Essen, Germany

    V Rebmann, A-K Ludwig, S Radtke, P A Horn & B Giebel

  4. Institute of Inorganic Chemistry, University of Duisburg-Essen, Essen, Germany

    J Ruesing & M Epple

  5. Department of Neurology, University Hospital, University of Duisburg-Essen, Essen, Germany

    T R Doeppner

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  1. L Kordelas
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Corresponding authors

Correspondence to D W Beelen or B Giebel.

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The authors declare no conflict of interest.

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Author contributions

PAH, DB and BG designed the study; LK and DB treated the patients; A-KL, SR and TRD raised cells and purified exosomes; VR, A-KL and JR characterized exosomes; VR performerd immune assays; LK, VR and BG wrote the paper; All authors discussed the data and the manuscript.

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A patent entitled ‘Use of preparations comprising exosomes derived from mesenchymal stem cells (MSCs) in the prevention and therapy of inflammatory conditions’ has been submitted.

Supplementary Information accompanies this paper on the Leukemia website

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Kordelas, L., Rebmann, V., Ludwig, AK. et al. MSC-derived exosomes: a novel tool to treat therapy-refractory graft-versus-host disease. Leukemia 28, 970–973 (2014). https://doi.org/10.1038/leu.2014.41

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