Supplemental Information
History of the patient
A 22-year old female patient received a first allogeneic stem cell transplantation (AlloSCT) at the
age of 13 due to a myelodysplastic syndrome with peripheral blood stem cells (PBSC) from a
HLA-identical female donor. After the first AlloSCT she developed moderate mucosal GvHD but
recovered hematologically with full donor chimerism. When she was 20, a secondary acute
myeloid leukemia was diagnosed. Therefore, a second AlloSCT with PBSC from a HLAidentical male donor following conditioning with hyper-fractionated total body irradiation with
12 Gy and cyclophosphamide was performed. GvHD prophylaxis was standard cyclosporine
(CSP) and a short course of methotrexate. The patient suffered a hyperacute GvHD of the skin
with general erythema and bullae, grade IV, which initially responded sufficiently to high-dose
steroid-therapy. Thus, the patient was discharged with a dual immunosuppression of CSP and
steroids. Unfortunately, the GvHD worsened after discharge and the patient had to be admitted to
hospital again 2 months after AlloSCT with exacerbated skin GvHD and severe intestinal
involvement. She had therapy-refractory nausea and vomiting, abdominal cramping and diarrhea
volumes reached up to five liters/day. The intestinal GvHD was repeatedly documented by
endoscopy and histological examination. There was no hepatic GvHD involvement.
The severe GvHD did not respond to a number of immunosuppressive lines. Immediately upon
re-admission the patient received high-dose steroids (5 mg/kg body weight [BW]) with no
improvement. Therefore, a course of anti-thymocyte globuline (10 mg/kg BW) was applied over
five days, which also did not lead to a response. Then, mycophenolate mofetil (MMF) and
subsequently tacrolimus were added. Since in some colon biopsies HHV-6, adenovirus and EBV
DNA were detectable, a therapy course with cidofovir was initiated. Unfortunately, all these
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measures did not lead to a relief in GvHD symptoms. Therefore, a course of infliximab (10
mg/kg BW once per week over a four week period) was administered and 19 sessions of
extracorporeal photopheresis were performed over four months. Throughout this time the basic
immunosuppression with steroids, mostly in combination with either tacrolimus or MMF, was
continued. A therapy attempt with sirolimus had to be discontinued due to increasing renal
insufficiency (a synopsis of immunosuppressive therapy is depicted in Figure S1).
Given the history of this therapy-refractory GvHD, we discussed in depth with the patient the
possibility of an experimental approach to control the GvHD. A concept for an individual
treatment attempt with MSC exosomes was developed and approved by the Legal Department of
the University Hospital Essen, Germany. The patient agreed to the proposed treatment and gave
her written consent.
Isolation and expansion of primary human mesenchymal stem cells
Human bone marrow (BM) was obtained from unrelated donors after informed consent according
to the Declaration of Helsinki. Primary human MSCs were raised from mononuclear cells
obtained by Ficoll (Biocoll Separating Solution, Biochrom AG, Berlin, Germany) density
gradient centrifugation of bone marrow. The mononuclear cells were cultured in tissue culture 6well plates at a density of 2 x 106 cells per well in MSC basal media (Pan Biotech, Aidenbach,
Germany) supplemented with 5% human thrombocyte lysate, 1% glutamine, and 1% penicillinstreptomycin (Invitrogen, Darmstadt, Germany) for 24 hours. After 24 hours non-adherent cells
were removed by medium exchanges. After confluence had been reached, usually within 14 days,
cells were continuously passaged after treatment with 0.25% Trypsin. Obtained MSCs were
characterized flow cytometrically, their osteogenic and adipogenic differentiation potential was
confirmed in vitro.
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Collection of MSC conditioned media
Starting at passage 3, MSC conditioned media (CM) were harvested every 48 hours and passed
through a 0.22 µm filter membrane (TPP, Trasadingen, Switzerland) to remove cell debris and
larger vesicles. CM were collected and stored at -20°C. After thawing CM were unified.
3 units/mL of heparin (Ratiopharm, Ulm, Germany) and 3 µg/mL Actilyse (Boehringer
Ingelheim, Ingelheim, Germany) were added. Supernatants were incubated for 3 hours at 37°C
and then filtered through 0.22 µm bottle top filters (TPP).
PEG-precipitation of exosomes from conditioned media of BM-MSCs
Exosomes were concentrated applying a polyethylene-glycol (PEG)-based purification strategy.
PEG 6000 (Sigma-Aldrich) and NaCl were added to filtered MSC supernatants to a final
concentration of 10% PEG 6000 (v/v) and 75 mM NaCl, respectively. Following incubation for
8-12 hours at 4°C, exosomes were precipitated and collected by centrifugation for 30 min at
1,500 g. The obtained pellets were resolved in 1 mL 0.9% NaCl (B. Braun Melsungen,
Melsungen, Germany), washed in a total volume of 45 mL 0.9% NaCl for 12 h at 4°C and
precipitated by ultracentrifugation for 2 hours at 100,000 g. The pellets were once again resolved
in 1 mL 0.9% NaCl and diluted with 0.9% NaCl to the applied working solutions. Working
solutions were stored as 1 mL aliquots at -80°C until usage.
Microbiological, virological and toxicological analyses of the applied exosome preparation
A 1:5 diluted sample of the exosome preparation was tested for bacterial contamination at the
Institute of Medical Microbiology (University Hospital Essen) and analyzed for infectivity via
PCR and infectious serology. The endotoxin concentration of the preparation was tested by the
Acila AG (Mörfelden-Walldorf, Germany).
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Nano particle tracking analysis (NTA)
NTA was performed by using the NanoSight® (NanoSight, Minton Park, GB) and the
ZetaView® (Particle Metrix, Meerbusch, Germany). Both these instruments track the Brownian
motion of laser beam illuminated particles over time and subsequently calculate particle sizes and
concentrations. As described previously, 1:500 or 1:1000 H2O diluted probes were measured in
triplicates (NanoSight®) and duplicates (ZetaView®) 1.
Western Blot analyses
Protein concentrations of obtained aliquots were determined by the micro BCA assay (Thermo
Fisher Scientific, Schwerte, Germany). To perform Western Blots 5 µg of the concentrated
exosomes were treated with sample buffer (DTT, 0.1% SDS, 0.1 M Tris HCl, pH 7.0) and boiled
for 5 min at 95°C. Samples were separated on 12% SDS-PAGE gels and transferred to PVDF
membranes (Merck Millipore, Darmstadt, Germany). Membranes were blocked for 1 hour in 5%
skimmed milk (Sigma-Aldrich) and phosphate buffered saline (PBS) and incubated with
antibodies either recognizing the exosomal marker proteins Tsg101 (Sigma-Aldrich) or CD81
(BD). For the detection of bound antibodies corresponding HRP-conjugated secondary antibodies
(Dianova, Hamburg, Germany) were used. HRP activities were visualized by enhanced
chemiluminescence (Thermo Fisher Scientific).
Peripheral blood samples
Peripheral blood mononuclear cells (PBMCs) and Heparin plasma samples were procured from
the GvHD patient before, during and after the MSC-exosome therapy. PBMCs were isolated from
the samples by Ficoll density gradient centrifugation (Invitrogen, Darmstadt, Germany) and
cryopreserved at -80°C until usage.
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Determination of Cytokines and HLA-G
ELISA technology was used to estimate the amount of soluble HLA-G (sHLA-G), cytokines and
apoptosis-inducing molecules in the exosome preparations and in patient’s Heparin plasma
samples obtained before, during and after the exosome therapy. Before the various ELISA
formats were performed, enriched exosomes (100 µg/mL) were solubilized in 1 mL PBS-Tween
(1%) and sonicated for 60 sec; plasma samples were analyzed undiluted. sHLA-G concentrations
were determined as described previously
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with minor modifications: The monoclonal antibody
(mAb) G233 (Exbio, Praha, Czech Republic) was used as the capture reagent, and bound sHLAG was detected by the biotinylated mAb w6/32 (Leinco Technologies, St. Louis, Missouri, USA)
followed by AMDEX™ Streptavidin HRP (Amersham, Freiburg, Germany). The detection limit
of sHLA-G ELISA was 0.1 ng/mL. Concentrations of pro-inflammatory (IFN-γ, TNF-α, IL-1b,
IL-2, IL-6, IL-8, IL-17a, IL-21), anti-inflammatory (IL-1RA, IL-4, IL-10, IL-13, TGF-1) and
apoptosis-inducing molecules (soluble FasL) were determined according to the manufacturers’
protocols (eBioscience, Frankfurt, Germany or BD Biosciences, San Jose, CA, USA). The assay
sensitivities ranged between 2.0 and 20 pg per mL.
Determination of the IL-1β, TNF-α and IFN-γ cytokine reactivity profile by ELISpot
After thawing PBMCs, NK cells were isolated by negative selection (Invitrogen). PBMCs and
NK cells were adjusted to concentrations of 1 x 106 cells per mL and used as effector cells. To
analyze impacts of MSC-exosomes on cytokine responses, 1 x 106 effector cells were mixed with
100 µg MSC-exosome preparations. Effector cell suspensions with and without exosomes were
used in cytokine stimulation experiments. HLA-E*01:03 or HLA-B27 stably transfected K562
cells and cells of the parental HLA class I negative K562 leukemic cell line were used as
allogeneic stimulator cells at densities of 1 x 105 cells per mL. ELISpot assays were performed in
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an effector to stimulator range of 6:1. For IL-1β and TNF-α ELISpot assays 1.25 x 105 effector
cells and 2,500 stimulator cells were used, for IFN- ELISpot assays 7.5 x 105 effector cells and
1.5 x 104 stimulator cells. Cells were seeded in 200 µL culture medium supplemented with 200
U/mL IL-2 and 10% fetal calf serum onto MultiScreen-HA ELISpot plates (MAIPS4510, Merck
Millipore) pre-coated with corresponding capture antibodies of the ELISpot kit (eBioscience,
Frankfurt, Germany). Effector cells without stimulator cells and stimulator cells without effector
cells served as controls. Each stimulation condition was performed in duplicate. After 24 hours
incubation at 37°C in 5% CO2 atmosphere, the numbers of cytokine producing effector cells were
estimated according to the manufacturer’s protocol. Spots were enumerated by the ELISpot
reader AID ELISpot Reader Systems (Autoimmun Diagnostika, Strassberg, Germany) using AID
ELISpot Software Version 6.
Statistics
The D’Agostino-Pearson omnibus normality test was applied to assess the Gaussian distribution
of individual data sets. In relation to that, the results of mixed lymphocyte reaction assays were
analyzed by the Mann-Whitney U test. The comparison of the cytokine response of the patient
derived PBMCs harvested before, during and after the exosome therapy was performed with the
Kruskal Wallis test. Statistical analyses were performed with GraphPad Prism version VI.
Overall, a 5% or lower p-value was considered to be statistically significant; only such p-values
are indicated within the text.
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Supplemental Figure Legends
Figure S1
Treatment regime of the 22-year old female GvHD patient following
allogeneic stem cell transplantation. ATG: anti-thymocyte globuline, MMF:
mycophenolate mofetil, ECP: extra corporal photophoresis.
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Figure S2
Analyses of the MSC exosome preparations. (A) Western Blot analyses of the
MSC exosome preparations for the exosomal marker proteins TSG101 and CD81
(5 µg protein lane). (B) Size distribution of nanoparticles within the MSC exosome
preparations (MSC1 Exo and MSC3 Exo were used as representatives and
measured on the NanoSight® platform). (C) Electron microscopic analyses of
exosome preparations (MSC1 Exo and MSC3 Exo were used as representatives;
scalbare 100 nm).
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1.
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of exosomes derived from human cells by nanoparticle tracking analysis and scanning
electron microscopy. Colloids Surf B Biointerfaces 2011 Oct 1; 87(1): 146-150.
2.
Schutt P, Schutt B, Switala M, Bauer S, Stamatis G, Opalka B, et al. Prognostic relevance
of soluble human leukocyte antigen-G and total human leukocyte antigen class I
molecules in lung cancer patients. Hum Immunol 2010 May; 71(5): 489-495.
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