Safety and Efficacy of the Intravenous Infusion of Umbilical Cord Mesenchymal Stem Cells in Patients With Heart Failure

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Measurements of Paracrine Factors
To compare the secretion levels of growth factors between BM MSCs and UC MSCs, 3X10 4 cells were plated in serum-free medium in 6-well plates. After 24 hours of incubation, the conditioned medium was collected, and the secreted levels of vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) were measured using the DuoSet ELISA Development System (R&D Systems, Minneapolis,MN . In addition, relative expression of TGF 1 was measured using the RNeasy mini kit (Qiagen, USA) to RNA extraction and complementary DNA was synthesized in a 20 μl reaction mixture using SuperScript III First-Strand Synthesis for RT-PCR (Invitrogen, USA). RT-qPCR was performed using SYBR Green Reagents (QPCR Master Mix, Agilent Technologies). All primer sets were previously screened for efficiency and their sequences were: B2M (F:5´ TCAGGTTTACTCACGTCATCC 3´, R:5' ACACGGCAGGCATACTCATC 3´), TGF 1 F 5 ACAATTCCTGGCGATACCTCAGCA3 , F 5 TGCAGTGTGTTATCCCTGCTGTCA3 .

IDO activity
UC-MSCs and BM-MSCs cells were stimulated with 20 ng/mL of IFN and 10 ng/mL of IL-1β or with 100 ng/mL of IFN during 48 hours in Dulbecco's modified Eagle's medium (DMEM) supplemented with 2% fetal bovine serum, 2 mM L-glutamine, 1% penicillin/streptomycin (all reagents from Gibco, Gran Island, USA) and 100 ug/mL of L-tryptophan (Sigma-Aldrich, St Louis, MO,USA). IDO enzyme activity was measured determining the kynurenine content in the cell supernatant as previously reported 1,2 .

3/R2
appropriate fluorescently labeled monoclonal antibody directed against lymphocyte surface markers (BD Biosciences, CA, USA), washed and resuspended in FACS buffer, and analyzed by the FACS Canto II cytometer using the FACS Diva software (BD Biosciences, CA, USA). The viability was determined using LIVE/DEAD®Fixable dead cell stain kit (Invitrogen, CA, USA) according to the manufacturer's protocol. The data acquired was analyzed using the FlowJo soſtware (Tree Star, Ashland, OR, USA).

T-Cell Subset Proliferation Assays
The proliferation of different T-cell subsets from 4 HFrEF patients included in this trial was performed in vitro to evaluate the immunomodulatory effect of UC-MSCs and BM-MSCs. Human peripheral blood mononuclear cells PBMCs were isolated by Ficoll-Paque Plus (GE Healthcare, Amersham, UK) (1.077 g/ml) density-gradient according to manufacturer's instruction. PBMCs were stained with carboxyfluorescein succinimidyl ester (CFSE; Life Technologies, Carlsbad, CA) following the manufacturer's protocol, and co-cultured with MSCs in 96-well plates at a 1:10 ratio (MSCs:hPBMC) in Roswell Park Memorial Institute (RPMI) medium (ThermoFisher, USA) supplemented with 10% FBS, 1% L-G, 1% nonessential amino acids (Sigma-Aldrich, USA), 100mM sodium pyruvate (Sigma, USA), 25 mMb-mercaptoethanol (Gibco, NY), and 15mg/ml phytohemagglutinin (PHA) (Sigma, USA). After 72 hours, PBMC were stimulated for 4 hours with 50 ng/ml phorbolmyristate acetate (PMA) (Sigma Aldrich) and 1μg/ml ionomycin (Sigma Aldrich) in the presence of Brefeldin A (Biolegend, San Diego, Ca, USA). For surface staining, cells were incubated with antibodies against human CD4, CD8, CD3 and CD25 (BD Biosciences, USA) in the dark at 4°C for 30 min. Intracellular staining was performed using the BD Cytofix/Cytoperm solution, according to the manufacturer's protocol with antibodies against human IL-17, IL4 and IFNγ (eBioscience, USA). For transcriptional factor evaluation, we assessed FoxP3 expression with staining buffer and specific antibodies (eBioscience, USA) according to the manufacturer's protocol. Cells were acquired using a FACS Canto II Flow cytometer (BD Biosciences) and analyzed with the FlowJo software (Tristar, Stanford) for phenotype and proliferation, calculated by the decrease in CFSE fluorescence.

Migration capacity
Cell migration assays were performed with the Transwell two-chamber cell culture method (Corning, Cambridge, MA) with an 8 μm pore polycarbonate membrane. The uppermost side of the Transwell membrane was coated with 0.1% gelatin in PBS (Sigma-Aldrich, St. Louis, MO) for 2 h at 37 . UC-MSCs or BM-MSCs were seeded at a density of 15.000 cells per 100 μl of DMEM 1% P/S, 0.1%FBS in the upper chamber of the Transwell apparatus. Cells were allowed to migrate toward medium (500 μl) in the lower chamber containing DMEM alone or supplemented with 5% of serum isolated from the HFrEF patients. The Transwell system was incubated for 16h at 37 in a humidified atmosphere containing 5% CO2. After incubation, non-migratory cells were carefully removed from upper face of the Transwell insert with a cotton swab. The attached cells remaining on the Transwell insert were fixed with 70% methanol and stained with 1% crystal violet in 20% methanol for 1 h. After washing, the stained cells that migrated from the upper to the lower side of the membrane were counted under an inverted bright-field microscope at 20X magnification. The number of migrated cells was expressed as the percent change from the control value (DMEM alone). Each experiment was performed in biological and experimental triplicate.

Cellular size
Parameters defined for the MSC setting of the Countess™ (Invitrogen) automated cell counter included control of circularity and maximum-minimum cell size (applied gate 10um -28um). The analysis of 26 therapy releases resulted in an average size measurement of UC-MSC´s of 17.1 3.5 m.

Doubling time
The doubling time (DT) data were collected from each of the cell culture passage performed under GMP conditions. The assessment of 50 samples resulted in an average DT of 32.6 8.8 hours.

Senescence-associated beta-galactosidase assay
UC-MSC and BM-MSC senescence according to the expression of SA-β-galactosidase was evaluated under standard culture conditions normalized for passage and culture duration. UC-MSC showed around 2 fold less senescent cells than BM-MSC´s (Online Figure II).

Quantification of IDO activity, IL6, TGF-β1, PGE2 and PDL-1 at basal and stimulated condition
The expression of these mediators involved in the regenerative and suppressive effects of MSCs was evaluated for both cell sources (BM or UC-MSCs) in baseline conditions and after proinflammatory stimulus with IFN and IL-1β at optimal conditions (20 and 10 ng/ml respectively). UC-MSCs expressed similar levels of PDL-1, HLA-G as well as IDO activity compared to BM-MSCs under the different culture conditions. Furthermore, both MSC sources responded to the IFN + IL-1β stimulated conditions by an increased protein expression levels of IL-6, TGF-β1 and PGE2 (Online Figure III). Of interest, UC-MSCs expressed higher constitutive levels of PGE2 and TGF-β1, a molecule known for the induction of T regulatory (Treg) CD4+ cells and inhibition of NK function.