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. 2019 Dec 31;14(12):e0227283. doi: 10.1371/journal.pone.0227283

Potential of mesenchymal- and cardiac progenitor cells for therapeutic targeting of B-cells and antibody responses in end-stage heart failure

Patricia van den Hoogen 1, Saskia C A de Jager 1,*, Emma A Mol 1,2, Arjan S Schoneveld 3, Manon M H Huibers 4,5, Aryan Vink 4, Pieter A Doevendans 6,7,8, Jon D Laman 9, Joost P G Sluijter 1
Editor: Federico Quaini10
PMCID: PMC6938331  PMID: 31891633

Abstract

Upon myocardial damage, the release of cardiac proteins induces a strong antibody-mediated immune response, which can lead to adverse cardiac remodeling and eventually heart failure (HF). Stem cell therapy using mesenchymal stromal cells (MSCs) or cardiomyocyte progenitor cells (CPCs) previously showed beneficial effects on cardiac function despite low engraftment in the heart. Paracrine mediators are likely of great importance, where, for example, MSC-derived extracellular vesicles (EVs) also show immunosuppressive properties in vitro. However, the limited capacity of MSCs to differentiate into cardiac cells and the sufficient scaling of MSC-derived EVs remain a challenge to clinical translation. Therefore, we investigated the immunosuppressive actions of endogenous CPCs and CPC-derived EVs on antibody production in vitro, using both healthy controls and end-stage HF patients. Both MSCs and CPCs strongly inhibit lymphocyte proliferation and antibody production in vitro. Furthermore, CPC-derived EVs significantly lowered the levels of IgG1, IgG4, and IgM, especially when administered for longer duration. In line with previous findings, plasma cells of end-stage HF patients showed high production of IgG3, which can be inhibited by MSCs in vitro. MSCs and CPCs inhibit in vitro antibody production of both healthy and end-stage HF-derived immune cells. CPC-derived paracrine factors, such as EVs, show similar effects, but do not provide the complete immunosuppressive capacity of CPCs. The strongest immunosuppressive effects were observed using MSCs, suggesting that MSCs might be the best candidates for therapeutic targeting of B-cell responses in HF.

Introduction

Cardiovascular disease (CVD) is the most common cause of death globally with almost 18 million deaths per year [1]. A prominent CVD-subtype is ischemic heart disease (IHD), which is characterized by myocardial cell death due to prolonged ischemia [2]. After subsequent reperfusion strategies, further myocardial damage is initiated by the release of cardiac proteins, which can induce an inflammatory response [3,4]. Activated T- and B lymphocytes significantly contribute to adverse cardiac remodeling via the production of pro-inflammatory cytokines and antibodies [5–7], which can progress to severe heart failure (HF) [6,8–10].

Currently, progenitor cell therapy is gaining a lot of interest in order to regenerate the damaged heart due to their regenerative properties and the ability to differentiate into other cell types [11–13]. Mesenchymal stromal cells (MSCs) improve cardiac function by reducing scar size and increasing left ventricular ejection fraction (LVEF) with 2–4% [14,15]. However, engraftment of these cells in the heart is relatively poor, where less than 10% of the injected cells remain at the site of injection [16,17]. In addition, the few remaining cells rarely differentiate into cardiac cells [18]. In addition to their regenerative capacity, MSCs have also been shown to suppress inflammatory responses, antibody production, and fibrosis, mostly in a paracrine manner [19,20]. Important paracrine mediators are extracellular vesicles (EVs), small lipid bi-layered vesicles containing lipids, small RNAs and proteins, which are able to influence many processes including inflammation [21,22]. Multiple studies investigated the therapeutic potency of MSCs and MSC derived EVs in cardiovascular disease [13,23,24]. MSC-derived EVs were found to reduce infarct size and infiltration of immune cells into the affected myocardium after myocardial infarction (MI) in animal models [25]. These findings suggest that the use of MSC-derived EVs might be a promising strategy to restore cardiac function, however, technical difficulties in large scale production and purification of MSC-EV are still limiting the translation to the clinic [19,26]. Considering the developmental origin of endogenous cardiac-derived progenitor cells (CPCs), these cells might prove better candidates for cell therapy for cardiac repair. Endogenous CPCs were previously tested in several clinical trials where they improved cardiac function [12,27], especially when combined with MSCs [28,29]. CPCs also have immunosuppressive properties, for example by inhibiting T-cell proliferation, which is partly mediated by paracrine factors [30]. CPC-derived EVs are proposed to be of great importance as paracrine mediators of these cells [31–33]. However, the immunosuppressive capacity of CPCs or CPC-derived EVs on B cells and antibody-mediated immune responses has not been elucidated yet. Therefore, we investigated the in vitro inhibitory actions of CPCs and CPC-derived EVs on lymphocyte proliferation and the production of immunoglobulin subclasses, using immune cells from healthy controls and end-stage HF patients.

Material and methods

Culture of human-derived progenitor cells

Human bone marrow-derived mesenchymal stromal cells (MSCs) and cardiomyocyte progenitor cells (CPCs) were obtained and isolated as described before [34,35]. MSCs were cultured in MEM-alpha (Gibco, 32561–037) supplemented with 10% fetal bovine serum (Gibco, 10270–106) + 1% PenStrep (Lonza, 17-602E) + 0.2 mM L-ascorbic acid-2-phospate (Sigma A4034) + 1 ng/ml bFGF (Sigma F0291). CPCs were cultured in SP++ (25% EGM-2 (Lonza CC-3156) + 75% M199 (Gibco 31150–022) supplemented with 10% fetal bovine serum + 1% PenStrep + 1% non-essential amino acids (Lonza 13–114). Cultures were incubated at 37°C (5% CO2 and 20% O2) and adherent cells were passaged when reaching 80–90% of confluency using trypsin digestion (0.25%, Lonza, CC-5012). MSCs and CPCs from fetal or adult donors were used in the co-cultures between passage 6–17.

Isolation of CPC-derived extracellular vesicles and Western blotting

CPC-derived EVs were isolated using size-exclusion chromatography (SEC), as previously described [36]. In brief, fetal-derived CPCs were cultured until they reached a confluency of 80–90%, after which the medium was replaced with serum-free medium (M-199, Gibco 31150–022).

After 24 h, conditioned medium (CM), containing the EVs, was collected, centrifuged at 2000g for 15 min, and filtered (0.45 μm) to remove dead cells and debris. Next, CM was concentrated using 100-kDA molecular weight cut-off Amicon spin filters (Merck Milipore) and loaded onto a S400 highprep column (GE healthcare, Uppsala, Sweden) using an AKTA start (GE Healthcare) containing an UV 280 nm flow cell. Fractions containing EVs were pooled and filtered (0.45 μm) before further concentration procedures. The number of particles and mean size distribution were measured using Nanoparticle Tracking Analysis (Nanosight NS500, Malvern) as described before [36]. Protein concentration was measured using microBCA protein assay kit (Thermo Scientific). Vesicle markers were assessed by Western blotting (WB) as previously described [36]. EV protein fractions were loaded on pre-casted Bis-Tris protein gels (ThermoFischer, NW04125BOX) and run for 1 h at 160V. Proteins were transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, IPVH00010) and stained for general EV markers [37] Alix (1:1000, Abcam, 177840), CD63 (1:1000, Abcam, 8219), CD81 (1:1000, Santa Cruz, Sc-166029), or Calnexin (1:1000, Tebu-bio, GTX101676). Proteins were detected using chemiluminescent peroxidase substrate (Sigma, CPS1120). Representative pictures S1 Fig.

Isolation of peripheral mononuclear cells

Peripheral blood mononuclear cells (PBMCs) were isolated from fresh whole blood samples of healthy controls or end-stage HF patients, in compliance with the declaration of Helsinki and under approval of the Medical Ethics Committee Utrecht (METC, reference number 12/387). Written informed consent for collection and biobanking of blood samples was obtained. End-stage HF derived PBMCs were obtained from blood samples prior to heart transplantation. PBMCs were isolated using Ficoll-plaque PLUS gradient (GE life sciences, 17-1440-03) according to the manufacturers protocol. A total of 2,5x105 PBMCs were added per well (48-wells plate) in RPMI-1640 medium (Lonza, BE12-702F) supplemented with 10% fetal bovine serum and 1% PenStrep.

PBMC stimulation and co-culture with progenitor cells or purified EVs

For the stimulation of PBMCs and subsequent antibody production, a combination of IL-2 (120 IU/ml, BD Pharmingen, 554563) and PMA (0.123 ng/ml, Sigma, P8139) was used as previously described [33]. PBMCs were co-cultured for 10 days, without medium change, in 48-well plates (2.5x105 PBMC/well) with MSCs, CPCs (5.0x104 cells/well), or CPC-derived EVs (1x 10 μg = 6.3x1010 particles), immediately upon co-culture or 3x 10 μg (6.3x1010 particles) added every 3 days of co-culture. After 10 days of co-culture, light-microscopic images were taken using an Olympus CKX41 microscope in combination with CellSense software. Non-adherent cells, containing the lymphocytes, were collected and processed for further analysis using flow cytometry (Gallios, Beckmann Coulter). Co-culture supernatant was collected, centrifuged at 500g for 10 min, aliquoted and stored at -80°C for immunoglobulin measurements.

Lymphocyte proliferation

Flow cytometry (Gallios, Beckmann Coulter) was used to assess lymphocyte proliferation. Prior to co-culture, PBMCs were labeled with 1.5mM carboxyfluorescein succinimidyl ester (CFSE, Sigma, 21888) as described previously [33]. In brief, PBMCs were incubated with CFSE for 10 min at 37°C in a dark shaking bath. After 10 minutes, 5% of FBS was used to prevent further uptake. After two washing steps with PBS, PBMCs were incubated for 30 min with fluorescent antibodies, including CD3 for T cells (Brilliant Violet 510, Biolegend, 317332) and CD19 for B cells (PE/Cy5, Biolegend 302210). After washing with PBS, PBMCs were incubated for 30 min with a fixable viability dye (eFluor506, Bioscience, 65-0866-14) to exclude dead cells. Prior to culture, general cell composition per donor was assessed by measuring the percentage of CD3+ T cells and CD19+ B cells, to ensure that the cell populations were similar between the different donors at baseline. Lymphocyte proliferation was calculated by measuring CFSE intensity and the number of cells present in each division as described before [33]. Since we encountered some donor variations in the absolute number of proliferating cells in the stimulation assays, the stimulated PBMC condition was considered as maximum response and defined as 100% proliferation (ratio = 1) and used for normalization of the data per donor and per experiment. Data was analyzed using Kaluza Analysis Software (Beckman Coulter, version 1.3).

Immunoglobulin multiplex

The levels of IgM and IgG subclasses (IgG1, IgG2, IgG3, IgG4) in the co-cultures (5x diluted) were measured using a Bio-Plex Pro™ human isotyping immunoassay 6-plex (Bio-Rad, 171A3100M) according to manufacturer’s instructions and were all within the detection limit of the assay. Immunoglobulin levels in the supernatant after co-culture with MSC/CPC or CPC-derived EVs were calculated using internal standards included in the assay. Immunoglobulin levels are represented as relative production, with the stimulated PBMC condition defined as 100% antibody production (ratio = 1) and used for normalization of the data per donor and experiment.

Statistics

Statistical analysis and data representation were performed using IBM SPSS Statistics 21 and Graphpad Prism (GraphPad Software Inc. version 8.01, San Diego CA, USA). Normal data distribution was tested using the Kolmogorov-Smirnov test. Group comparison was performed by a one-way ANOVA or Kruskal-Wallis test, corrected for multiple comparison testing. Each individual PBMC donor is considered as an independent individual experimental number (n), ranging from 2–8 donors per experiment. Data was considered significant with two-tailed p-values <0.05 and is presented as mean ± SEM.

Results

Progenitor cells suppress lymphocyte proliferation upon cell-cell contact

To investigate the immunosuppressive effects of progenitor cells on the proliferation of lymphocytes, a co-culture using MSCs or CPCs was performed (Fig 1). To represent normal lymphocyte activation by antigen-presenting cells, the total PBMC population was used. After 10 days of co-culture, large clusters of proliferating T cells were visible upon stimulation with IL-2 and PMA. These large clusters were smaller or even absent when PBMCs were cultured in the presence of MSCs or CPCs (Fig 1A). Flow cytometry was used to measure CFSE intensity and to assess lymphocyte proliferation (Fig 1B and 1C). FACS plots clearly showed active cell proliferation upon stimulation with IL-2 and PMA and suppression of proliferation when PBMCs were cultured with MSCs or CPCs. Quantification showed that both MSCs and CPCs significantly decreased proliferation of lymphocytes by 64±18.6% and 19±12.5% respectively (MSC p<0.0001, CPC p<0.05).

Fig 1. Progenitor cells suppress lymphocyte proliferation.

Fig 1

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Lymphocyte proliferation was measured after 10 days of co-culture of PBMCs with MSCs or CPCs. A) Representative microscopic images after 10 days of co-culture. Upon PBMC stimulation, large clusters of proliferating cells were observed. These large clusters were absent in the presence of MSCs or CPCs. B) PBMCs were labeled with CFSE and lymphocyte proliferation was assessed by measuring CFSE intensity using flow cytometry. FACS plots of non-stimulated lymphocytes show one peak of undivided cells, whereas upon stimulation, lymphocytes start to divide. C) Quantification of lymphocyte proliferation, where stimulated lymphocytes were used as normalization. Both MSCs and CPCs show a significant decrease of lymphocyte proliferation upon co-culture. Strongest effects were observed using MSCs, where proliferation was inhibited towards 36% compared to CPCs (81%). PBMC: pheripheral blood mononuclear cells, MSC: mesenchymal stromal cell, CPC: cardiac progenitor cell. Per condition n = 4. Line bar indicates 200μm, magnification 4x. Significance was determined using one-way ANOVA, * p<0.05, **** p<0.0001.

Production of IgM and different IgG subclasses is suppressed by cardiac-derived progenitor cells

Next to reduced cell proliferation, MSCs are also able to inhibit several immune cell functions, such as antibody secretion [38]. To examine whether this also holds true for CPCs, we collected the supernatant after 10 days of co-culture and measured the levels of different immunoglobulin subclasses (Fig 2A). Since it is known that the age of the donor can affect their inhibitory potency [39,40], both fetal and adult MSCs and CPCs were included. Adult and fetal-derived MSCs significantly inhibited antibody production from stimulated PBMCs (Fig 2B–2F). Fetal and adult MSC significantly reduced the production of IgM (aMSC = 0.005±0.0 fMSC = 0.02±0.0; p<0.0001), IgG1 (aMSC = 0.24±0.06, fMSC = 0.28±0.06; p<0.0001), IgG3 (aMSC = 0.19±0.06, fMSC = 0.25±0.09; p<0.0001) and IgG4 (aMSC = 0.29±0.07; p<0.01, fMSC = 0.43±0.1; p<0.05). In addition, also CPCs showed strong suppressive effects on the production of mainly IgM (aCPC = 0.02±0.0; p<0.0001, fCPC = 0.38±0.16; p<0.001), IgG1 (aCPC = 0.12±0.02; p<0.001, fCPC = 0.55±0.18; p<0.05) and IgG3 (aCPC = 0.06±0.0; p<0.0001, fCPC = 0.44±0.14; p<0.001). For CPCs, the strongest immunosuppression was observed using adult CPCs.

Fig 2. Progenitor cells suppress the production of immunoglobulins.

Fig 2

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Antibody production was measured after 10 days of co-culture with fetal or adult-derived MSCs or CPCs. A) Experimental design of the co-culture. B) Both fetal and adult MSCs showed strong immunosuppressive effects on the production of different immunoglobulin isotypes and subclasses. IgM, IgG1, IgG3, and IgG4 levels were significantly decreased upon co-culture with MSCs. For CPCs, strongest effects were observed in cultures using adult-derived CPCs, where the production of IgM, IgG1, IgG3, and IgG4 was significantly suppressed. aMSC: adult-derived mesenchymal stromal cell, fMSC: fetal-derived mesenchymal stromal cell, aCPC: adult-derived cardiac progenitor cell, fCPC: fetal-derived cardiac progenitor cell. For aMSC, fMSC and fCPC n = 7, for aCPC n = 3. Significance was determined using One-way ANOVA, * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.

CPC-derived extracellular vesicles suppress antibody production, but are not as effective as direct cell-cell interaction when using CPCs

To explore whether the suppressive capacity of CPC on antibody production is mediated by paracrine factors, we assessed the potential of CPC-derived EVs (Fig 3). We experienced that it is technically challenging to obtain sufficient MSC-derived EVs using SEC. Therefore, we only included CPC-derived EVs in our co-cultures. Prior to co-culture, EVs were characterized based on size distribution and the presence or absence of protein markers [37]. Isolated EVs showed a representative size distribution profile with the highest peak at approximately 90 nm (Fig 3A). In line with previous findings [36], WB analysis showed that CPC-derived EVs were enriched for the typical EV proteins Alix, CD81, and CD63. Calnexin was only detectable in the cell lysate, thereby confirming the absence of contaminations with other membrane compartments (Fig 3B). An amount of 1x10 μg or 3x10 μg (every 3 days of co-culture) was added to the PBMC cultures (Fig 3C). After 10 days of co-culture, antibody secretion was significantly suppressed by EVs (Fig 3D). The production of IgM, IgG1, and IgG4 was significantly decreased using the 3x dose of CPC-derived EVs (IgM = 0.35±0.05; p<0.05, IgG1 = 0.57±0.03; p<0.05, and IgG4 = 0.66±0.0; p = 0.03), thereby indicating that long term suppression is more effective than a single dose of EVs. However, the inhibitory effect was most robust when adding CPCs and not CPC-derived EVs, with strongest suppressive effects on the release of IgG1 (0.59±0.1; p<0.05), IgG2 (0.23±0.06; p = 0.02), IgG4 (0.53±0.03; p = 0.01) and IgM (0.17±0.03; p<0.01).

Fig 3. Immunosuppressive capacity of CPC-derived EVs on immunoglobulin production.

Fig 3

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To assess whether CPC-derived paracrine factors can be used, EVs were isolated and used in the PBMC co-cultures. A) EVs with a size of approximately 90 nm were isolated using SEC. B) WB of EVs and CL with general EV markers and calnexin. C) Experimental setup of the co-culture model, where either CPCs or CPC-derived EVs with a total of 1x 10 μg or 3x 10 μg (every 3 days of co-culture) was added to PBMCs. D) CPC-derived EVs showed a significant decrease of immunoglobulin production, especially when administered for a longer period of time. Levels of IgM, IgG1, and IgG4 were significantly decreased when, every 3 days of co-culture, 10 μg of EVs were added to stimulated PBMCs. However, the strongest inhibition of antibody production was observed when CPCs were used. CPC: cardiac progenitor cell, EV: extracellular vesicles, CL: cell lysate, SEC: size-exclusion chromatography, PBMC: pheripheral blood mononuclear cells. For each condition n = 2, Significance was determined using One-way ANOVA, * p<0.05, ** p<0.01.

MSCs show the strongest immunosuppressive effects and are more likely to be used as cell therapy in end-stage HF patients

Since we observed that CPC-derived EVs do not give the same degree of immunosuppression as CPCs, we decided to continue with CPCs to examine their potential suppressive effect on antibody-mediated immune responses in end-stage HF patients. PBMCs were isolated from end-stage HF patients and cultured with or without MSCs/CPCs. (Fig 4). At baseline culture, non-stimulated PBMCs derived from end-stage HF patients produced similar amounts of IgGs with the exception of IgG3, which is, slightly but not significantly, increased compared to PBMCs derived from healthy controls (Fig 4A). Upon co-culture of patient-derived PBMCs with MSCs or CPCs, antibody production was significantly suppressed (Fig 4B and 4C). Mainly MSCs showed strong suppressive effects, as they significantly decreased the production of IgM (0.02±0.0; p<0.0001), as well as all IgG subclasses (IgG1 = 0.25±0.08; p = 0.001, IgG2 = 0.03±0.02; p<0.0001, IgG3 = 0.25±0.08; p = 0.009, IgG4 = 0.19±0.07; p = 0.0006). Co-cultures using CPCs showed similar suppressions, albeit at a lower level and the differences were only statistically significant for IgM (0.20±0.06; p<0.0001) and IgG2 (0.31±0.14; p = 0.0003).

Fig 4. Inhibition of immunoglobulin production by progenitor cells in end-stage HF.

Fig 4

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The immunosuppressive actions of CPCs on antibody production in end-stage HF was investigated using patient-derived PBMCs. A) Baseline antibody levels of unstimulated PBMCs in culture were measured and compared to end-stage HF-derived PBMCs. Before co-culture with MSCs/CPCs, HF patients showed high levels of IgG1 and IgG3 compared to healthy controls. B) Experimental set-up of the co-culture. C) Levels of IgM and IgG1-IgG4 were significantly decreased upon co-culture of patient-derived PBMCs with MSCs. CPCs were able to significantly suppress IgM and IgG2, however, were not as potent as MSCs. MSC: mesenchymal stromal cell, CPC: cardiac progenitor cell, HF: heart failure, PBMC: pheripheral blood mononuclear cells. Per condition n = 8. Significance was determined using a Kruskal Wallis test or One-way ANOVA, ** p<0.01, *** p<0.001, **** p<0.0001.

Discussion

The post-MI immune response is an important contributor to adverse cardiac remodeling and the development of HF [41–44]. The release of cardiac proteins upon MI can trigger antibody-mediated immune responses, which further induce cardiac damage and heart failure [45–47]. Stem cell therapy using progenitor cells, such as MSCs or CPCs, showed promising reparative effects on cardiac function despite poor engraftment in the myocardium [17,48]. This indicates that paracrine mediators, secreted by progenitor cells, can be of great importance. MSCs and MSC-derived EVs also have immunosuppressive properties, for example by lowering antibody production in vitro [49,50]. However, the immunosuppressive capacity of endogenous CPCs and CPC-derived EVs on B cells and antibody production has not been elucidated yet. Consequently in this study, we investigated the immunosuppressive effects of CPCs and CPC-derived paracrine mediators on antibody production using immune cells of both healthy controls and end-stage HF patients.

In line with previous findings, we showed that both MSCs and CPCs significantly suppressed proliferation of lymphocytes [30,32,38]. The suppressive effects of MSCs were more effective than CPCs. The suppressive effects of MSCs and CPCs on effector and regulatory T cells have been described before, where several studies show T cell inhibition via PDL-1/PD1 in a direct cell communication manner [32,51]. Moreover, both MSCs and CPCs are also able to suppress CD4+ T helper cell-mediated immune responses[52]. However, the interaction of progenitor cells with B cells is still controversial and this issue has recently gained more interest[53–55]. MSCs can inhibit plasma cell formation and subsequent IgG production in a cell-cell contact dependent as well as in an independent manner [38,55]. It is not known whether CPCs are also able to suppress antibody production in vitro. We demonstrated that, similar to MSCs, CPCs effectively suppress antibody production in vitro. We showed that both adult- and fetal-derived CPCs significantly inhibit the levels of IgM, IgG1, and IgG3, of which IgM was most efficiently suppressed, despite variation between different donors. These findings are in line with the effects of MSC, where MSC are known to exert an inhibitory effect on T helper cells, B-cell differentiation and class switching into IgG-producing cells [56,57]. Therefore, we could speculate that CPC might use a similar mechanism, in which IgG production might be suppressed either by inhibiting T-helper cell responses, thereby influencing B-cell activation and antibody production, or by directly influencing B-cell differentiation and subsequent class-switching.

To facilitate clinical translation, we examined if the strong immunosuppressive effects of CPCs and MSCs on antibody production using healthy donors, can be confirmed for IgG production using HF patient-derived PBMCs. MSCs were able to significantly inhibit the production of IgM and all IgG subclasses. For CPCs, the immunosuppressive effects were not as potent compared to MSCs, where CPCs only significantly lowered the production of IgM and IgG2. In end-stage HF, chronically activated immune cells progressively worsen cardiac function, for example by the production of cardiac antibodies [58,59]. Our findings indicate that progenitor cells, preferably MSCs, might be used as therapeutic agents to suppress antibody-mediated immune responses as observed in end-stage HF. However, mimicking the physiological immune response in vitro, as observed in end-stage HF patients, is still complicated. Therefore, these findings still have to be validated in vivo.

Part of the immunosuppressive properties of MSCs is mediated by paracrine factors, such as EVs [19,21]. The advantage of using EVs is that they can be used as a cell-free approach, thereby increasing safety, and allowing a longer duration of the treatment [19,26]. However, high variability in quantity and quality in the scaling and production process of MSC-derived EVs has been a limitation [26]. CPC-derived EVs might provide a promising alternative, not only due to their regenerative and immune modulating capacities [60], but also for their culture scalability. CPC-derived EVs have immunosuppressive effects on T cells [30,60], however, the effects on B cells and antibody-mediated responses is not clear. Our findings showed that CPC-derived EVs lower the different immunoglobulin isotypes and subclasses, such as IgM, IgG1, and IgG4. However, the number of EVs needed to reach similar suppressive effects compared to CPCs, remains challenging. In this study, we were only able to test EVs produced by fetal CPCs due to technical difficulties in obtaining sufficient numbers of EVs from adult CPCs. Fetal-derived progenitor cells might exert different effects than adult-derived cells, where, for example, adult-derived MSCs show stronger immunosuppressive capacities relative to fetal-derived MSCs [61]. For CPCs, it has been described that fetal- and adult-derived CPCs have different developmental potentials, and adult CPCs may be more effective in cardiac repair [62,63]. In addition, fetal-derived CPCs are highly proliferative as compared to adult-derived cells. Due to this proliferative state CPCs may secrete a different palette of paracrine factors that are more associated to cell cycle rather than immunomodulation. Therefore, the effects of EVs from adult CPCs may differ from fetal–derived CPCs and have to be investigated in future studies. Nonetheless, from our data, it is clear that EVs can be used as immunosuppressive mediators, but do completely cover the strong immunosuppressive effect of CPCs.

In conclusion, we demonstrated immunosuppressive actions of both MSCs and CPCs on lymphocyte proliferation and antibody production, with strongest effects observed when using MSCs. These are partly mediated by EVs, in a time-dependent matter. Lastly, we showed that CPCs and especially MSCs were able to suppress antibody production by patient-derived cells, thereby indicating the therapeutic potential of progenitor cells in HF. Currently, cell therapy using MSCs is no longer the holy grail for true cardiac regeneration and cell replacement therapy, however, MSCs might be promising candidates for targeting the post-MI immune response and HF progression. Future studies should focus on the identification of the cardiac antigens which are targeted by the produced IgGs and on the potential of combination therapies, using both MSCs and CPCs, to simultaneously target cardiac regeneration and antibody-mediated immune responses.

Supporting information

S1 Fig. Extracellular vesicle markers.

Proper isolation of extracellular vesicles (EV) was determined by the presence of CD63, CD81 and Alix and absence of the cellular marker Calnexin by Western Blotting. Cell lysates (Cl) were used as controls.

(TIF)

Click here for additional data file. (2.2MB, tif)

Acknowledgments

The authors gratefully acknowledge Erica Siera-de Koning, Joyce van Kuik, Frederieke van den Akker, and Sander van de Weg for their excellent technical support.

Data Availability

All relevant data are within the manuscript and its Supporting Information files.

Funding Statement

This work was supported by CVON2011-12 HUSTCare grant from the Netherlands CardioVascular Research Initiative (CVON): The Dutch Heart Foundation, Dutch Federation of University Medical Centers, the Netherlands Organization for Health Research and Development, the Royal Netherlands Academy of Science, the ZonMW Translational Adult Stem Cell grant 1161002016, and by Horizon2020 ERC-2016-COG EVICARE (725229). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Decision Letter 0

Federico Quaini

31 Oct 2019

PONE-D-19-25026

Potential of mesenchymal- and cardiac progenitor cells for therapeutic targeting of B-cells and antibody responses in end-stage heart failure

PLOS ONE

Dear Ms van den Hoogen,

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Additional Editor Comments:

The study is of high interest for the scientific community because embraces different features of cardiac repair including immune mediated mechanisms. Data, however, need to be implemented to support results and conclusion. When replying to each reviewer criticism, Authors should pay attention to the major issues raised involving methodological and conceptual aspects of the work.

In addition, most of the suggested implications of your finding are related to tissue repair or cell engraftment. However, in terms of pathogenetic mechanism of heart failure, could you speculate on which target antigen these Ig are produced? For example, do you have any data on IgG or IgM against alfa- myosin heavy chain?

Although the aim of the study was to investigate humoral immunity and/or antibody mediated immune response, the possibility that changes in Th (CD4 helper) compartment could affect antibody production should be acknowledged.

The point made by reviewer #2 on patient-specific properties of CPC and MSC is of high relevance.

In line with a point made by reviewer #1, the finding that adult MSC display more immunosuppressive effect than fetal cells is understandable, while the observed similar difference in CPC is more difficult to interpret. Could you please comment on this finding? Also the observation that fetal CPC preparations are more reach in EV than the adult one might be a well known outcome although requires a comment.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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Reviewer #1: Partly

Reviewer #2: Partly

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: I Don't Know

Reviewer #2: Yes

**********

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Reviewer #1: No

Reviewer #2: Yes

**********

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Reviewer #1: Yes

Reviewer #2: Yes

**********

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Reviewer #1: No: PONE-D-19-25026

Potential of mesenchymal- and cardiac progenitor cells for therapeutic targeting of B-cells and antibody responses in end-stage heart failure

The manuscript is well written, the subject is relevant, the “story” well composed, and out-put a contribution to the basic understanding of MSC and CPC MoA and a next step for others to build on.

Data however need more detail to allow validation of results and conclusion.

I suggest the paper is published after revision, - provided the currently missing data upon prober presentation in fact support the claimed results.

Specific comments/questions:

Methods:

Line 113-123: Is co-culture running for 10 days with-out media change?

Line 125-138: Timing of measures is a bit blurry…. I assume CFSE labelling is performed before co-culture…

CD3 and CD19 labelling is described, - what happened to these measures, - no further evaluation is found.

Flow cytometry should be described in more detail; no gating strategy is presented

Results:

An over-all concern in the results section: I have no idea how many replicates data are based on….- and data is completely missing…. only significance levels are presented…

Which absolute/relative values were obtained?

What is the sensitivity and ranges of the IgG/M assay used, - are data in fact with-in these ranges?

Discussion:

A few more words on the difference between IgG and IgM; IgM is most efficiently suppressed. How do you interpret this finding? Any suggestions on why, and what the consequence of this finding may be?

Adult CPC are more efficient that fetal; - can this be explained?

Reviewer #2: This is a very interesting study with novel findings. However, the study has the major limitation of showing only in vitro/ex vivo data. While it is clear that requesting an in vivo study would be totally off the mark, it is however essential that at least the in vitro findings are technically undisputable to back the conclusions made.

On this premise, it would be essential that the comparison among the two progenitor cell populations was made using cells derived from the same human donor (same HLA type). From the methods section this doesn't seem to be the case for the presented experiments.

Mixing the two cell types (or their derivatives) enhances the effect of the single cell type alone? This is important because it would indirectly hypothesize whether the mechanism of action on B-cell target by the MSCs and CPCs is similar or unique to each of them.

**********

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Reviewer #1: No

Reviewer #2: No

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PLoS One. 2019 Dec 31;14(12):e0227283. doi: 10.1371/journal.pone.0227283.r002

Author response to Decision Letter 0

13 Dec 2019

First of all, we would like to thank the editor for the opportunity to improve our manuscript. We feel that the raised points are of additional value for our work. We have thoroughly addressed all the raised points and our response is listed below.

Editor's Comments to Author:

1) The study is of high interest for the scientific community because embraces different features of cardiac repair including immune mediated mechanisms. Data, however, need to be implemented to support results and conclusion. When replying to each reviewer criticism, Authors should pay attention to the major issues raised involving methodological and conceptual aspects of the work.

Response: We thank the editor for the constructive remarks. We replied to each reviewer’s criticism below.

2) In addition, most of the suggested implications of your finding are related to tissue repair or cell engraftment. However, in terms of pathogenic mechanism of heart failure, could you speculate on which target antigen these Ig are produced? For example, do you have any data on IgG or IgM against alfa- myosin heavy chain?

Response: We thank the editor for this remark. We are currently working to identify the target antigens to which the IgGs are produced, which will be part of a separate manuscript. Therefore, we are currently performing a discovery epitope screening, including 26,000 antigens known in cardiovascular disease. This is still preliminary data, but depending on the different etiologies of heart failure, we identified that structural components of cardiomyocytes and cell-adhesion proteins might be affected. One of the epitopes is indeed alfa-myosin heavy chain.

We are still validating these findings in larger numbers of patients, and at this point we could only speculate that upon cardiac damage, intracellular proteins are released and exposed to the immune system, which leads to an antibody-mediated immune response against different cardiac-specific proteins, which might induce additional cardiac damage and accelerates heart failure progression. We also added an additional sentence in the coclusion:

Future studies should focus on the identification of the cardiac antigens which are targeted by the produced IgGs and on the potential of combination therapies, using both MSCs and CPCs, to simultaneously target cardiac regeneration and antibody-mediated immune responses.

3) Although the aim of the study was to investigate humoral immunity and/or antibody mediated immune response, the possibility that changes in Th (CD4 helper) compartment could affect antibody production should be acknowledged.

Response: We agree that differences in CD4 Th responses between donors may have directly altered antibody production.

It has been established that MSCs (and CPCs) can also affect CD4 T cell responses and as a consequence B cell mediated antibody production. We cannot exclude that antibody production in our experiment was indirectly altered by differences in CD4 T cell compartment. Although we did not assess CD4 T cell numbers after 10 days of co-culture, we did assess CD4-T cell numbers at baseline. We did not observe differences in the number of CD4 T cells between donors at baseline, which may suggest the CD4 T compartment will also remain similar upon stimulation. We have now touched upon this in the discussion (line 250-268):

In line with previous findings, we showed that both MSCs and CPCs significantly suppressed proliferation of lymphocytes (30,32,38). The suppressive effects of MSCs were more effective than CPCs. The suppressive effects of MSCs and CPCs on effector and regulatory T cells have been described before, where several studies show T cell inhibition via PDL-1/PD1 in a direct cell communication manner (32,51). Moreover, both MSCs and CPCs are also able to suppress CD4+ T helper cell-mediated immune responses(52). However, the interaction of progenitor cells with B cells is still controversial and this issue has recently gained more interest(53–55). MSCs can inhibit plasma cell formation and subsequent IgG production in a cell-cell contact dependent as well as in an independent manner (38,55). It is not known whether CPCs are also able to suppress antibody production in vitro. We demonstrated that, similar to MSCs, CPCs effectively suppress antibody production in vitro. We showed that both adult- and fetal-derived CPCs significantly inhibit the levels of IgM, IgG1, and IgG3, of which IgM was most efficiently suppressed, despite variation between different donors. These findings are in line with the effects of MSC, where MSC are known to exert an inhibitory effect on T helper cells, B-cell differentiation and class switching into IgG-producing plasma cells(56,57). Therefore, we could speculate that CPC might use a similar mechanism, in which IgG production might be suppressed either by inhibiting T-helper cell responses, thereby influencing B-cell activation and antibody production, or by directly influencing B-cell differentiation and subsequent class-switching.

4) The point made by reviewer #2 on patient-specific properties of CPC and MSC is of high relevance.

Response: we agree with the editor and reviewer #2. We completely understand and agree with the comment of the reviewer, that using cells derived from the same donor would be the most optimal approach to make the best comparison between the different cell types. Unfortunately, due to ethical reasons, we are not allowed to harvest the two types of progenitor cells (MSC and CPC) from the same donor. The CPCs are harvested upon coronary artery bypass grafting (CABG), which is an invasive procedure that in itself is already challenging for both the patient and surgeon. The CPCs are directly harvested form the heart, which is technically very challenging and comes with additional risk for the patient and is only obtained from planned operations and considered waist material.

5) In line with a point made by reviewer #1, the finding that adult MSC display more immunosuppressive effect than fetal cells is understandable, while the observed similar difference in CPC is more difficult to interpret. Could you please comment on this finding?

Response: In contrast with fetal CPC, which are obviously isolated from fetal tissue, adult CPC are harvested from patients with ischemic heart disease that are eligible for coronary artery bypass grafting (CABG). The CPCs are operatively collected during the CABG procedure. We could therefore speculate that these cells are already primed, as a consequence of chronic inflammation associated to atherosclerosis or oxygen deprivation due to ischemia. The priming will result in activated state that may lead to increased secretion of paracrine factors, which in this case, could result in a stronger immunosuppressive capacity as compared to fetal-derived CPC. Moreover, fetal-derived cells are highly proliferative as compared to adult-derived cells. Due to this proliferative state CPCs may secrete a different palette of paracrine factors that are more associated to cell cycle rather than immunomodulation. This is still subject for further study. We have now touched upon this in the discussion (line 295-298):

For CPCs, it has been described that fetal- and adult-derived CPCs have different developmental potentials, and adult CPCs may be more effective in cardiac repair (62,63). In addition, fetal-derived CPCs are highly proliferative as compared to adult-derived cells. Due to this proliferative state CPCs may secrete a different palette of paracrine factors that are more associated to cell cycle rather than immunomodulation. Therefore, the effects of EVs from adult CPCs may differ from fetal–derived CPCs and have to be investigated in future studies.

6) Also the observation that fetal CPC preparations are more reach in EV than the adult one might be a well-known outcome although requires a comment.

Response: We thank the Editor for this remark. We believe this may be directly related to the point raised above. Fetal-derived CPC are highly proliferative and grow must faster than adult-derived CPC. This could also influence the production of extracellular vesicles and the secretion of paracrine factors. We observed higher yields of EV production by fetal-derived cells, which may suggest that fetal-derived CPC can cope with medium starvation better compared to adult CPC.

 

Reviewer(s)' Comments to Author:

Reviewer: 1

We thank reviewer 1 for his/her constructive remarks. Answers to the questions are displayed in Italics. All changes and additions to the manuscript are underlined.

Comments to the Author

The manuscript is well written, the subject is relevant, the “story” well composed, and out-put a contribution to the basic understanding of MSC and CPC MoA and a next step for others to build on. Data however need more detail to allow validation of results and conclusion. I suggest the paper is published after revision, - provided the currently missing data upon prober presentation in fact support the claimed results.

Methods:

1) Line 113-123: Is co-culture running for 10 days with-out media change?

Response: Indeed, the co-culture is running for 10 days, without media change to make sure that all immunoglobulins which are produced are in the supernatant upon collection. We choose 10 days, based on the fact that the production of immunoglobulins normally takes place after 7-10 days after initial B-cell activation. We added some additional words in our methods section:

PBMCs were co-cultured for 10 days, without medium change, in 48-well plates (2.5x105 PBMC/well) with MSCs, CPCs (5.0x104 cells/well), or CPC-derived EVs (1x 10 µg= 6.3x1010 particles), immediately upon co-culture or 3x 10 µg (6.3x1010 particles) added every 3 days of co-culture.

2) Line 125-138: Timing of measures is a bit blurry…. I assume CFSE labelling is performed before co-culture…

Response: The reviewer is right, the CFSE labeling is performed after PBMC isolation and before the co-culture. We added this to our method section:

Flow cytometry (Gallios, Beckmann Coulter) was used to assess lymphocyte proliferation. Prior to co-culture, PBMCs were labeled with 1.5mM carboxyfluorescein succinimidyl ester (CFSE, Sigma, 21888) as described previously (33)

3) CD3 and CD19 labelling is described, - what happened to these measures, - no further evaluation is found. Flow cytometry should be described in more detail; no gating strategy is presented

Response: We understand the reviewer’s remark, indeed the data about CD3+ and CD19+ lymphocytes is not incorporated in our manuscript. After isolation and prior to co-culture, we stained the cells for CD3 and CD19 in order to assess the relative contribution of T- and B- cells to the lymphocyte population of each donor. We assessed whether the cell populations are within normal range and similar between the different donors, just to make sure we started with the same number B cells at the start of the co-culture and the differences in immunoglobulins after 10 days cannot be explained by differences in the number of B cells prior to co-culture. We did not specifically stain for CD3 and CD19 at termination of the co-culture. We added an additional sentence in our method sections:

After two washing steps with PBS, PBMCs were incubated for 30 min with fluorescent antibodies, including CD3 for T cells (Brilliant Violet 510, Biolegend, 317332) and CD19 for B cells (PE/Cy5, Biolegend 302210). After washing with PBS, PBMCs were incubated for 30 min with a fixable viability dye (eFluor506, Bioscience, 65-0866-14) to exclude dead cells. Prior to culture, general cell composition per donor was assessed by measuring the percentage of CD3+ T cells and CD19+ B cells , to ensure that the cell populations were similar between the different donors at baseline. Lymphocyte proliferation was calculated by measuring CFSE intensity and the number of cells present in each division as described before (33).

Moreover, we added an informative figure below, including the gating strategy to identify the number of T- and B cells, and PBMC cell composition and the percentage of T and B cells at baseline prior to co-culture. If the reviewer feels that this figure is of great value for our manuscript, we can include this as a supplementary figure in our manuscript.

Results

4) An over-all concern in the results section: I have no idea how many replicates data are based on….- and data is completely missing…. only significance levels are presented…

Response: With respect, we don’t understand which data the reviewer refers to when stating data is completely missing. All the data related to this work is part of the manuscript or supplemental data. We do apologize that the number of replicates was not described clearly enough. In the original paper we touch upon that in the figure legends, were we stated the number of PBMC donors per experiment. Each individual PBMC donor is considered as independent experimental number (n), ranging from 2-8 donors per experiment. We now also added this in our statistics section:

Statistical analysis and data representation were performed using IBM SPSS Statistics 21 and Graphpad Prism© (GraphPad Software Inc. version 8.01, San Diego CA, USA). Normal data distribution was tested using the Kolmogorov-Smirnov test. Group comparison was performed by a one-way ANOVA or Kruskal-Wallis test, corrected for multiple comparison testing. Each individual PBMC donor is considered as an independent individual experimental number (n), ranging from 2-8 donors per experiment. Data was considered significant with two-tailed p-values <0.05 and is presented as mean ± SEM.

5) Which absolute/relative values were obtained?

Response: Due to donor differences in the absolute values of immunoglobulin levels, we decided to use relative values and normalize our data per donor. PBMCs stimulated with IL-2 and PMA, without addition of MSCs, CPCs or EVs, served as our positive control and was defined as maximal stimulation (100%). This condition was used as a reference to normalize the other conditions per donor and per experiment. Meaning for each individual donor a maximum response was determined and the suppressive capacity of MSC, CPCs or EVs is depicted relative (as ratio) to this maximal response. We clarified this in our method section and in addition to the significance levels, we also added the relative values in our results section:

Production of IgM and different IgG subclasses is suppressed by cardiac-derived progenitor cells.

Next to reduced cell proliferation, MSCs are also able to inhibit several immune cell functions, such as antibody secretion (38). To examine whether this also holds true for CPCs, we collected the supernatant after 10 days of co-culture and measured the levels of different immunoglobulin subclasses (Fig 2A). Since it is known that the age of the donor can affect their inhibitory potency (39,40), both fetal and adult MSCs and CPCs were included. Adult and fetal-derived MSCs significantly inhibited antibody production from stimulated PBMCs (Fig 2B-F). Fetal and adult MSC significantly reduced the production of IgM (aMSC=0.005±0.0 fMSC=0.02±0.0; p<0.0001), IgG1 (aMSC=0.24±0.06, fMSC=0.28±0.06; p<0.0001), IgG3 (aMSC=0.19±0.06, fMSC=0.25±0.09; p<0.0001) and IgG4 (aMSC=0.29±0.07; p<0.01, fMSC=0.43±0.1; p<0.05). In addition, also CPCs showed strong suppressive effects on the production of mainly IgM (aCPC=0.02±0.0; p<0.0001, fCPC=0.38±0.16; p<0.001), IgG1 (aCPC=0.12±0.02; p<0.001, fCPC=0.55±0.18; p<0.05) and IgG3 (aCPC=0.06±0.0; p<0.0001, fCPC=0.44±0.14; p<0.001). For CPCs, the strongest immunosuppression was observed using adult CPCs.

CPC-derived extracellular vesicles suppress antibody production, but are not as effective as direct cell-cell interaction when using CPCs

The production of IgM, IgG1, and IgG4 was significantly decreased using the 3x dose of CPC-derived EVs (IgM=0.35±0.05; p<0.05, IgG1=0.57±0.03; p<0.05, and IgG4=0.66±0.0; p=0.03), thereby indicating that long term suppression is more effective than a single dose of EVs. However, the inhibitory effect was most robust when adding CPCs and not CPC-derived EVs, with strongest suppressive effects on the release of IgG1 (0.59±0.1; p<0.05), IgG2 (0.23±0.06; p=0.02), IgG4 (0.53±0.03; p=0.01) and IgM (0.17±0.03; p<0.01).

MSCs show the strongest immunosuppressive effects and are more likely to be used as cell therapy in end-stage HF patients

Upon co-culture of patient-derived PBMCs with MSCs or CPCs, antibody production was significantly suppressed (Fig. 4B-C). Mainly MSCs showed strong suppressive effects, as they significantly decreased the production of IgM (0.02±0.0; p<0.0001), as well as all IgG subclasses (IgG1=0.25±0.08; p=0.001, IgG2=0.03±0.02; p<0.0001, IgG3=0.25±0.08; p=0.009, IgG4=0.19±0.07; p=0.0006). Co-cultures using CPCs showed similar suppressions, albeit at a lower level and the differences were only statistically significant for IgM (0.20±0.06; p<0.0001) and IgG2 (0.31±0.14; p=0.0003).

6) What is the sensitivity and ranges of the IgG/M assay used, - are data in fact with-in these ranges?

Response: We made use of the Bio-plex Pro Human Isotyping assay. In this assay, a quality control is included to ensure whether the assay was performed correctly and whether the measured values were in the normal ranges according to the assay. Proper sample dilution was first tested in each donor, ensuring all samples were in the linear part of the standard curve and thus within the detection range of the assay.

We added the assay performance according to the manufacturer below. We can confirm that our data was in fact within these ranges, when samples were diluted 5 (we also added this in the method section).

The levels of IgM and IgG subclasses (IgG1, IgG2, IgG3, IgG4) in the co-cultures (5x diluted) were measured using a Bio-Plex Pro™ human isotyping immunoassay 6-plex (Bio-Rad, 171A3100M) according to manufacturer’s instructions and were all within the detection limit of the assay.

Biorad: https://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_6344.pdf

Discussion

7) A few more words on the difference between IgG and IgM; IgM is most efficiently suppressed. How do you interpret this finding? Any suggestions on why, and what the consequence of this finding may be?

Response: We thank the reviewer for this remark. With the approach we now used, we mainly aimed to assess if antibody production by B cells could be altered by MSC or CPCs we therefore chose to stimulate PBMC with PMA, which is a general activator of lymphocytes and also stimulates B-cell division and antibody production. IgM is the first antibody produced in great amounts upon activation, which in vivo is followed by the production of antigen-specific IgG after class-switching. Since we use a non-antigen specific stimulus (PMA) in our opinion it is not un-logical that we observe the most robust effect on IgM production. We expect the effect on IgGs may have been more pronounced when stimulating the cells with cardiac derived antigens, which a). are likely different between donors and b) have not been fully elucidated yet. In patients with heart failure we observe that mostly IgG is increased in the circulation and in the heart of end-stage heart failure. Obviously, more work is needed to determine patient specific epitope signatures (beyond the scope of this manuscript), but IgG in general and more specifically antigen specific IgGs may be considered as biomarkers for disease progression or even targets for therapeutic interventions in end-stage HF.

Indeed, the levels of IgM appear to be more efficiently suppressed in this in vitro setting compared to IgG. We did not look into the exact effects of progenitor cells on B-cell differentiation and class switching in this manuscript, and we could therefore only speculate that these findings might indicate that our progenitor cells might already have suppressive effects on B-cell differentiation and class switching, which in the end also results in lower levels of IgG. We added some additional sentences in the discussion of our manuscript:

We demonstrated that, similar to MSCs, CPCs effectively suppress antibody production in vitro. We showed that both adult- and fetal-derived CPCs significantly inhibit the levels of IgM, IgG1, and IgG3, of which IgM was most efficiently suppressed, despite variation between different donors. These findings are in line with the effects of MSC, where MSC are known to exert an inhibitory effect on B-cell differentiation and class switching into IgG-producing cells (56,57). Therefore, we could speculate that CPC might use a similar mechanism, in which IgG production might be suppressed either by inhibiting T-helper cell responses, thereby influencing B-cell activation and antibody production, or by directly influencing B-cell differentiation and subsequent class-switching.

8) Adult CPC are more efficient that fetal; - can this be explained?

Response: In contrast with fetal CPC, which are obviously isolated from fetal tissue, adult CPC are harvested from patients with ischemic heart disease that are eligible for coronary artery bypass grafting (CABG). The CPCs are operatively collected during the CABG procedure. We could therefore speculate that these cells are already primed, as a consequence of chronic inflammation associated to atherosclerosis or oxygen deprivation due to ischemia. The priming will result in activated state that may lead to increased secretion of paracrine factors, which in this case, could result in a stronger immunosuppressive capacity as compared to fetal-derived CPC. Moreover, fetal-derived cells are highly proliferative as compared to adult-derived cells. Due to this proliferative state CPCs may secrete a different palette of paracrine factors that are more associated to cell cycle rather than immunomodulation. This is still subject for further study. We have now touched upon this in the discussion (line 295-298):

For CPCs, it has been described that fetal- and adult-derived CPCs have different developmental potentials, and adult CPCs may be more effective in cardiac repair (62,63). In addition, fetal-derived CPCs are highly proliferative as compared to adult-derived cells. Due to this proliferative state CPCs may secrete a different palette of paracrine factors that are more associated to cell cycle rather than immunomodulation. Therefore, the effects of EVs from adult CPCs may differ from fetal–derived CPCs and have to be investigated in future studies.

Reviewer: 2

We thank the reviewer for his/her constructive remarks. Answers to the questions are displayed in Italics. All changes and additions to the manuscript are underlined.

1) This is a very interesting study with novel findings. However, the study has the major limitation of showing only in vitro/ex vivo data. While it is clear that requesting an in vivo study would be totally off the mark, it is however essential that at least the in vitro findings are technically undisputable to back the conclusions made.

Response: We agree with the reviewer. Validating our findings in an in vivo model is also in our opinion the next step and we are currently working on the details and future experiments.

2) On this premise, it would be essential that the comparison among the two progenitor cell populations was made using cells derived from the same human donor (same HLA type). From the methods section this doesn't seem to be the case for the presented experiments.

Response: we agree with the editor and reviewer #2. We completely understand and agree with the comment of the reviewer, that using cells derived from the same donor would be the most optimal approach to make the best comparison between the different cell types. Unfortunately, due to ethical reasons, we are not allowed to harvest the two types of progenitor cells (MSC and CPC) from the same donor. The CPCs are harvested upon coronary artery bypass grafting (CABG), which is an invasive procedure that in itself is already challenging for both the patient and surgeon. The CPCs are directly harvested form the heart, which is technically very challenging and comes with additional risk for the patient and is only obtained from planned operations and considered waist material.

3) Mixing the two cell types (or their derivatives) enhances the effect of the single cell type alone? This is important because it would indirectly hypothesize whether the mechanism of action on B-cell target by the MSCs and CPCs is similar or unique to each of them.

Response: We thank the reviewer for this suggestion. We agree with the reviewer, however, we encountered technical difficulties in mixing the same cell types in our in vitro co-culture. MSC and CPC both have their own specific medium and culture protocols, therefore, combining these two cell types in one well cannot be performed without creating and validating a new culture protocol, to make sure that the functional and paracrine effects of both cell types are not altered or influenced. Moreover unfortunately, it is not possible to obtain MSCs and CPCs from the same human donor. Therefore, as a consequence, combining MSCs and CPCs would result in mixing two different HLA types/ donors, which in our opinion is not favorable.

Mixing two types of extracellular vesicles (EVs) is a nice suggestion and would be a good addition to our paper. However, we encountered technical difficulties in obtaining sufficient numbers of MSC-derived EVs. In order to purify our EV population, we cultured our MSCs in serum-free culture media before EV collection and isolation. However, MSCs cultured in serum-free medium, in our hands, fail to produce sufficient numbers of EVs for further analysis. Therefore, it is not possible to combine MSC-EV with CPC-EVs.

The use of cell-derived conditioned medium is in our opinion, the only possible way to combine the derivatives of the two cell types. However, we expect this will not be directly comparable with our EV data, since conditioned medium contains many other (un)identified soluble paracrine factors secreted by the cells next to EVs.

Therefore, we feel the suggestion is of great additional value and was therefore also suggested as potential future therapeutic direction in our discussion, but due to the technical limitations described above, we cannot answer this question at the moment.

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Decision Letter 1

Federico Quaini

17 Dec 2019

Potential of mesenchymal- and cardiac progenitor cells for therapeutic targeting of B-cells and antibody responses in end-stage heart failure

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Reviewers' comments:

Acceptance letter

Federico Quaini

20 Dec 2019

PONE-D-19-25026R1

Potential of mesenchymal- and cardiac progenitor cells for therapeutic targeting of B-cells and antibody responses in end-stage heart failure

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    Supplementary Materials

    S1 Fig. Extracellular vesicle markers.

    Proper isolation of extracellular vesicles (EV) was determined by the presence of CD63, CD81 and Alix and absence of the cellular marker Calnexin by Western Blotting. Cell lysates (Cl) were used as controls.

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