Human pluripotent stem cell-derived cardiomyocytes as a target platform for paracrine protection by cardiac mesenchymal stromal cells

Ischemic heart disease remains the foremost cause of death globally, with survivors at risk for subsequent heart failure. Paradoxically, cell therapies to offset cardiomyocyte loss after ischemic injury improve long-term cardiac function despite a lack of durable engraftment. An evolving consensus, inferred preponderantly from non-human models, is that transplanted cells benefit the heart via early paracrine signals. Here, we tested the impact of paracrine signals on human cardiomyocytes, using human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) as the target of mouse and human cardiac mesenchymal stromal cells (cMSC) with progenitor-like features. In co-culture and conditioned medium studies, cMSCs markedly inhibited human cardiomyocyte death. Little or no protection was conferred by mouse tail tip or human skin fibroblasts. Consistent with the results of transcriptomic profiling, functional analyses showed that the cMSC secretome suppressed apoptosis and preserved cardiac mitochondrial transmembrane potential. Protection was independent of exosomes under the conditions tested. In mice, injecting cMSC-conditioned media into the infarct border zone reduced apoptotic cardiomyocytes > 70% locally. Thus, hPSC-CMs provide an auspicious, relevant human platform to investigate extracellular signals for cardiac muscle survival, substantiating human cardioprotection by cMSCs, and suggesting the cMSC secretome or its components as potential cell-free therapeutic products.


Results
Suppression of cell death by mouse cMSc in human cardiomyocytes derived from pluripotent stem cells. To identify mechanisms underlying the reduction of infarct size and prevention of heart failure conferred by intramyocardial injection of cMSC previously described, we used cells that were prospectively isolated using the same markers, Sca1 + SP cells 5 . Specifically, a pro-survival paracrine effect on cardiomyocytes was postulated. For a direct assessment of cMSC's paracrine effects in a relevant human context, a co-culture system was established using human ventricular cardiomyocytes derived from pluripotent stem cells (vCor.4U, Ncardia) 20,21 , plated at the bottom of a two-chamber system, with Sca1 + SP stromal cells above (Fig. 1a, top). By this means, the "target" and "secreter" cell populations were separated by a microporous membrane that prevents cell migration but allows the transit of secreted factors. Co-cultures were exposed to menadione, a naphthoquinone that induces endogenous reactive oxygen species (ROS) 32 , instrumental mediators of ischemia-reperfusion injury as in myocardial infarction ( Supplementary Fig. S1) 23,33,34 . As shown by the 56.5% inhibition of DRAQ7 uptake, co-culture with cMSCs protected the human cardiomyocytes from cell death after oxidative stress (p = 0.0007; Fig. 1a, bottom).
Previously, we showed that SP cells are enriched for PDGFRα which is synergistic with the SP status as a determinant of clonal growth and together define the highly clonogenic cardiac stromal cells with cardiovascular differentiation capacity after grafting 5 . As expected, PDGFRα was enriched in the expanded Sca1 + SP cells used for the present experiments ( Supplementary Fig. S2A). Importantly, to exclude fortuitous effects from the genotoxicity of Hoechst 33342 and ultraviolet irradiation required for preparative sorting of SP cells, we also tested short-term pools of PDGFRα + CD31 − Sca1 + adult mouse cardiac cells, expanded for 5-10 passages. cMSCs produced by this alternative protocol were highly enriched for the SP phenotype ( Supplementary Fig. S2B,C) and provided comparable protection in co-culture to hPSC-CMs ( Supplementary Fig. S2D).
To explore cMSC's paracrine signalling further, the cells were plated at increasing densities, and the respective media were added to vCor.4U cardiomyocytes at three concentrations at the time of menadione treatment (Fig. 1b, top). As shown by the dose-dependent reduction of DRAQ7 + uptake, cMSC-conditioned media suppressed human cardiac muscle cell death, in proportion to donor cMSC number and the conditioned media concentration, reaching > 85% inhibition of cardiomyocyte death at ~ 50,000 cMSC/cm 2 (p < 0.0001; Fig. 1b,  bottom). In a direct side-by-side comparison, the conditioned media from tail tip fibroblasts conferred no statistically significant protection (Fig. 1c). In contrast to this potent reduction of cardiomyocyte death, the (a) cMSC protect vCor.4U human ventricular myocytes from lethal oxidative stress in trans-well co-culture. Above, schematic representation and timeline. Middle, representative images of nuclear DRAQ7 staining after a menadione challenge. Below, bar graph of DRAQ7 uptake; n = 6. cMSC were prospectively sorted using Sca1 and the SP phenotype, and were 90% PDGFRα + .  Fig. S2E).
To test whether cMSC-conditioned media might disrupt human cardiomyocyte death more generally, vCor.4U hPSC-CMs were treated with the cardiotoxic anthracycline, doxorubicin 22 . As with menadione, cardiomyocyte death triggered by doxorubicin was markedly suppressed by cMSC-conditioned media (p < 0.0001; Fig. 1d).
Next, IMR90-derived cardiomyocytes 35 were likewise tested as a target for cardiac protection, as an independent, complementary hPSC-CM line, derived from a different human donor, tissue source, method of generation, method of differentiation, and maintenance medium. Immunostaining against the cardiac-specific marker α-actinin, demonstrated a purity of 96.2 ± 2.6% in IMR90-CMs ( Supplementary Fig. S3A). RNA-seq analysis of the IMR90-CM transcriptome indicates a level of maturity at least comparable to hPSC-CMs previously used for translational studies 22,35 (Supplementary Fig. S4; Table S5): (1) no residual expression of pluripotency and cardiac mesoderm genes (NANOG, SOX2, POU5F1, EOMES, TBXT, KDR, PDGFRA); (2) highly robust expression of sarcomeric genes (ACTC1, MYH7, TNNC1,TNNT2); (3) presence of transcripts encoding for SERCA2 (ATP2A2); (4) expression of major adult L-type calcium channels (e.g. CACNA1C) as well as the major sodium (SCN5A) and potassium (KCNH2) cardiac channels; (5) absence or low expression of fibroblast markers (DCN, DDR2) and (6) expression of typical cardiac transcription factors such as NKX2-5 ( Supplementary Fig. S3B-D). Furthermore, hPSC-CMs under these conditions of culture, have progressed to a mature, mitogen-resistant state, consistent with the reported post-mitotic phenotype in other studies, even in 2D culture 36,37 . Consistent with this empirical finding, RNA-seq analysis shows: (1) high expression of the tumor suppressor gene RB1 versus its relative RBL1 (p107), which underlies the developmental switch to a post-mitotic phenotype 38-40 ; (2) high expression of CDKN1A encoding for Cdk inhibitors p21 and the transcript for p27, CDKN1B ( Supplementary  Fig. S3E). Exploratory studies suggested a possible trophic effect of conditioned medium on hPSC-CMs as shown by the increase in NPPA expression, however, this increase was only two-fold, much smaller than in bona fide hypertrophy; also NPPB expression increased just 1.5 fold. These changes were not accompanied by downregulation of MYH6 nor induction of ACTA1 and MYL4 ( Supplementary Fig. S3F) [41][42][43][44] .
Consistent with the results in vCor.4U cardiomyocytes, cMSC-conditioned media protected IMR90-derived CMs from cell death triggered by either menadione or doxorubicin, as well as by imatinib, a cardiotoxic anticancer tyrosine kinase inhibitor (Fig. 1e,f; Supplementary Fig. S6A). Partial protection was seen, even when giving cMSC-conditioned medium one hour after oxidative stress ( Supplementary Fig. S6B). Thus, mouse cMSCs confer a potent pro-survival benefit in human cardiomyocytes derived independently from two pluripotent cell lines, and three independent death signals. Taken together, these data support the conclusion that cMSC-dependent cytoprotection is generalisable to clinically relevant compounds, beyond just the use of menadione as a model for oxidative stress.
cMSc-conditioned medium down-regulates apoptosis-related genes and preserves cardiac structural genes in lethally stressed human cardiomyocytes. To explore the protection of hPSC-CMs from menadione mediated by cMSC-conditioned medium, unbiased RNA-sequencing was performed on the following groups of IMR90-derived CMs: (1) untreated, (2) treated for 24 h with menadione, (3) treated with cMSC-conditioned medium and (4) treated together with menadione and cMSC-conditioned medium (Fig. 2a). A total of 3,628 genes were differentially expressed across the four groups (log 2 fold change > 2 and p value < 0.05), with the majority of these changes occurring in the transcriptome of menadione-treated myocytes relative to their basal expression (2,719 differentially expressed genes; Fig. 2a). Gene Ontology (GO) analysis of the transcripts induced in stressed myocytes identified categories directly relevant to the observed protection including "regulation of cell death" (153 genes, FDR 7.56E−19), along with other GO categories relevant to paracrine circuits like "response to cytokine" (126 genes, FDR 9.66E−19), "cell activation" (138 genes,  Table S7). Changes related to cell death included the induction of genes encoding proteins with proven adverse functions in cardiac muscle cell survival, such as death domain receptors (TNFRSF8, TNFRSF10D, TNFRSF18) 45 and DNA damage-inducible proteins and growth arrest mediators (GADD45B, GADD45G) 46,47 . Notable was the upregulation of PMAIP1/NOXA, whose product regulates mitochondrial membrane permeabilization and the release of apoptogens 48 . Given that cardiomyocyte function is severely affected under conditions of oxidative stress, it was not surprising to detect downregulation of genes related to GOs of "heart contraction"' (FDR 5.66E−08) and "metal ion transport" (FDR 5.82E−07), encompassing transcripts encoding major cardiac ion channels (CACNA1G, KCNJ4, KCNJ5, SCN1A, SCN5A), transporters (ATP1A2), and sarcomeric genes (MYH7, TNNI2) ( Fig. 2c; Supplementary Table S7) 46,47 .
Treatment with cMSC-conditioned media broadly interfered with menadione-dependent gene expression in the lethally-stressed cardiomyocytes, with 2,421 transcripts affected (Fig. 2a). Conditioned medium prevents the expression of ~ 80% of the menadione-induced genes and it rescues > 40% of the transcripts that were downregulated in the stressed cardiomyocytes (Fig. 2b). Specifically, the cMSC secretome prevented the expression of most genes associated with the GO cluster "regulation of cell death" (FDR 8.48E−15), while preserving expression of genes in GOs related to cardiac function such as "muscle contraction" (FDR 4.92E−05) ( Fig. 2d; Supplementary  Table S7). Intriguingly, treating menadione-stressed myocytes with the cMSC secretome inhibited the expression of transcripts for oxidative stress response factors (ETS1, MMP3, FOS, CYP1B1, ANXA1) 46,47,49 , pro-death signals (TNFRSF8, TNFRSF10D, TNFRSF12A) 45,47 and Toll-like receptors (TLR4, TLR6), which are part of the innate immunity circuit in cardiac muscle mediating some forms of cardiomyocyte death. Also, notable was interference with several transcripts encoding pro-apoptotic proteins known to affect cardiomyocytes (ALK7, BCL10, GADD45B, PHLDA) 46   www.nature.com/scientificreports/ summary, transcriptomic analysis and, specifically, the down-regulation of death-and apoptosis-related genes by cMSC-conditioned medium suggests that the potent rescue of human cardiomyocyte survival depends on attenuation of one or more transcriptional circuits for apoptosis.
the mouse cMSc secretome blocks apoptosis and the dissipation of mitochondrial potential induced by menadione. Given the transcriptomic prediction above, cardiac DNA fragmentation was evaluated by the TUNEL assay to test empirically both the prevalence of apoptosis following menadione treatment and whether this is blocked by cMSCs. Human cardiomyocytes treated with menadione ± conditioned media at three different concentrations were compared to untreated cells and tested by TUNEL assay after 24 h. cMSCconditioned media reduced DNA fragmentation to the basal levels seen in unstressed cells, even with 33% conditioned media, the lowest concentration tested (p < 0.0001; Fig. 3a). As the classically defined "intrinsic" apoptotic pathway hinges on mitochondrial depolarisation, we monitored mitochondrial membrane potential using the cell-permeant dye, tetramethylehodamine methyl ester perchlorate (TMRM; Fig. 3b; Supplementary Fig. S6C).
In the absence of oxidative stress, vCor.4U and IMR-90 cardiomyocytes showed abundant active mitochondria, as expected. Following oxidative stress, loss of the TMRM signal was confirmed. Conversely, cMSC-conditioned media preserved mitochondrial membrane potential in each of these two independent human cardiomyocyte lines (p = 0.0004 and p < 0.0001, respectively, in vCor.4U-and IMR-90-derived cardiomyocytes). Given known actions of menadione in both the cytosol and mitochondrial compartments 54 , and the role of mitochondria in feedback loops that amplify ROS production, we next tested whether cMSC-conditioned media can attenuate the formation of ROS induced by menadione. CellROX staining demonstrated that cMSC-conditioned media strongly reduces whole-cell ROS production in hPSC-CMs ( Fig. 3c; Supplementary Fig. Fig. S6D,E), resembling the impact of bone marrow-or iPSC-derived MSC signalling to rodent cardiomyocytes 55 . Using MitoSOX, which detects mitochondrial superoxides specifically, we further showed that cMSC-conditioned media blocks superoxide production within the mitochondrial compartment (Fig. 3d). These data suggest that cMSC-conditioned media impinge on the mitochondria-dependent apoptotic pathway in human cardiomyocytes, at least in part through interference with sustained ROS accumulation.
An exosome-independent mechanism mediates the observed protection by mouse cMScs. In several noteworthy studies, the paracrine benefits of cell therapy products like certain reported mouse and human cMSCs, cardiosphere-derived cells, MSCs, and PSC-CMs have been attributed to cell-cell communication by exosomes 56 . To assess whether exosomes from the well-characterised cMSCs used here mediate the observed protection of cardiomyocytes, hPSC-CMs challenged with menadione were treated with exosomedepleted or -enriched cMSC-conditioned medium, versus unfractionated cMSC-conditioned medium as the control. Exosomes were isolated by ultracentrifugation and their resulting depletion or enrichment was validated by flow cytometry 57 : isolated exosomes were captured using CD63-coated latex beads and detected with a fluorescently conjugated antibody against CD9 (Fig. 4a,b). As previously reported for this procedure 57 , exosomes were enriched more than 20-fold, comparing the respective fractions (Fig. 4b,c). Whereas exosome-depleted medium conferred protection at least equal to that of the unfractionated medium control, myocyte survival was not improved by exosome-enriched medium compared to the unfractionated media and slightly worsened compared to the standard medium (Fig. 4d). Similar results were obtained by complementary procedures, using an independent spin-column-based method for exosome precipitation and size-fractionation ( Supplementary  Fig. S8). Thus, under the specific conditions tested here, mechanisms alternative to exosomes likely mediate the observed protection of human cardiomyocytes. Consequently, the possibilities include growth factors, cytokines, chemokines, non-vesicular RNAs, lipids and low-molecular weight metabolites. Therefore, to obtain inferences on the nature of the factors mediating the protective effects, thermal stability testing was performed. Paracrine protection depends on thermolabile molecules and on molecules exceeding 3 kDa in size, for both hPSC-CM lines (Fig. 4e,f; Supplementary Fig. S6F,G). The increase in cell-death in heat-treated conditioned-medium is likely due to the depletion of essential nutrients. Together, these results provide evidence against a metabolite as the responsible mediator and are consistent, instead, with protection by one or more protein factors. An exploratory screening of the cardioprotective cMSC secretome was performed using cytokine membrane arrays pre-printed with capture antibodies against 308 mouse proteins. Conditioned medium from cMSC was compared to non-protective TTF and IMR90 basal medium. Statistically significant enrichment was seen for just 7 proteins (DTK, GDF-8, IGFBP-2, IL-15Rα, M-CSF, OPN and TRAIL) in cMSC-conditioned medium compared to the two non-protective controls ( Fig. 4g; Supplementary Fig. S9). Of these, M-CSF, IL-15Ra and IGFBP2 have each been implicated in pro-survival signalling in the heart and specifically have been shown to inhibit cardiomyocyte apoptosis [58][59][60] . GDF-8 best known as an inhibitor of skeletal muscle growth, regulates energy homeostasis in the mouse heart, prevents cardiac hypertrophy and inhibits cardiomyocyte proliferation in zebrafish, with no demonstrated impact on apoptosis 61,62 . The DTK receptor tyrosine kinase was recently shown to undergo a cleavage step and have an anti-apoptotic effect in cancer cells, though the role of the released extracellular domain is still unclear 63 . Two pro-apoptotic mediators that commonly promote cell death but have highly variable consequences in cardiomyocytes, including suppressive effects on cell death (OPN, TRAIL) 45,[64][65][66] were also enriched in the cMSC secretome. Together, our results suggest that a combination of factors is likely mediating paracrine signals whose net effect is cardio-protection.   68 . As predicted, expression of PDGFRα and MSC markers (CD29, CD44, CD73, CD105) was detected by flow cytometry (Fig. 5b) and by single-cell qPCR (Fig. 5c). Cardiac transcription factors were co-expressed uniformly (GATA4, GATA6, HAND2, MEF2A, MEF2C, TBX20), but no later differentiation markers were observed ( Fig. 5c; Supplementary Fig. S10). Typically, human dermal fibroblasts, used as controls, were enriched for CD90 and did not express GATA4, GATA6 or TBX20 ( Fig. 5c; Supplementary Fig. S10). Hence, by these combined criteria, these human cells strongly resemble the mouse cells conferring cardiomyocyte protection in Figs. 1, 2, 3 and 4 5 . In addition, they resemble certain human cardiac mesenchymal cells reported previously 11 , albeit without need of histone deacetylase depletion, and correspond better, by this signature, to the cardiac-resident MSC 27 and closely related populations 6,13 . Samples from all five patients tested suppressed cell death induced by menadione in the two human cardiomyocyte lines (p < 0.0001; Fig. 5d). Human dermal fibroblasts, used as an irrelevant control, had no effect. In summary, we isolated and expanded a population of human cardiac stromal cells with surface properties, molecular signatures, side population phenotype, and protective potency substantially comparable to those of the cardioprotective mouse cMSCs. To our knowledge, this is the first evidence that paracrine signals from human cardiac stromal cells can suppress human cardiac muscle cell death.

Human cardiac stromal cells protect human cardiomyocytes. PDGFRα
Conditioned media from mouse cMSC is sufficient to suppress local cardiomyocyte apoptosis after myocardial infarction in mice. In order to test whether cMSC-conditioned medium, as a cell-free product, has a protective effect on cardiomyocytes in vivo, we performed permanent ligation of the left coronary artery inducing myocardial infarction in C57Bl/6 mice 5 . Conditioned media produced by cMSC and unconditioned control media were concentrated and injected into the border zone at the time of injury (Fig. 6). To confirm intramyocardial delivery and spatial distribution, the conditioned and control media were co-injected with a lipophilic fluorescent carbocyanine dye ( Supplementary Fig. S11). Hearts were collected 24 h post-MI, when cell death is virtually complete 69 , and cardiac DNA fragmentation detected by terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) was used as a selective measurement of cell death through apoptosis (Fig. 6). As expected, a significant reduction (> 70%) of cardiomyocyte apoptosis was detected in the injected border zone (p = 0.02), with no significant reduction in the core region of infarction, distant from the injection site. In summary, conditioned media from cMSC is sufficient, not only, to promote human cardiomyocyte survival, but also, as proof of principle in vivo, to suppress local cardiomyocyte death in mouse myocardial infarction.

Discussion
Organ-level benefits that have been ascribed, provisionally, to paracrine signals from grafted cells include scar regression, myocyte proliferation, resident stem cell activation, immunomodulation, and cardiomyocyte protection, among others 5,7-10, 16 . As with cardiac target validation for other pathobiological and therapeutic circuits, these premises will likely benefit from rigorous testing in a human context, not animal models alone 20,21,70 . The irregular availability of human cardiomyocytes from biopsies or explanted hearts and the inability of cardiomyocytes from these sources to expand in culture have made the human cardiac phenotype inaccessible for routine and scalable experimental therapeutics. The advent of hPSC-CMs has overcome this long-standing impasse, albeit applied thus far more to safety pharmacology 21 , drug-induced cardiomyopathies 22 , and hereditary disorders 71 than to the globally most prevalent forms of acquired heart disease including ischemic heart disease. Targeting cardiac muscle cell number to reduce infarct size-further preventing cell death as an adjunct to reperfusion-is an urgent therapeutic need to improve survival post-MI 16 , as infarct size predicts the risk of heart failure and 1 year all-cause mortality 72 . Thus, considering the past failures of promising interventions for cardiomyocyte protection, all based on animal models alone, we emphasize the use of a human model, instead, to establish preclinical efficacy and explore the mechanisms of benefit.
Here, we demonstrate the utility of hPSC-CMs as a human platform to investigate paracrine signals that underpin cardioprotection by cardiac cell therapy 16 . Cell death induced by oxidative stress, apoptotic DNA Figure 4. An exosome-independent mechanism mediates the observed protection of human cardiomyocytes by mouse cMSC. (a) Schematic of exosome capture and detection using anti-CD63-conjugated latex beads plus FITC-anti-CD9. (b,c) Bead-exosome complexes were analysed by flow cytometry. (b) Representative contour plots are shown. The gate defines the CD63 + CD9 + exosomes. The top row shows controls for staining and the bottom row the proportion of exosomes in unfractionated conditioned media versus the depleted (exodepleted) and enriched (exo-enriched) fractions. FSC forward scatter. More than 5,000 beads were scored for each condition shown. (c) Bar graph of CD9 + depletion and enrichment; n = 3. (d) DRAQ7 uptake in vCor.4U human ventricular myocytes after menadione ± cMSC-conditioned media or the indicated fractions. Above, representative images. Scale bar, 50 μm. Below, bar graph of DRAQ7 uptake; n = 10. (e) Thermostability testing of cMSC-conditioned media. Bar graph of DRAQ7 uptake; n = 9. (f) Size-fractionation of cMSC-conditioned media. Bar graph of DRAQ7 uptake; n = 6. For all panels, data are shown as the mean ± SEM. *p < 0.05; **p < 0.001; ***p < 0.0001. (g) Bar graphs of cytokine levels, using low-density membrane arrays, for the factors enriched in cMSC-conditioned medium vs medium only or TTF-conditioned medium. Results are image analysis of integrated density, normalised to the average of anti-streptavidin and anti-HRP controls for each membrane. Enlarged versions of the arrays can be found in Supplementary Fig. S9. Data are mean ± SEM; n = 2; *p < 0.05; **p < 0.01; ***p < 0.001. Unpaired one-tailed t test between cMSC and medium or cMSC and TTF. Graphics were created using Servier Medical Art website, a free medical image database with a licence under Creative Commons Attribution 3.0 Unported License (https ://creat iveco mmons .org/licen ses/by/3.0/).  www.nature.com/scientificreports/ fragmentation, the dissipation of mitochondrial membrane potential, and mitochondrial superoxide formation were all suppressed by cMSC-conditioned medium, using murine cMSC that prevent adverse remodelling and heart failure in infarcted mice. As further confirmation of the cMSC-dependent paracrine protective effect, conditioned medium was shown to reduce cardiomyocyte apoptosis in mouse myocardial infarction, within the injected region. Intramyocardial delivery of conditioned media results in a localised diffusion as shown by the tracking agent that was co-injected. Therefore, to test improvement of cardiac function, tissue engineering approaches will be required to ensure broader and controlled delivery of conditioned media in space and time.
Notably, cMSC-conditioned media also protected human cardiomyocytes from death induced by two clinically relevant cardiotoxic drugs (doxorubicin and imatinib; Fig. 1d,f; Supplementary Fig. S6A), suggesting that the cytoprotecive effect might involve multiple pro-survival mechanisms or, perhaps more likely, a shared mediator such as whole-cell ROS. Consistent with the hypothesis that protein factors mediate the paracrine protection, conditioned media were concentrated using columns with a cutoff of 3 kDa for in vivo experiments, following substantiation of this fraction's effect in the human cardiomyocytes (Fig. 4f).
Although hPSC-CMs represent a scalable human platform for cardiac drug discovery reproducing many relevant features of cardiomyocytes' function, a few cautionary notes need to be highlighted. Cells used here include vCor4U that enrich for ventricular cardiomyocytes 23 , the relevant cells in a model of ischemic injury. Our RNA-seq data indicate that IMR-90 express cardiomyocytes' structural and functional genes in the absence of reprogramming genes, a proliferation signature or fibroblast markers. Notably, recent work demonstrates the predictive power of hPSC-CMs even in routine 2D culture 21,23,24,73 . Nevertheless, improvement of structural and functional maturity, as well as chamber specificity in vitro remains a highly sought goal. Currently improvement in maturation and specificity is achieved by the use of 3D human engineered heart tissue, mechanical or electrical conditioning, and heart-on-chip technologies although throughput and/or accessibility of these methods remain limiting factors [74][75][76][77][78] . Interestingly, low sensitivity to hypoxia/reoxygenation was previously reported for hPSC-CMs 23,79 , and in our experimental setting, although differences were observed between the lines studied in their sensitivity to menadione, the paracrine protective effect was consistent and comparable. Interestingly, higher susceptibility of hPSC-CM to ischemic damage was linked to a polymorphism of ALDH2 and, unsurprisingly, screening a single commercial line failed to capture the known cardiotoxicity of rosiglitazone 80,81 . Thus, it is plausible that patient-specific susceptibility might contribute to the differences in response to oxidative stress together with variability in the levels of maturity and/or chamber specificity. Future translational drug and target validation will need to include more ample demographic features such as gender and ethnic background. Studying cMSCs' paracrine effects in vitro provides a clean model to dissect cardiomyocyte-specific protective mechanisms, however, as in any in vitro system the effects on the cellular landscape and intercellular networks in the tissue will not be captured by this model. Notably, here, we provided evidence that the protective effect of cMSCs-conditioned media on cardiomyocytes is reproduced in the myocardium.
Of immediate benefit here, we validate a workable model to investigate extracellular signals that augment human cardiac muscle survival, demonstrating that the secretomes of mouse and human cMSC drastically suppress human cardiomyocyte death. This provides an accessible, scalable, translationally relevant platform toward future therapeutic cell-free strategies, based on the use of conditioned media but also towards more refined interventions via the identification of specific extracellular factors or their downstream mediators. The magnitude of protection conferred to human pluripotent cell-derived cardiomyocytes under these reductionist conditions, while just a first step, provides the most direct support yet for the seminal hypothesis that heart-derived cells protect human cardiomyocytes from lethal stress in clinical settings 16 .
Moreover, the assays detailed here are well-suited to high-throughput, high-content imaging with limited numbers of human myocytes per well and consequently to the large scale needed to deconvolute conditioned media in future gain-and loss-of-function studies. More comprehensive sample collection and deep, unbiased profiling should help inform the quest for a consensus secretome that tracks with human cardiac muscle cell protection and whether this changes in cardiac disease.
Under our experimental settings, we see an exosome-independent mechanism of protection. The implication of exosomes in the cardio-protective effects of our secretor cMSCs has not been previously tested and no study to our knowledge has shown exosome-mediated protection of human cardiomyocytes from apoptotic death. This might also explain the slight increase observed in hPSC-CM death in the presence of the exosome-enriched fraction.
In addition, the data are consistent with the possibility that conditioned media, if suitably optimized, could be used as a cell-free product to suppress cardiomyocyte death induced by ischemic injury, as a potential adjunct to merely restoring blood flow. The potential use of conditioned media as cell-free therapy has several noteworthy advantages over cells in terms of translatability, including most obviously, the lack of histocompatibility issues.  cMSc isolation. Hearts from C57Bl/6 adult male mice (8-13 weeks old; Charles River) were dissociated using 100 μg/ml Liberase and 50 μg/ml DNAse I (Roche) 5 . The cardiomyocyte-depleted preparation was filtered through a 70 μm mesh (BD Falcon). Hematopoietic lineage (Lin) depletion was performed using an AutoMACS Pro Separator (Miltenyi Biotec). To resolve SP and non-SP cells, staining with 5 μg/ml Hoechst 33342 (Sigma-Aldrich) was performed 5 . Adult mouse cardiac Lin -/Sca1 + /SP cells (Supplementary Fig. S1) and Lin -/Sca1 + / PDGFRα + /CD31cells ( Supplementary Fig. S1) were selected by FACS 5 .
Conditioned media production and modification. cMSC were seeded in maintenance medium onto collagen-coated 6-well plates at 20,000-100,000 cells/cm 2 . At 24 h, the medium was aspirated, plates washed, and the respective hPSC-CM medium was added. Conditioned media were collected 20-24 h later, passed through a 0.2 μm filter (GE Healthcare). Conditioned media were injected at the time of coronary artery ligation in vivo and were added to hPSC-CMs at the time of stress induction, unless otherwise indicated. Pierce PES columns with a cutoff of 3 kDa (Thermo Fisher) were used to concentrate the media for in vivo experiments and to discriminate between low and higher molecular weight factors. Thermolability testing was performed by heating at 90 °C for 10 min.

Myocardial infarction.
Coronary artery ligation was performed as described previously 5,23 . Briefly, induction of general anaesthesia was performed with 4% isoflurane, then maintained at 2% in 100% O 2 . Mice received 0.024 mg buprenorphine subcuteneously (i.e. average dose, 1.1 mg/kg; Vetergesic, Alstoe Animal Health, UK), and were intubated and ventilated with a tidal volume of 250 μl and respiratory rate 150 breaths/min (Hugo-Sachs MiniVent type 845; Harvard Apparatus Ltd., Kent, UK). Throughout the surgery heart rate, ECG and core body temperature were constantly monitored. A left thoracotomy was performed in the fourth intercostal space followed by removal of the pericardium. A 6-0 polypropylene suture was used to permanently occlude the left anterior descending (LAD). Myocardial ischemia was confirmed by blanching of the myocardium downstream of the suture and clear ST-segment elevation on ECG tracings. Concentrated conditioned media or vehicle were immediately injected into the infarct border zone (2 injections of 10 μl each). After surgery, mice were allowed to recover in a heated chamber for 20 min, then moved to a normal holding cage with supplemental heat if necessary. Twenty-four hours after MI, the hearts were collected for immunohistochemistry. Briefly, general anaesthesia was induced with 5% isofluorane and maintained at 2.5% with 100% O 2 . Animals were subsequentally perfused via intracardiac injection with PBS for 30 s and 4% PFA in PBS for 60 s prior to heart removal. immunohistochemistry and confocal imaging. Isolated hearts were fixed in 4% PFA solution for 2 h.
Subsequently, the organs were incubated in cryoprotective solutions containing gradually higher concentrations of sucrose for the indicted periods of time: 10% (~ 16 h), 20% (8 h) and 30% (~ 16 h) sucrose in distilled H 2 O. Hearts were then frozen in OCT and stored at − 80 °C until sectioning.
For tissue staining, 10 μm sections were washed and permeabilized using 0.5% Tween-20 in PBS. Apoptosis staining was performed using the Click-iT TUNEL Alexa Fluor 594 Imaging Assay (Invitrogen, C10246). After TUNEL staining, sections were blocked in 5% donkey serum and 1% BSA for 30 min, washed twice in PBS for 3 min and incubated with primary antibodies (Table S1 in Supplementary Data) overnight at 4 °C. The following day, sections were washed twice in PBS for 3 min and incubated with secondary antibodies for 2 h at 4 °C. Sections were then washed twice in PBS for 3 min, mounted in Vectashield antifade mounting medium with DAPI (Vector Laboratories), and kept at 4 °C until scanned. Slides were raised to room temperature 15 min prior to image acquisition.
Confocal imaging was performed with a Zeiss LSM-780 inverted microscope, using a EC Plan Neofluor 40×/1.3 oil objective. Images were acquired as a z-stack, ranging from 17-21 optical sections at 1.5 μm intervals, with a pinhole aperture of 1.3 μm. For each heart, we quantified 2 sections per region and 3 regions per mouse encompassing 18-24 fields each, with 10% overlap, for the infarct area, border zone and remote area (interventricular septum). Higher resolution images were acquired as representative images of the TUNEL staining by acquiring z-stacks consisting of 50 optical sections at a 0.36 μm interval, with a pinhole aperture of 1.3 μm and 2 × zoom. Post-acquisition processing was performed using Fiji (ImageJ2). TUNEL staining in cardiomyocytes (an average of ~ 3,400 cardiomyocytes counted per heart) was scored manually by an individual blinded to the experimental conditions. co-culture. HTS Transwell 96-well permeable supports with polyethylene terephthalate membranes and 1.0 micron pores were used (Corning). Cardiomyocytes were seeded onto the bottom well at a density of 100,000 cells/cm 2 in Cor.4U Complete Culture Medium. One day before starting co-cultures, cMSC were seeded at a density of 100,000 cells/cm 2 on the apical side of inserts coated with 50 μg/ml collagen in cMSC-mantainance medium. Seven days after human cardiomyocytes' seeding, inserts with cMSC were added on top of the cardio-Scientific RepoRtS | (2020) 10:13016 | https://doi.org/10.1038/s41598-020-69495-w www.nature.com/scientificreports/ myoctes and menadione (Sigma-Aldrich) treatment was started (Fig. 2a). For the control, the stressor was added to cardiomyocytes cultures in the absence of cMSC.
cell staining and high-throughput imaging. To monitor cardiomyocyte death, hPSC-CMs were stained with DRAQ7 (Biolegend) at a final concentration of 3 μmol/l and counter-stained with 8 μmol/l Hoechst 33342 (Sigma-Aldrich). Mitochondrial depolarisation was visualised using 20 nmol/l TMRM (Thermo Fisher) for 30 min at 37 °C; 0.01 mmol/l carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP; Abcam), 0.001 mmol/l oligomycin (Abcam) and 0.001 mmol/l rotenone (Abcam), 15 min before scanning, were used as the control for mitochondrial depolarization. CellROX Green (Invitrogen) and MitoSOX Red (Invitrogen) were used to detect cytosolic reactive oxygen species (ROS) and mitochondrial superoxide, after incubation with 5 μmol/l dye at 37 °C for 30 or 10 min, respectively. ROS-Glo H 2 O 2 assay (Promega) was used to moderate the production of H 2 O 2 in human IMR-90 after menadione stress. Click-iT EdU HCS assay (Invitrogen) was used to detect cardiomyocytes in S phase for the proliferation assay after incubation with the cells for 24 h and subsequent click-reaction staining. Nuclei were counterstained with Hoecsht 33342 as above.
After live-cell image acquisition, cells were fixed in 3.7% formaldehyde in PBS for 15 min at room temperature and stored at 4 °C. DNA fragmentation was detected using the Click-iT Plus TUNEL Alexa Fluor 594 assay for in situ apoptosis detection (Invitrogen) and Cellomics ArrayScan VTI.
Bulk RnASeq. RNA isolation from human IMR90-cardiomyocytes cultured at 100,000 cells/cm 2 in 6-well plates was performed using DirectZol RNA Miniprep kit (Zymo Research). Samples included four groups: (1) untreated cells; (2) cells treated with 20 μM menadione; (3) cells maintained in cMSC-conditioned media and (4) cells treated with menadione and cMSC-conditioned media. Cells were harvested for RNA isolation at 24 h after the treatments were started. Quality control was checked by RNA Qubit and TapeStation before and after library preparation and sequencing (Genewiz, UK). Sequencing was performed using Illumina HiSeq 2 × 150 bp configuration. Raw data were downloaded in FASTQ format for review of the QC results and downstream analysis. Trim Galore (https ://www.bioin forma tics.babra ham.ac.uk/proje cts/trim_galor e/) was used to trim the adapter sequences and reads were aligned to the human genome (GRCh38) using STAR 86 . Data was then imported to SeqMonk (https ://www.bioin forma tics.babra ham.ac.uk/proje cts/seqmo nk/) to quantify, explore and generate heatmaps. Specifically, quantitation was performed using the RNA-seq pipeline, correcting for genomic DNA contamination and merging transcripts isoforms. The DESeq2 R pipeline was used to identify differentially expressed transcripts between the four samples 87 . Data mining was performed using the ToppFun function in the ToppGene Suite tool to identify Gene Ontologies (GO) relevant to the highly significant differentially expressed genes between samples. Data were deposited on SRA public repository with accession number PRJNA629893. exosome isolation and characterization. Exosomes were enriched and depleted from conditioned medium by ultracentrifugation. Medium was centrifuged at 4 °C for 30 min at 2,500×g (Thermo Fisher, Heraeus Multifuge 3SR+). The supernatant was centrifuged at 4 °C for 35 min at 4,565×g, passed through a 0.2 μm filter, and centrifuged at 4 °C for 2 h at 110,000×g in an Optima XPN ultracentrifuge with a SW32 Ti rotor (Beckman Coulter). The cleared supernatant was the exosome-depleted fraction; pellets were resuspended in Cor.4U medium as the exosome-enriched fraction. For exosome identification, 75,000 4 μm aldehyde/sulphate latex beads (Thermo Fisher) were resuspended in 0.025 mol/l MES buffer [2-(N-Morpholino)ethanesulfonic acid; Sigma-Aldrich], coated with 8 μg CD63 (Biolegend) or rat IgG2α (Biolegend), incubated on a rotator for 20 min in a final volume of 50 μl PBS, then incubated for 30 min with 100 mmol/l glycine (Sigma-Aldrich) to occupy unreacted sites. Media were incubated with the antibody-coated beads in a final volume of 250 μl for 15 min. Exosome-bead complexes were stained with 10 μg/ml FITC labelled anti-CD9 (Biolegend), with gentle agitation for 30 min in the dark. Results were visualised using an LSRII flow cytometer (Becton Dickinson) equipped with five lasers 5 , and were analysed using FlowJo (v10, FlowJo). cytokine arrays. High density array membranes (RayBiotech) with 308 mouse proteins were used to screen mouse cMSC-conditioned media for the presence of secreted factors. Basal medium and non-protective TTFconditioned medium were also tested as controls. Following collection of conditioned media from cells seeded at 20,000/cm 2 , samples were dialysed overnight in PBS to remove small molecular weight molecules including salts and ions. Protein concentration in each sample was determined using the 660 nm assay (ThermoScientific) before and after dialysis and used to calculate the amount of biotin-labeling reagent to add per sample (as per manufacturer's protocol). Samples were incubated with biotin-labeling reagent at room temperature for 30 min with gentle shaking prior to addition of 5 μl stop solution to prevent further biotin binding. Spin columns and centrifugation were then used to remove unbound biotin and elute the samples. Membranes were first blocked using the provided blocking buffer for 2 h at room temperature with gentle shaking. Each sample was then diluted five-fold in blocking buffer and added onto the blocked membranes for incubation overnight at 4 °C with Scientific RepoRtS | (2020) 10:13016 | https://doi.org/10.1038/s41598-020-69495-w www.nature.com/scientificreports/ gentle shaking. Subsequently, the membranes were washed using the solutions provided by the kit and incubated with HRP-conjugated streptavidin for 2 h at room temperature with gentle shaking. After washing, the membranes were incubated with detection buffer for 2 min. Signal was captured on X-ray films (Amersham). Results were analysed using Microsoft Excel and Fiji and Java scripts were used for automation of analysis in ImageJ.
flow cytometry and sorting. Flow cytometry and sorting were performed using a FACS Aria II (Becton Dickinson) equipped with 355 nm ultraviolet, 405 nm violet, 488 nm blue, 561 nm yellow-green and 640 nm red lasers and analysed using FlowJo v10. Antibodies and dyes are detailed in Table S1.
Human subjects. Adult human heart samples were obtained from the NIH-funded National Disease Research Interchange (NDRI), with protocols reviewed and approved annually by the University of Pennsylvania Institutional Review Board. Human cardiac stromal cells were obtained by four rounds of 7 min enzymatic digestions with 90 μg/ml Liberase (Roche) and 50 μg/ml DNase I (Roche), followed by filtering through 70 μm sterile mesh. The cells were then purified by Percoll gradient centrifugation, to remove debris and red blood cells.
Single cell qRt-pcR. For single cell qRT-PCR single viable cells were sorted directly into 96-well plates containing 10 μl of the reaction mixture for pre-amplification, using CellDirect One-Step qRT-PCR kits (Invitrogen) 5 . Pre-amplification was performed for 22 cycles (Veriti Thermal Cycler, Applied Biosystems, Thermo Fisher). Non-template samples were included in each run at the pre-amplification stage as negative controls. Quantitative amplification was performed using Dynamic Array chips for 96 assays × 96 samples on the Bio-Mark HD system (Fluidigm). For TaqMan primer/probe sets (ABI, Thermo Fisher) see Supplementary Table S1. Ct values were calculated and exported using Fluidigm software (v4; Fluidigm). Data analysis was performed based on cycle threshold (Ct) values obtained by the Fluidigm software, using an R pipeline developed in house. Outliers were identified from quantile-quantile plots of cycle threshold (Ct) values for an internal control gene (Ubc), using a quality control step based on robust standardized expression fractions and expression levels 88 . Ct values were normalised on a per-sample basis, using Ubc as control, expressing all Ct values as their difference to the Ct value of Ubc for the same sample (ΔCt sample, gene = Ct sample, gene − Ct sample, Ubc ); ΔCt values were subsequently centered on the sample mean (ΔCt centered = ΔCt sample, gene − Ct sample mean ). Opposite values (− ΔCt centered ) were used for visualisation and further analyses. Euclidean distances among samples and among genes were used to compute hierarchical clustering, using the complete linkage method. Differential expression was determined using ANOVA with Tukey's test, using a significance level of p < 0.05. Dimensionality reduction was performed by principal component analysis using FactoMineR 89 . All plotting was performed using R base graphics and ggplot2.
Statistics. GraphPad Prism (versions 6 and 7) was used to perform statistical analyses. Results are shown as the mean ± SEM (p < 0.05). Data were analyzed by two-way ANOVA, using the Bonferroni correction for multiple comparisons and Welch's t test for pairwise comparisons. The immunohistochemistry data were analysed using an unpaired non-parametric one-tail Mann-Whitney test.