Extracellular vesicles derived from induced pluripotent stem cells mediate anti- inflammatory effects in primary human macrophages

Extracellular vesicles derived from induced pluripotent stem cells mediate anti- inflammatory effects in primary human macrophages | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Extracellular vesicles derived from induced pluripotent stem cells mediate anti- inflammatory effects in primary human macrophages Stephen Fitzsimons, Silvia Oggero, Eugène T. Dillon, María Muñoz-San Martín, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7436803/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 3 You are reading this latest preprint version Abstract Extracellular vesicles derived from induced pluripotent stem cells (iPSC EVs) have immunoregulatory potential with the ability to alter the macrophage phenotype. Modulating the macrophage phenotype towards an anti-inflammatory, pro-resolving state may be beneficial in the treatment of chronic inflammatory diseases. The contents of iPSC EVs and their effects on macrophages are poorly understood. Here iPSC EVs were characterized and analysed by mass-spectrometry based proteomics and a targeted microRNA (miR) panel and their immunomodulatory effects on primary human macrophages were assessed. Podocalyxin-like protein 1 (PODXL1) and Insulin (INS) were the most abundant proteins unique to the iPSC EVs while miR-302d-3p was the most abundant miR. Notably, thioredoxin- and peroxiredoxin-related proteins were detected. iPSC EVs increased the anti-inflammatory associated Mannose Receptor C-Type 1 ( MRC1 ) and miR-21, while monocyte chemoattractant protein 1 (MCP-1) and IL-8 were decreased. Proteomics revealed that treated macrophages had decreased levels of chemoattractant proteins, Azurocidin 1 (AZU1), Growth Differentiation Factor 15 (GDF15), and Ribosomal Protein S19 (RPS19). Conditioned media from macrophages treated with iPSC EVs inhibited monocyte transmigration, a key component in the propagation of inflammation. This study provides insights into the protein and miR cargo of iPSC EVs and highlights their capacity to inhibit chemotactic proteins in macrophages. Biological sciences/Cell biology Biological sciences/Immunology Biological sciences/Molecular biology Biological sciences/Stem cells Extracellular vesicles iPSCs macrophages monocytes inflammation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Induced pluripotent stem cells (iPSCs) and mesenchymal stem cells (MSCs) have a novel therapeutic potential in multiple diseases owing to their multilineage differentiation potential, immunomodulatory, anti-inflammatory, regenerative and neuroprotective properties 1 – 3 . The therapeutic and anti-inflammatory effects of stem cells are mediated in part through paracrine effects including the secretion of extracellular vesicles (EVs) 4 – 6 . EVs are nanometre sized lipid membranous spherical particles, secreted from all cells and can contain lipids, proteins, DNA, mRNA and miRNA. EVs have the capacity to transfer biologically functional cargo to recipient cells to mediate cell signalling. Tumour derived EVs are known to function in pre-metastatic niches in target organs 7 . Endothelial cell derived EVs decreased proinflammatory macrophage and increased anti-inflammatory macrophages, in part through a decrease in transfer of miR-155 8 . In contrast to the use of viable stem cells as a potential therapeutic, the EVs derived from such cells are acellular and have been proposed as a safer alternative for therapeutic application. For example, in a murine model of myocardial infarction (MI), injection of murine derived allogenic iPSC-EVs did not induce teratoma formation which was in contrast to the effect observed following injection of iPSCs 9 . Specifically, cardiac tumors developed in over 50% of the iPSC-treated group representing a substantial concern that could prevent clinical translation of direct iPSC injections. Importantly, in this model, iPSC-EVs were more effective in enhancing left ventricular (LV) ejection fraction and in reducing LV mass post MI when compared to iPSCs 9 . This suggests that EVs may be a safer alternative due to the concerns of live cell therapy such as tumorigenesis 9 and embolism 10 – 12 . The ability of stem cell-derived EVs to modulate the macrophage phenotype has been extensively reviewed 13 , 14 . Macrophages are phagocytic cells and EVs are up taken into cells via phagocytosis, caveolin- and clathrin- mediated endocytosis 15 . Some EVs, such as epithelial cell derived EVs, and their associated miRNA contents have been shown to promote a pro-inflammatory M1-like macrophage phenotype 16 , whereas stem cell derived EVs enhanced a pro resolving M2-like macrophage phenotype, associated with a reduction in inflammation 17 , 18 . In a model of myocardial ischaemia reperfusion injury, mesenchymal stem cell (MSC)-derived EVs alleviated infarct size and inflammation through their ability to shift the macrophage from an M1 like phenotype to an M2 like phenotype 19 . In addition, MSC-derived EVs decreased atherosclerosis development in ApoE knockout mice via promotion of an M2-like phenotype 20 . There is now a growing body of evidence demonstrating the efficacy of iPSC-EVs in vitro and in vivo . Previous studies have shown that iPSC-EVs decreased NF-κB inflammatory signalling and markers of fibrosis in TGFbeta stimulated mouse mesangial cells 21 . iPSC-EVs also decreased fibrotic markers, chemotaxis and proliferation of TGF-beta stimulated hepatic stellate cells and had an anti-fibrotic effect in a murine model of hepatic fibrosis and cholestatic liver fibrosis 22 . iPSC-EVs have shown neuroprotective effects when combined with electroacupuncture in murine models of ischemic stroke 23 . Furthermore, EVs from iPSC derived neural stem cells decreased IL-6 secretion in LPS stimulated macrophages. In this study in vivo administration also decreased MCP-1, TNF-α and IL-1β in the hippocampus after acute seizure 24 . In vivo , EVs from human iPSC-derived mesenchymal stomal cells decreased hepatic injury coincident with reduced inflammation, apoptosis and enhanced antioxidant markers 25 . Although the proteome of murine iPSC EVs has previously been described 9 , in this study we analysed the proteome of human iPSC EVs and 216 human miRs within these EVs. EVs from a pluripotent carcinoma cell line were used as a control to identify the unique proteins and miRs within iPSC EVs. We also assessed the inflammatory impact of iPSC EVs on primary human macrophages and whether they have a functional relevance on monocyte transmigration, a key component in the propagation of inflammation. Results Characterisation of iPSC EVs EVs were isolated from the supernatant of iPSCs by differential centrifugation. Several methods were employed to extensively characterise the isolated particles as recommended in the MISEV guidelines 26 , 27 . These isolated EVs were analysed by TEM (Fig. 1 A and Supplemental Fig. 4 ). EVs within the nanometre size range appeared to be intact with cup shaped morphology, a common artefact of the TEM preparation process. Super resolution microscopy was used to demonstrate that among the iPSC-EV population, EVs were positive for the exosome markers CD9, CD63 and CD81 and were within the nanometre size range (Fig. 1 B). Some EVs were single positive or double positive for these markers and the full diversity of the EV population can be observed in Supplemental Fig. 5 . To assess the size of the isolated iPSC EVs en masse, nanoparticle tracking analysis (NTA) was utilised (Fig. 1 C). The mode size was 97.5nm ± 29.9nm with an average total number of particles of 2.6x10 10 ± 0.6x10 10 . NTA was also used to calculate the concentration of particles in solution and to calculate the different EV amounts to be applied to macrophages. Bodipy membrane labelling followed by flow cytometry was used to identify particles with a lipid membrane that were then gated for CD63 positivity (Fig. 1 E). There was an average of 5.4x10 7 particles positive for CD63, representing approximately 72% of the overall membrane labelled population. These particles could also be visualized by the ImageStream flow cytometer (Fig. 1 F). Collectively, the particles isolated from iPSCs have a size, surface proteins and a morphology that is indicative of an EV population. iPSC EVs induce upregulation of MRC1 and miR-21 in human macrophages. Macrophages are phagocytotic cells and readily take up EVs, however the effects of stem cell EVs on macrophage phenotype has not been fully elucidated. Here we investigated that effect of iPSC EVs on macrophage gene and miRNA expression, using human derived macrophage which were characterized prior to treatment ( Supplemental Fig. 2 ). Primary human macrophages were treated with two concentrations of iPSC EVs (1x10 5 and 5x10 5 ) for 24hr. LPS was used as a pro-inflammatory stimulus for the final 4hr and ATP was added for the final hour to ensure IL-1beta secretion. Gene expression analysis showed no changes in TNFA expression however there was a dose dependent increases in MRC1 expression and a trend towards increased IL10 (Fig. 2 A-C). To investigate whether the increase in MRC1 was iPSC EV mediated, Triton X-100 diluted in PBS, was applied to the iPSC EVs to disrupt the membrane and resulted in a decrease in the mode particle size (Fig. 2 D). While PBS treated iPSC EV increased MRC1 expression in macrophages, application of Triton X100 exposed iPSC EVs did not, suggesting the induction of MRC1 relies on undisrupted or intact EVs (Fig. 2 E). EVs are known carriers of microRNAs, here we analysed two anti-inflammatory miRs, miR-21 and Let-7c, and a pro-inflammatory miR, miR-155. There was a significant increase in miR-21 in macrophages exposed to the iPSC EVs (Fig. 2 F). To determine if this increase was due to the presence of the EVs or transfer of their miR content, the RNA content of the EVs was analysed for the three miRs. Let-7c was undetected while miR-21 and miR-155 had similar Ct values (Fig. 2 G) in iPSC EVs. Thus, the miR-21 increase in macrophages exposed to EVs is likely due to upregulation of this miR within the macrophage rather than transfer from the EVs. iPSC EVs decrease MCP-1 secretion from macrophages and decrease the chemotactic properties of the macrophage secretome. Next we investigated if iPSC EVs alter inflammatory cytokine secretion in primary human macrophages. iPSC EVs decreased pro-inflammatory IL-1β secretion (Control 100% vs 5x10 8 EVs 87%+/-1.9%) and IL-8 secretion (Control 100% vs 5x10 8 EVs 68%+/-17.5%) from unstimulated macrophages (Fig. 3 A). There was no change in the gene expression of CXCL8 which encodes IL-8 (Fig. 3 A-B). IL-8 and IL-1β cytokines had the highest basal levels of secretion however the effects of the iPSC-EVs on these cytokines was not observed in the presence of LPS ( Supplemental Fig. 6 ). There was a significant decrease in MCP-1 secretion (Control 132.8%+/-36.8% vs 5x10 8 EVs 74%+/-16.6%) from iPSC-EV treated macrophages in the presence of LPS which was mirrored by a trending decrease at the gene level in CCL2 (Fig. 3 C-D). Given that iPSC EVs decreased this chemoattractant in macrophages, a transmigration assay was used identify any functional significance of this reduction as further recruitment of inflammatory cells is a critical step in sustaining chronic inflammation (Fig. 3 E). Conditioned media (CM) from the human macrophages was used to establish its chemotactic effects on THP-1 monocytes. There was a statistically significant decrease in monocyte migration toward the CM from macrophages treated with iPSC EVs (CM-EV) when compared to CM from the untreated controls (CM-CTRL). The CM from LPS stimulated macrophages (CM-CTRL + LPS) increased monocyte migration which was significantly reduced in the presence of CM-EV + LPS (Fig. 3 F-G ). Representative 5X images were also obtained ( Supplemental Figs. 6–7 ). In summary, iPSC-EVs decreased the macrophage secretion of MCP-1, and decreased the chemotactic properties of the macrophage secretome as demonstrated by reduced monocyte migration, suggesting that iPSC-EVs mediate an anti-inflammatory effect on monocyte/macrophage cells. The iPSC EV proteome contains EV associated proteins and has greater diversity than the NT-2 proteome In order to investigate the proteins in iPSC EVs that may mediate the observed anti-inflammatory effects, mass spectrometry based proteomics was performed. EVs from the pluripotent human embryonal carcinoma cell line NTERA-2 (NT-2) were isolated and characterised by TEM ( Supplemental Fig. 8 ) and NTA and used as an EV control. Here the objective was to identify iPSC EV specific proteins and effects through comparison with a pluripotent tumour cell line. Approximately 572 proteins were detected in iPSC EVs compared to 252 identified in NT-2 EVs suggesting a greater diversity and abundance of proteins within the iPSC EV proteome (Fig. 4 A). Notably, 89% of iPSC EV proteins detected had been previously identified in EVs as reported in the ExoCarta Mass Spectrometry Database. The proteins common to both iPSC EV and NT-2 EVs (220) were considered to be generic EV associated proteins. ‘Extracellular exosome’, ‘extracellular vesicle’ and ‘extracellular organelle’ were among the most significant terms identified by PANTHER Cellular Component Analysis of the iPSC- and NT-2 EV proteins (Fig. 4 B and Supplemental Fig. 9A ). We further identified specific proteins overlapping with the top 20 EV-proteins (ExoCarta). Some of the classical EV-related markers, CD9, CD81, Alix (PDCD6IP), HSP70 and HSP90 identified here, were previously reported in iPSC-EV proteomics 28 (Fig. 4 C). Histone proteins, actin, tubulin and ribosomal proteins were the most highly abundant proteins identified in both EV groups. Filtering the proteins unique to each group of EVs identified the sialomucin, podocalyxin (PODXL) as the most abundant in iPSC EVs followed by Insulin (INS) and SLC2A3 (GLUT3) (Fig. 4 D and Supplemental Fig. 9B). PODXL and Lin-28 Homolog A (LIN28A) were present only in the iPSC-EV group and have been previously identified as being specifically enriched in iPSC-EVs 28 . Independently, PODXL has been identified as a glycoprotein ligand on iPSC EVs 29 . Comparison of the iPSC- and NT-2 EV proteomes identified 298 proteins enriched in iPSC EVs and 76 proteins enriched in NT-2 EVs (Fig. 4 E and Supplemental Fig. 9C ). Ingenuity pathway analysis (IPA) of the unique iPSC EV proteins categorised 31% of the proteins as enzymes among them were the thioredoxin and peroxiredoxin associated proteins. Ezrin (EZR), and basic fibroblast growth factor (FGF2) were highlighted given their known role in M2-like macrophage polarization 30 , 31 . IPA of iPSC EV proteins also identified a list of transcription factors and upstream regulators predicted to be activated, Transforming Growth Factor Beta 1 (TGFB1) was among them ( Supplemental Fig. 9D-E ). Proteins unique for iPSC EVs and unique for NT-2 EVs were analysed by Proteomaps to highlight their known involvement in functions and pathways (Fig. 4 G and Supplemental Fig. 10 ). iPSC EVs decrease chemotactic proteins in human macrophages. Next iPSC-EVs and NT-EVs were incubated with macrophages and mass-spectrometry based proteomics was performed. 71 proteins were significantly changed in macrophages stimulated with iPSC-EVs in comparison to the control (untreated) macrophages. Incubation with NT-2 EVs altered 205 proteins in comparison to the control (Fig. 5 A). The iPSC EV proteome was intersected with these datasets to identify proteins that may be increased due to physical transfer/presence of the EVs. A heatmap was generated with annotation to show the proteins commonly regulated by both iPSC EV and NT-2 EVs in macrophages and which were considered to be ‘generic EV effects’. Elimination of these proteins left 40 unique proteins (as marked by asterisks) that were considered to represent an iPSC EV specific effect (Fig. 5 B). Analysis of these proteins identified several processes in macrophages altered by the iPSC EVs in comparison to the untreated control (Fig. 5 C). iPSC EVs decrease macrophage secretory proteins; Azurocidin 1 (AZU1), Growth Differentiation Factor 15 (GDF15), Ribosomal Protein S19 (RPS19), Leucyl And Cystinyl Aminopeptidase (LNPEP), Complement 9 (C9) and Complement C1q B Chain (C1QB). In concert, a mediator of SNARE exocytosis, STX4, was also decreased. They also altered the mitochondrial associated proteins; Inner Membrane Mitochondrial Protein (IMMT), Translocase Of Inner Mitochondrial Membrane 50 (TIMM50), Acyl-CoA Synthetase Family Member 2 (ACSF2) and D-Glutamate Cyclase (DGLUCY). iPSC EVs also enhanced the anti-oxidant proteins: Apurinic/Apyrimidinic Endodeoxyribonuclease 1 (APEX1) and Glutathione S-Transferase Zeta 1 (GSTZ1). AZU1 proteins levels were significantly decreased in macrophages treated with iPSC-EVs (Fig. 5 D) notably, the mRNA expression of AZU1 was unchanged ( Supplemental Fig. 11A ). The effects of iPSC EV on macrophages were compared to the NT-2 EV effects. Plexin Domain Containing 2 (PLXDC2) and TGFBI was among the proteins increased by iPSC EVs and notably inflammatory associated Intercellular Adhesion Molecule 1 (ICAM1) and Interferon Induced Protein With Tetratricopeptide Repeats 1 (IFIT1) were decreased (Fig. 5 E). GDF15 was among the 8 proteins that was decreased in both dataset comparisons (iPSC EV vs Ctrl GDF15 FC = -6.25, p-value = 0.0053, iPSC EV vs NT-2 EV GDF15 FC -4.59, p-value = 0.024). Collectively, IPA analysis of iPSC-EV versus NT-2 EV treated macrophages predicted inhibition of pro-inflammatory signalling molecules including IL1A, IL1B, TNF and IFNG ( Supplemental Fig. 11B ). IPA analysis of the effects of iPSC EVs on macrophages versus untreated macrophages identified the most significantly decreased functions as ‘Microtubule dynamics’, ‘Organization of cytoplasm’ and ‘Organization of cytoskeleton’. Given our previous findings that the iPSC EV treated macrophage secretome decreased chemotaxis of myeloid cells it was of interest to highlight that ‘chemotaxis of phagocytes’ were predicted to be inhibited (Fig. 5 F). The key proteins involved in these pathways were AZU1, Mitogen-Activated Protein Kinase 1 (MAPK), Rac Family Small GTPase 2 (RAC2) and RPS19 which as a dimer has monocyte chemoattractant properties 32 , 33 . In addition, ‘Degranulation of Phagocytes’ was identified. Peroxisome Proliferator Activated Receptor Alpha (PPARA) was among the upstream molecules predicted to be activated in macrophages stimulated with iPSC EVs when compared to untreated control (Fig. 5 G). The upstream regulators, predicted by IPA, based on analysis of the EV proteome were intersected with the proteins that were significantly changed by iPSC EVs in macrophages. TGFBI was the only molecule predicted to be activated that was significantly increased in iPSC-EV treated macrophages (Fig. 5 H). In summary, iPSC EVs alter the macrophage proteome decreasing chemotactic proteins secreted by macrophages. iPSC EV miR content and miR targets altered in treated macrophages The miR content of the iPSC EVs and NT-2 EVs was also analysed. The most abundant miR was miR-302d-5p, which was previously reported to be among the most abundant miRs in iPSC EVs 22 and MSC-EVs 34 (Fig. 6 A). miR-221-3p, also identified in iPSC EVs, was the most abundant in NT-2 EVs (Fig. 6 B). Intersection of the top miRs in iPSC EVs identified in this study with previously published data on the top miRs identified in iPSC EVs 22 and MSC-EVs 34 revealed that miRs in common included miR-302d-5p, miR-92b-3p, miR-191-5p, miR-99b-5p, miR-23a-3p and miR-25-3p (Fig. 6 C). A comparison of iPSC EVs and NT-2 EVs revealed that 13 miRs were differentially expressed between the two groups. Notably, iPSC EVs had less expression of miR-Let-7c, miR-27a-3p and miR-146a-5p (Fig. 6 D). The miR profile of the iPSC EVs was more diverse than that of the NT-2 EVs. The miRs specific to iPSC EVs were tabulated showing those detected in all 3 independent experiments and absent in NT-2 EVs (Fig. 6 E ). The miRs marked with an asterisk were previously identified in iPSC derived EVs by Povero et al . 22 . Further experiments are required to identify if any of these miRs have an effect on the recipient macrophages. Bioinformatic analysis was used to identify miR-protein interactions using two datasets: 1) miRs increased in iPSC EVs vs NT-2 EVs and 2) proteins decreased in macrophages treated with iPSC EVs vs NT-2 EVs. The circos plot illustrates the interactions of the nine upregulated miRs and their known targets downregulated in iPSC EV treated macrophages (Fig. 6 F). Discussion This study highlights that iPSC EVs decrease macrophage chemotactic proteins and secretory proteins and increase anti-inflammatory associated MRC1 and miR-21. In addition, iPSC EVs alter the macrophage secretome to the extent that the chemoattractant capacity to induce monocyte migration was impaired. Here we also provide insights into the iPSC EV proteome which included anti-oxidant proteins and the miRs that are detectable in iPSC EVs. Understanding of the key signalling molecules within iPSC EVs may facilitate mimicry and concentration of such molecules for therapeutic application. Recruitment of inflammatory cells to the area of damage is critical for repair and resolution of inflammation. However under certain circumstances, when resolution of inflammation is impaired there is sustained inflammatory cell recruitment and activation mediated via secretion of chemoattractants and pro-inflammatory cytokines. M1 like, pro-inflammatory macrophages secrete Il-1β, TNF-α and chemoattractants and hence modulation of their secretome may reduce cyclic low grade chronic inflammation that underlies multiple diseases. We have shown in this study that MCP-1 ( CCL2 ), a potent chemoattractant, is downregulated by iPSC EVs. MCP-1 is frequently used as a chemoattractant stimulus in monocyte transmigration assays. Here, monocyte transmigration towards iPSC EV macrophage conditioned media was significantly decreased. Reduction in monocyte transmigration may be beneficial in decreasing the pro-inflammatory response in chronic inflammatory conditions. It must be noted that other known chemoattractants, Il-8, AZU1 35 , GDF15 (also known as Macrophage Inhibitory Cytokine 1 (MIC-1)) and RPS19 32,33 were also significantly decreased by iPSC-EV treatment. In contrast, parasite derived EVs increased IL-8 in unstimulated THP-1 macrophages 36 . Notably, iPSC EVs were carriers of RPS19 and RPS6 but crucially these were downregulated in the recipient macrophages, indicative of regulation rather than transfer. RPS6 (protein with the greatest fold change decrease) was previously linked with IL-8/ CXCL8 where Ang et al. showed that induction of rps6 phosphorylation at S235/236 enhanced the translation of CXCL8 in macrophages and that both rsp6 and CXCL8 could be attenuated by ERK1/2 inhibitors 37 . IL-8 and AZU1, known to be stored in secretory vesicles, were decreased but unchanged at the transcript level suggesting the iPSC EVs modulate translation or affect vesicle granule release and/or degradation initiated via the endosomal pathway. This decrease in chemotactic vesicle proteins may be due to inhibition of degranulation as our proteomics analysis identified several altered proteins (STX4, RAC2, RAB14) associated with ‘Degranulation of Phagocytes’. EVs are frequently taken via endocytosis in an energy dependent process 15 and macrophages can phagocytose EVs 38 . It could be hypothesized that the addition of iPSC-EVs and their complexity activates the endosomal lysosomal pathway within the recipient macrophages, supresses exocytosis/degranulation via STX4 inhibition and in tandem drives the degradation of endogenous vesicles containing secretory molecules such as IL-8 and AZU1. Additionally, the cell may receive signals that there is an abundance of vesicles in the immediate microenvironment resulting in a negative feedback inhibiting further vesicle production/secretion of endogenous vesicles. The mechanism by which iPSC EVs alter these proteins remains to be elucidated and was beyond the scope of this study. In vivo studies have demonstrated that stem cell derived EVs alter the macrophage phenotype to an anti-inflammatory, M2-like phenotype however the mechanism and full spectrum of their effects remains to be elucidated 20 , 39 . Here, iPSC EVs increased macrophage expression of MRC1 , PLXDC2, TGFB1 and miR-21 which are indicative of an anti-inflammatory phenotype. This is in keeping with previous studies which found that administration of MSC EVs led to increased CD206 ( MRC1 ) in heart tissue in a model of myocardial I/R injury 19 . Additionally, adipose-MSC derived EVs enhanced CD206 and Arginase 1 in PBMC-derived macrophages 40 . Macrophage PLXDC2, induced by iPSC EVs, was previously shown to correlate with M2 macrophage associated genes 41 . Previously, PLXDC2 was identified as the lead immunoregulatory target in bone marrow derived macrophages stimulated with Helicobacter pylori . In in vivo models, loss of Plxdc2 in macrophages was associated with increased M1 markers (TNFa, iNOS/Arg1 and IL6), increased inflammation and disease severity 42 . Although miR-21 and miR-155 were present within the EVs, only miR-21 was significantly upregulated, suggesting activation of endogenous macrophage transcription of miR-21. MiR-21 is a key mediator of the inflammatory response and resolution in macrophages and is upregulated by pro-inflammatory stimuli to modulate inflammation 43 . Macrophage efferocytosis of apoptotic cells induces miR-21 expression which in turn supresses pro-inflammatory signalling 44 . iPSC EVs were carriers of EZR and FGF2, both of which have previously been shown to induce an M2 macrophage phenotype 30 , 31 . Specifically EZR in EVs induce an M2-like phenotype while knockdown induced an M1 phenotype in the context of pancreatic ductal adenocarcinoma 31 . Notably, in this study peroxiredoxins ( PRDX2, PRDX3, PRDX4) and thioredoxins (TXN, TXNDC5, TMX1) were unique to the iPSC EV proteome. These are anti-oxidant enzymes which scavenge reactive oxygen species. iPSC EVs may drive degradation or release of IRAP ( LNPEP ) from the cell. IRAP ( LNPEP ) is primarily found in the endosomal vesicles and can re-locate to the cell membrane acting as a receptor for angiotensin IV 45 . Notably, IRAP ( LNPEP ) expression was enhanced in pro-inflammatory M1 like macrophages compared to M2-like macrophages 46 . Indeed, IRAP inhibitors have been proposed as an anti-inflammatory strategy. The ability of MSCs and their derivatives (apoptotic and membrane particles) to modulate macrophage phenotype through phagocytosis has been concisely reviewed by Lu et al. 47 . MSCs in direct contact with macrophages enhanced CD206 ( MRC1 ) and increased phagocytotic activity 48 . MSCs phagocytosed by lung macrophages induced an M2-like phenotype 49 . Exposure of macrophages to apoptotic MSCs also altered the macrophage secretome and application of this macrophage supernatant to hypoxic cardiomyocytes enhanced viability and survival time 50 . MSC-EVs mediated macrophage anti-inflammatory effects via mitochondrial transfer 51 . MSCs package mitochondria into EVs and when transfered to macrophages resulted in enhanced bioenergetics (oxygen consumption rate) 52 . It is not just the exposure of macrophages to EVs or any spherical particles that triggers the change in phenotype as here we observed that equivalent amounts of NT-2 EVs did not have the same effect on secretory proteins as those mediated by iPSC EVs. Additionally, macrophages exposed to silica particles induced mitochondrial ROS production which was mitigated when combined with MSC-EVs 52 . It is evident that the content and/or unique membrane of the EVs is a key mediator. EVs are known to shuttle functional miRNA content between cells 53 , 54 . Whether the amount of miRs within the iPSC EVs is sufficient to illicit a biological shift remains to be determined. However, assessment of the content and use of mimics of the most efficacious miRs may be a viable future therapeutic strategy. The most abundant miR in iPSC EVs was miR-302d-3p which was not detected in NT-2 EVs. Previously, MSC-EVs loaded with miR-302d-3p mimic inhibited its target, BCL6, downregulated the NF-κB pathway and reduced inflammation and cardiac dysfunction in a murine model of acute myocardial dysfunction 55 . miR-302d-3p regulates IFN signalling via IRF9 suppression 56 and was identified as a crucial signalling molecule in MSC-EVs that inhibits migration and lymphangiogenesis of lymphatic endothelial cells 57 . Notably miR-25-3p was another one of the most abundant miRs in iPSC-EVs. miR-25-3p mimic induces an M2-like macrophage phenotype ( ARG1 , CD163 , IL10 ) while downregulating M1-like makers ( TNFA and NOS2 ) 58 . Similarly, bone marrow MSC-EVs loaded with miR-25-3p enhance the M2-like macrophage phenotype ( IL10 , ARG1 ) and decreased the M1-like phenotype ( IL1B and IL6 ) 59 . However, it is challenging to identify the specific miRs or proteins that are altering the macrophage phenotype. Future studies would require knock out or suppression of these miRs or proteins in iPSCs to elucidate the effects of the EV derivatives on recipient macrophages. Our findings describe key proteins and miRs that were associated with iPSC EVs. iPSC EVs are more biologically complex than NT-2 EVs as they consist of a wider array of proteins and miRs. In addition, we have observed that these iPSC-EVs promote an anti-inflammatory macrophage phenotype and decrease the abundance of secretory proteins some of which have known chemotactic properties. Here we showed that the alterations in the secretome of macrophages treated with EVs is of functional relevance as it decreased monocyte transmigration, a key process in the propagation of inflammation. Further work will be necessary to determine the key signalling molecules within the iPSC EVs, the diversity of iPSC EVs across iPSC lines and critical EV surface proteins responsible for cell uptake. Here we have identified several potential anti-inflammatory mediators and mechanisms which may be amenable to mimicry and enhance the efforts to use stem cell derived EVs as anti-inflammatory therapeutics. Methods Cell Culture of Human iPSCs The human iPSC line WTSli028-A from the European Bank for Induced Pluripotent Stem Cells (EBISC) was used to generate EVs. These iPSCs were derived from the dermal fibroblasts of a healthy female (50–54 year old) of white-British descent. Non-integrating Sendai virus to induce transient overexpression of KLF4, C-Myc, Sox2 and Oct-4 was used to derived these cells. iPSC were cultured on 6-well plates coated with Vitronectin XF (10 µg/mL) (StemCell Technologies, Cat No. 07180) for 1hr at room temperature before use. Essential 8™ Flex Media (Gibco, Cat no. A28858501) was used to maintain iPSCs incubated at 37°C in 5% CO2. iPSCs were subcultured using a passage ratio of 1:6. Supernatant was collected from iPSCs upon reaching 70–80% confluency and frozen at -80°C. iPSC were then passaged via ReLeSR™ Passaging Reagent (StemCell Technologies, Cat no. 100–0483). iPSCs were passaged upon reaching confluency into Essential 8™ Flex Media supplemented with ROCK Inhibitor, Y-27632, (10 µM) (Tocris, Cat No. 1254) and maintained in this media for 24h. Colonies had classical morphology as observed by brightfield microscopy, had high expression of pluripotency genes SOX2 , NANOG and OCT4 and were fluorescently positive for OCT4 ( Supplemental Fig. 1 ). Cell Culture of NT-2 Cells NT-2 cells also known as NTERA-2 cells (ATCC, Cat. No. CRL-1973) are a human pluripotent embryonal carcinoma cell line from the testis. NT-2 cells were used to generate control EVs to compare the EV content to that of iPSC EVs. NT-2 cells were cultured in T75 flasks in high glucose Dulbecco's Modified Eagle Medium (DMEM) (4.5g/L glucose) supplemented with 10% (v/v) Fetal Bovine Serum (Hyclone), 100U/ml penicillin and 100U/ml streptomycin (Gibco). Cells were subcultured using 0.25% Trypsin-EDTA (Invitrogen). Confluent NT-2 cells were cultured in serum-free DMEM for 24hr prior to passaging of cells. The supernatant/conditioned media was collected and centrifuged at 1,200 rpm for 5mins and frozen at -80°C prior. Extracellular Vesicle Isolation Extracellular vesicles were isolated from iPSC and NT-2 cell supernatant via a series of differential centrifugations (Eppendorf Centrifuge 5417R) at 4°C. Samples were centrifuged in 1.5mL Eppendorfs at 2,000g for 20min, the supernatant was then centrifuged at 20,000g for 90min, the EV pellet was washed in double filtered phosphate buffered saline (DF-PBS) and then the centrifugation step of 20,000g was repeated and the EVs were resuspended in i) neat M199 media (Analab, Dublin, Ireland) or ii) DF-PBS. For nanoparticle tracking analysis (NTA) and RT-PCR, the supernatant was then centrifuged at 24,000g for 60min, the EV pellet was washed in PBS and then the centrifugation step was repeated and the EVs were resuspended in DF-PBS or TRIzol. For flow cytometry and transmission electron microscopy (TEM), the supernatant was then centrifuged at 20,000g for 90min, the EV pellet was washed in DF-PBS and then the centrifugation step was repeated and the EVs were resuspended in PBS. For NTA and flow cytometry analysis 4 EV pellets were pooled and resuspended in 300µL of DF-PBS. For TEM 4 EV pellets were pooled and resuspended in 50µL of double filtered sterile DF-PBS. For RT-PCR analysis, 4 pellets were pooled in 500µL of TRIzol reagent. For disruption of EVs; 0.1% Triton X-100 in DF-PBS was added to 7.5x10^8 EVs and incubated at 37°C for 15mins. DF-PBS was used as the control. Samples were centrifuged at 20,000g for 90mins and resuspended in 100µL of DF-PBS with 50µL used for NTA and 50µL remaining for the treatment of macrophages. Macrophages were treated with 500 EVs per cell. Transmission Electron Microscopy (TEM) of EVs TEM was performed as previously described 60 . In brief, formvar carbon coated copper (G200-Cu) (EMS, Pennsylvania, USA), grids were used to mount the EVs. EVs were fixed using 2.5% glutardialdehyde (Merck, Darmstadt, Germany) and then incubated with 2% uranyl acetate (Agar Scientific, Essex, UK). Grids containing EVs were treated with a solution of 1.8% methyl cellulose (Sigma-Aldrich, Dorset, UK) and 0.4% uranyl acetate followed by air drying. An FEI Tecnai 12 Transmission Electron Microscope with the acceleration voltage set at 120 kv was used to image the EVs. Images were obtained at a magnification of 43,000X and 135,000X. Super Resolution Microscopy of EVs The EV Profiler Kit (ONI, Oxford, Cat. no. EV-MAN-1.0) was used as per manufacturer’s instructions to assess EVs by super resolution microscopy. EVs were immobilized on the profiler chip and stained for the presence of the tetraspanin antibodies, CD9, CD63 and CD81. Positive control EVs (supplied by ONI, Oxford) and negative controls were also included. Profiler chips with EVs were analysed on the Nanoimager (ONI, Oxford) with temperature (32°C) and illumination angle (53°) set. 100nm TetraSpeck™ Microspheres (Thermofisher, UK) were used for channel mapping calibration. Direct stochastic optical reconstruction microscopy (dSTORM) analysis was applied. For each field of view (FOV) acquisition 1000 images were captured using the 488nm and 561nm laser and 500 images were captured using the 640nm laser. The exposure (100ms) and the frequency (10Hz) was set. 3–4 FOV were captured per sample with Z-lock applied. Images were visualised using the Collaborative Discovery (CODI) Software with drift correction applied. Nanoparticle Tracking Analysis of EVs The concentration and size of the EVs was measured by nanoparticle tracking analysis using the NanoSight NS300 (Malvern Panalytical Ltd, UK) which uses light scattering and Brownian motion to assess the size distribution and concentration of particles suspended in DFPBS ( Supplemental Fig. 2 ). EV samples were diluted 1 in 100 in DFPBS and delivered into the sample chamber via automated injection. Particles in suspension scatter the light of a laser beam that is passed through the solution which is visualized by a 20X microscope and recorded by a camera. Fifteen videos of 1 minute duration were recorded for each sample using the appropriate camera level and focus. Videos were analysed using the Nanosight NTA 3.1 software using 10nm sized bins, a set detection threshold of 8 and then the concentration was calculated accounting for the dilution factor. Data from each sample were expressed as the mean ± SD of the fifteen recordings. Graphs were generated using R Studio and GraphPad Prism. Human Peripheral Blood Monocyte Cell Isolation and Differentiation to Macrophages Peripheral venous blood was donated by healthy volunteers to the Irish Blood Transfusion Service (IBTS), National Blood Bank located in St. James’s Hospital, James’s Street, Dublin 8 for transfusion and research purposes. Written informed consent that was obtained from all volunteers. Written approval was granted by the IBTS which provides de-identified blood components, pro-bono, to academic researchers. IBTS approval number: 001-03-19. Peripheral blood monocyte cells (PBMCs) were isolated as previously described 61 . In brief, whole blood was centrifuged at 190xg for 15min to remove the platelet rich plasma and then diluted 1:1 in PBS. This mix was layered onto Lymphoprep™ (Alere Ltd, UK) and centrifuged at 450xg for 30min. The PBMC layer was removed and washed in PBS three times. Cells were re-suspended in M199 media (Analab, Dublin, Ireland) supplemented with 10% human serum (Sigma), L-glutamine (2mM) (Gibco) and antibiotic (100U/mL Penicillin and 100µg/mL Streptomycin) (Bio-sciences, Dublin, Ireland) and incubated on 14cm petri dishes for 2hr at 37°C, 5% CO 2 . Adhered cells were considered monocytes and were re-seeded at a density of 1x10 6 cells/mL in 12 well plates (day 0). On day 4 the 1mL of M199 media was added to each well. On day 7 the M199 media was discarded and replaced with 1mL of M199 media to allow for further macrophage differentiation ( Supplemental Fig. 3 ). On day 10, the M199 media on the macrophages was changed to serum free M199 media containing the EV treatments. EV treatments of 1x10 8 EVs per well (equivalent to 100 EVs per cell) and 5x10 8 EVs per well (equivalent to 500 EVs per cell) were added. Macrophages were treated with EVs for 24h in total. Lipopolysaccharide (LPS) (InvivoGen, Toulouse, France) (50ng/mL) was added for the final 4h and adenosine triphosphate (ATP) (5mM) was added for the final hour before harvesting for RNA and protein. Gene/miR Expression Analysis by qRT-PCR RNA was isolated from EVs and cells using the TRIzol™ Reagent (Thermo Fisher Scientific) protocol. Messenger RNA was reverse transcribed using SuperScript™ III Reverse Transcriptase (Thermo Fisher Scientific, Cat no. 18080-044). Samples were analysed using SYBR™ Green Universal Master Mix and Universal Taqman PCR Mix on the QuantStudio™ 7 Flex Real-Time PCR System (Applied Biosystems, ThermoFisher Scientific). GAPDH was used as the endogenous control. For miRNA synthesis the Applied Biosystems TaqMan microRNA reverse transcription kit (Applied Biosystems, Thermo Fisher Scientific) was used. TaqMan™ microRNA Assays (Applied Biosystems) were used for reverse transcription and miR expression analysis was performed using Universal Taqman PCR Mix on the QuantStudio™ 7 Flex Real-Time PCR System. U6 snRNA was used as the endogenous control for miRNA expression analysis. Supplemental Table 1 consists of a list of assays used for mRNA/gene and microRNA expression. RT-PCR results were analysed via the 2(-Delta Delta C(T)) method 62 . Cytokine/Chemokine Analysis and MesoScale Discover Assay Supernatants from stimulated macrophages were analysed for MCP-1 via Enzyme-linked Immunosorbent Assay (ELISA). MCP-1 ELISA (ThermoFisher Scientific) was performed as per manufacturer’s instructions. A CLARIOStar Microplate Reader was used to read plate absorbance at 450nm. Supernatants from stimulated macrophages were analysed using Mesoscale Discovery Assays Multiplex V7 (Meso Scale Discovery) which included IL-8. Electrochemiluminescence signal was analysed on a Meso™ QuickPlex SQ 120 (Meso Scale Discovery). Values outside of the standard curve range were excluded from the downstream analysis. Results were expressed as a percentage relative to the unstimulated and untreated macrophage control sample. Monocyte Transmigration Assay A transmigration assay was used to assess the chemotactic efficacy of the conditioned media/supernatant derived from macrophages treated with and without iPSC EVs. Pro-inflammatory macrophages frequently release chemokines and inflammatory cytokines to recruit additional monocytes and propagate the inflammatory process. Thus, human THP-1 monocytes cells (ATCC® TIB-202™, Middlesex, UK) were used as to assess the chemotactic efficacy of the macrophage conditioned media. THP-1 monocytes were maintained in in RPMI 1640 medium supplemented with GlutaMAX, 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin antibiotic. THP-1 monocytes were seeded in 125µL of serum free RPMI at a concentration of 1x10^5 cells per polycarbonate membrane transwell insert (5µM pores) (Corning, Cat. no. 3421). 250µL of macrophage conditioned media was added to 250µL of serum free media and added to the lower chamber. Transwell inserts with THP-1 monocytes were lowered into the lower chamber and incubated for 2 hr at 37°C, in 5% CO 2 . THP-1 monocytes migrate through the pores to the underside of the transwell membrane insert. The negative control consisted of THP-1 cells in the upper well and serum free media only in the lower chamber. Transwell inserts were removed and fixed in 3% formaldehyde (Fisher). Media was removed from inside the transwell chamber and the inner membrane insert was swabbed with a cotton bud. Transwells were washed in PBS and then stained with Hoechst 33342, trihydochloride, trihydrate (ThermoFisher Scientific, Cat no. H3570). The membrane insert was cut and mounted on a slide with the underside of the transwell membrane facing upwards. Fluorescence microscopy was performed using a Zeiss Axio Imager M1 Microscope (Zeiss). 12 random images at 20X were obtained per insert. The presence of Hoechst positive nuclei were considered as migrated cells and results were shown as average number of migrated cells/field of view. Three independent experiments consisting of three different passages of THP-1 monocytes and conditioned media from three independent experiments with iPSC EV treatment were performed using biological duplicates for all conditions. Proteomics on EVs Samples were processed for mass spectrometry analysis as described by Howard et al. 2022 63 . In brief, EV samples in PBS (~ 20uL) were diluted in a 40µL solution of 8M urea and 50mM Tris HCL and then sonicated. The protein samples were reduced using dithiothreitol (DTT) (8mM) (BioLabs, B7705S) followed by incubation in a thermomixer (1000 rpm) for 30mins at 30°C. Idoacetamide (IAA) (20mM) was added to each sample and incubated in a thermomixer (1000 rpm) for 30mins at 30°C (samples were protected from light). The samples were diluted in 50mM Tris HCL to decrease the concentration of urea to < 2M. Sequencing Grade Modified Trypsin (Promega, Cat: V511C) was added to each sample in a ratio of 1:20, trypsin enzyme to substrate. Samples were incubated overnight in a thermomixer (1000 rpm) at 37°C. Trypsin digestion was stopped using formic acid (diluted to 1% of the final concentration). Samples were cleaned using HyperSep™ SpinTips C18 (Thermo Scientfic, Cat. 60109-412) and eluted in a solution of 80% acetonitrile (ACN), 0.1% trifluoroacetic acid in LC/MS grade water. Samples were evaporated in a SpeedVac Concentrator for ~ 90mins at 30°C and peptides were resuspended in LC/MS grade water containing 0.5% acetic acid + 2.5% ACN. Peptides from iPSC EVs were quantified and then analysed on a Q-Exactive mass spectrometer (Thermo Scientific) which was fitted with a reversed-phase NanoLC UltiMate 3000 high performance liquid chromatography (HPLC) system (Thermo Scientific) as previously described 64 . MaxQuant 65 , 66 (version 2.0.3.0) was used to process the raw data from the Q-Exactive through incorporation of the Andromeda search engine 67 . In order to identify the peptides ad proteins, the UniProt Homo Sapiens database (Version 2021_03) containing a total of 78,120 entries was used to match the mass spectrometry/mass spectrometry spectra detected. Mass spectrometry data and processing protocol have been made available at the PRIDE 68 Database (EMBL-EBI) with the dataset identifier PXD067567. Reviewer access details: Log in to the PRIDE website using the following details: Project accession: PXD067567 Token: 5O3Mmyynluyl. Alternatively, reviewer can access the dataset by logging in to the PRIDE website using the following account details: Username: [email protected] Password: tzg6x5Ar49vn Proteomics on Macrophages Macrophages were washed with PBS and then lysed in a 40µL solution of 6M urea in 50mM Ammonium Bicarbonate. 50µg of protein was reduced with DTT (final concentration of 4mM) and incubated for 30mins at 60°C. IAA (8mM) solution in 50mM Ammonium Bicarbonate was added and the samples were incubated in the dark for 40mins. The samples were diluted in 50mM Tris HCL to decrease the concentration of urea to < 2M. Sequencing Grade Modified Trypsin (Promega, Cat: V511C) was added to each sample in a ratio of 1:15, trypsin enzyme to substrate. Samples were incubated overnight in a thermomixer (1000 rpm) at 37°C. Trypsin digestion was stopped using acetic acid (diluted to 1% of the final concentration). Samples were cleaned using C18 Stage tips prepared as per Rappsilber et al. [34]. Following stage tip activation, peptides were loaded, washed and eluted in a solution of 80% acetonitrile (ACN), 0.1% trifluoroacetic acid in LC/MS grade water. Peptides were evaporated in a SpeedVac Concentrator for ~ 90mins at 45°C and peptides were resuspended in LC/MS grade water containing 0.5% acetic acid + 2.5% ACN. 1.5mg/mL of peptide solution was loaded into mass spectrometry vials. Samples were analysed on a timsTOF Pro mass spectrometer (Bruker Daltonics, Bremen, Germany) coupled to a nanoELute (Bruker Daltonics, Bremen, Germany) ultra-high pressure nanoflow chromatography system. MaxQuant 65 , 66 (version 1.6.17.0) was used to process the raw data from the timsTOF through incorporation of the Andromeda search engine 67 . In order to identify the peptides ad proteins, the UniProt Homo Sapiens database (Version 2020_09) containing a total of 75,777 entries was used to match the mass spectrometry/mass spectrometry spectra detected. Mass spectrometry data and processing protocol have been made available at the PRIDE 68 Database (EMBL-EBI) with the dataset identifier PXD067612. Reviewer access details: Log in to the PRIDE website using the following details: Project accession: PXD067612 Token: HXbf9mooxar4. Alternatively, reviewer can access the dataset by logging in to the PRIDE website using the following account details: Username: [email protected] Password: jpJvDE3OXvkO Proteomic Analysis Heatmaps were generated using Perseus Software [65] . Proteins detected in all 3 independent experiments were analysed using the PANTHER Classification System (PANTHER19.0) 69 . Specifically, ‘Cellular Component Analysis’ was performed with Fisher’s Extact T-test and FDR applied. Data was filtered for the top EV markers using the Vesiclepedia database and a heatmap was generated showing the relative protein quantification. The LFQ values of the most abundant proteins unique to iPSC EVs and unique to NT-2 EVs were graphed. Univariate analysis was performed comparing iPSC EV proteins with NT-2 proteins using imputation of data in the case of absence in either group. A scatter plot showing enrichment of iPSC EV proteins and NT-2 EV proteins were generated using GraphPad Prism. All proteins identified in iPSC and NT-2 EVs and the proteins unique to each group were analysed via Proteomaps 70 , 71 . Ingenuity pathway analysis (IPA) (Qiagen) was used to identify the EV proteins that are upstream regulators and transcription regulators. microRNA Analysis by TaqMan™ OpenArray™ Human Advanced microRNA Panel A customised TaqMan™ OpenArray™ Human Advanced microRNA Panel was used to analyse the microRNAs that were isolated from the EVs. This panel contained 216 miRs as listed in Supplemental Table 2 . The TaqMan OpenArray microRNA Panel was analysed using a QuantStudio™ 12K Flex instrument. The relative threshold (Crt) was determined for all miRs with lower values indicative of higher miR abundance. The results were filtered for valid values in all 3 independent experiments in at least one group. The fold change was determined for miRs that were detected in both groups (3/3 valid values) and the statistically significant results were graphed. miRs detected in one group (3/3 valid values) but not the other were tabulated. IPA software (Qiagen) was applied to identify the microRNAs upregulated in the iPSC EVs with the ability to target specific mRNA (proteins used in this case) that were downregulated in macrophages following iPSC EV exposure. The results were visualised using a circos plot 72 . Statistical Analysis Statistical analysis was performed using GraphPad Prism Software. The normality of the data was assessed. A one-way ANOVA comparing multiple selected columns was used for comparisons of more than two groups. For comparisons of two groups, a t-test was performed. Individual dots are representative of independent experiments. Error bars are representative of ± standard error of the mean (SEM). Declarations Funding Declaration This project was funded by the UCD Strategic Support Wellcome Trust Fund (17533). Acknowledgements UCD Conway Microscopy Core. ONI Nanoimaging Company. Irish Blood Transfusion Service. Author Contributions S.F.-conception and design of experiments, data collection, data analysis and interpretation, drafting of manuscript. O.B.-design of experiments, data interpretation, drafting of manuscript. S.O. and M.M.S.M.-data collection and data interpretation. J.C., S.C., C.K. and J.D.- data collection. D.A.- design of experiments. ETD- data analysis. All authors reviewed the manuscript. Competing interests The author(s) declare no competing interests. Data availability statement Mass spectrometry data and processing protocol have been made available at the PRIDE 68 Database (EMBL‐EBI) with the dataset identifiers PXD067567 and PXD067612. Additional supporting data can be found in the supplemental tables and figures. Reviewer access details: Log in to the PRIDE website using the following details: Project accession: PXD067612 Token: HXbf9mooxar4. Alternatively, reviewer can access the dataset by logging in to the PRIDE website using the following account details: Username: [email protected] Password: jpJvDE3OXvkO Reviewer access details: Log in to the PRIDE website using the following details: Project accession: PXD067567 Token: 5O3Mmyynluyl. 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Immunol. 11 , 576516. https://doi.org/10.3389/fimmu.2020.576516 (2020). de Gaetano, M., Alghamdi, K., Marcone, S. & Belton, O. Conjugated linoleic acid induces an atheroprotective macrophage MΦ2 phenotype and limits foam cell formation. J. Inflamm. 12 , 15. https://doi.org/10.1186/s12950-015-0060-9 (2015). Livak, K. J. & Schmittgen, T. D. Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2 – ∆∆CT Method. Methods 25 , 402–408. https://doi.org/https://doi.org/10.1006/meth.2001.1262 (2001). Howard, J. et al. A comparative analysis of extracellular vesicles (EVs) from human and feline plasma. Sci. Rep. 12 , 10851. https://doi.org/10.1038/s41598-022-14211-z (2022). Fitzsimons, S. et al. Inhibition of pro-inflammatory signaling in human primary macrophages by enhancing arginase-2 via target site blockers. Mol. Ther. Nucleic Acids . 33 , 941–959. https://doi.org/10.1016/j.omtn.2023.08.023 (2023). Tyanova, S. et al. 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Proceedings of the National Academy of Sciences 111, 8488–8493 (2014). https://doi.org/doi:10.1073/pnas.1314810111 Otto, A. et al. Systems-wide temporal proteomic profiling in glucose-starved Bacillus subtilis. Nat. Commun. 1 , 137. https://doi.org/10.1038/ncomms1137 (2010). Krzywinski, M. I. et al. Circos: An information aesthetic for comparative genomics. Genome Res. https://doi.org/10.1101/gr.092759.109 (2009). Additional Declarations No competing interests reported. Supplementary Files SupplementaryInformationiPSCEVEffectsonMacrophages.docx floatimage1.png Graphical Abstract: iPSC-derived EVs were characterized and the protein and microRNA (miR) content was analysed. Human PBMC derived macrophages were treated with iPSC EVs which increased anti-inflammatory associated proteins and miR-21 and decreased chemoattractant proteins. The conditioned media from these iPSC-EV treated macrophages inhibited transmigration of human THP-1 monocytes. Asterisk (*) is used to indicate a statistically significant difference in the iPSC EVs when compared to the NT-2 EVs. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 02 Sep, 2025 Submission checks completed at journal 27 Aug, 2025 First submitted to journal 27 Aug, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7436803","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":506373806,"identity":"b4e4973b-90f8-4529-a747-31d659df3115","order_by":0,"name":"Stephen Fitzsimons","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/ElEQVRIiWNgGAWjYDAC5oMNQFICzD7woQIkwtyAXwtbYmMDVAvjwxlnQFoYCWlJgKtgNuZtA2vFr0W+jbn94Y8/FtH80u3PJGfOq43mbwdq+VGxDacWg2OMjQ2SbRK5M+ecMZP4uO147ozDjA2MPWdu49Yi39jYYNggkbvhRg6b5Mxtx3IbgFqYGdtwa5FvA9qS8AekJf2ZNO+cY7nzCWlhADnsABtIS4KxMW9DTe4GQlpAfpnZCPLLjBzDhzOOHcjdCNRyEJ9f5NvYH3z88acut18i/cGBDzV1ufPOHz744EcFHoehgcNg8gDR6oGgjhTFo2AUjIJRMEIAAPnnYz4Hz/3MAAAAAElFTkSuQmCC","orcid":"","institution":"University College Dublin","correspondingAuthor":true,"prefix":"","firstName":"Stephen","middleName":"","lastName":"Fitzsimons","suffix":""},{"id":506373807,"identity":"6ed768d3-8096-4b85-8be4-000915cbc3c3","order_by":1,"name":"Silvia Oggero","email":"","orcid":"","institution":"William Harvey Research Institute, Barts and the London School of Medicine, Queen Mary University of London","correspondingAuthor":false,"prefix":"","firstName":"Silvia","middleName":"","lastName":"Oggero","suffix":""},{"id":506373808,"identity":"81c2ffd8-966e-4e11-aaa0-ab1c4e42b727","order_by":2,"name":"Eugène T. Dillon","email":"","orcid":"","institution":"University College Dublin","correspondingAuthor":false,"prefix":"","firstName":"Eugène","middleName":"T.","lastName":"Dillon","suffix":""},{"id":506373809,"identity":"509e8bf8-ffac-4cff-beff-8d60d3528164","order_by":3,"name":"María Muñoz-San Martín","email":"","orcid":"","institution":"Royal College of Surgeons in Ireland","correspondingAuthor":false,"prefix":"","firstName":"María","middleName":"Muñoz-San","lastName":"Martín","suffix":""},{"id":506373811,"identity":"ac1da38c-38b0-4090-8d5a-cb88322250e0","order_by":4,"name":"Shane Clerkin","email":"","orcid":"","institution":"University College Dublin","correspondingAuthor":false,"prefix":"","firstName":"Shane","middleName":"","lastName":"Clerkin","suffix":""},{"id":506373813,"identity":"fbdcf8a5-afb5-465d-b8ec-48cb97d1ad1f","order_by":5,"name":"Ciarán Kennedy","email":"","orcid":"","institution":"University College Dublin","correspondingAuthor":false,"prefix":"","firstName":"Ciarán","middleName":"","lastName":"Kennedy","suffix":""},{"id":506373814,"identity":"cea7352f-c828-416d-8f92-6d1108584d08","order_by":6,"name":"Jessica Davis","email":"","orcid":"","institution":"University College Dublin","correspondingAuthor":false,"prefix":"","firstName":"Jessica","middleName":"","lastName":"Davis","suffix":""},{"id":506373815,"identity":"23dfa66f-7b87-4af1-94c3-529dfc7f34db","order_by":7,"name":"Darrell Andrews","email":"","orcid":"","institution":"University College Dublin","correspondingAuthor":false,"prefix":"","firstName":"Darrell","middleName":"","lastName":"Andrews","suffix":""},{"id":506373817,"identity":"b102dd0a-d098-49f6-8bec-516ac97573f9","order_by":8,"name":"John Crean","email":"","orcid":"","institution":"University College Dublin","correspondingAuthor":false,"prefix":"","firstName":"John","middleName":"","lastName":"Crean","suffix":""},{"id":506373819,"identity":"a2e2bb8a-dc71-4a31-88bd-5175a8b22be4","order_by":9,"name":"Orina Belton","email":"","orcid":"","institution":"University College Dublin","correspondingAuthor":false,"prefix":"","firstName":"Orina","middleName":"","lastName":"Belton","suffix":""}],"badges":[],"createdAt":"2025-08-22 18:23:07","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7436803/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7436803/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":90173936,"identity":"a6772cb7-a9e4-44cb-bf3a-a0dd57e5a607","added_by":"auto","created_at":"2025-08-29 11:57:11","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1069745,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCharacterisation of iPSC derived EVs.\u003c/strong\u003e \u003cstrong\u003eA)\u003c/strong\u003eTransmission electron microscopy (TEM) of EVs isolated from the supernatant of iPSCs. \u003cstrong\u003eB)\u003c/strong\u003e Super resolution microscopy of the exosome markers CD9, CD63 and CD81 on iPSC EVs. \u003cstrong\u003eC)\u003c/strong\u003e Size distribution of iPSC EVs analysed by nanoparticle tracking analysis (n=8). Total particle count and the mode size of EVs was determined. \u003cstrong\u003eD)\u003c/strong\u003e Flow cytometry on iPSC EVs labelled with Bodipy and CD63. Samples were initially gated for Bodipy positive events and then gated for CD63 positive events. \u003cstrong\u003eE)\u003c/strong\u003e Number of CD63 positive particles per mL and the percentage of CD63 positive events relative to Bodipy (n=2). \u003cstrong\u003eF)\u003c/strong\u003eRepresentative images of CD63 (FITC labelled) and Bodipy (Texas Red Labelled) positive iPSC EVs.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-7436803/v1/09ebc921bb0e2dd92b7a4b9b.png"},{"id":90173938,"identity":"7331e41c-41c2-4d24-aff1-8ffacf4522fb","added_by":"auto","created_at":"2025-08-29 11:57:11","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":148813,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eiPSC EVs induce upregulation of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eMRC1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and miR-21 in human macrophages.\u003c/strong\u003e Primary human PBMC-derived macrophages were treated with increasing amounts of iPSC EVs (1x10\u003csup\u003e5 \u003c/sup\u003eand 5x10\u003csup\u003e5\u003c/sup\u003e) for 24hr. LPS (50ng/mL) was added for the final 4hr and ATP (5mM) was added for the final hour. EVs were re-suspended in media thus media was used as the control. Gene expression analysis was performed on the treated macrophages investigating \u003cstrong\u003eA) \u003c/strong\u003e\u003cem\u003eTNFA \u003c/em\u003e\u0026nbsp;\u003cstrong\u003eB) \u003c/strong\u003e\u003cem\u003eIL10 \u003c/em\u003eand \u003cstrong\u003eC) \u003c/strong\u003e\u003cem\u003eMRC1 \u003c/em\u003eusing \u003cem\u003eGAPDH \u003c/em\u003eas the endogenous control (3 independent experiments were performed for each condition, n=3). \u003cstrong\u003eD) \u003c/strong\u003eiPSC-EVs were treated with 0.1% Triton X-100 or PBS and then washed, re-centrifuged and analysed by NTA to determine the mode particle size. iPSC EVs from three independent passages were treated and analysed. \u003cstrong\u003eE) \u003c/strong\u003eMacrophages were exposed to the Triton X-100- and PBS- treated EVs (5x10\u003csup\u003e8\u003c/sup\u003e) and \u003cem\u003eMRC1\u003c/em\u003e expression was assessed (n=3). \u003cstrong\u003eF) \u003c/strong\u003eExpression of miRs, miR-21, miR-155 and Let-7c were analysed in macrophages treated with a total of 5x10\u003csup\u003e8\u003c/sup\u003e EVs, U6 snRNA was used as the endogenous control (n=3). \u003cstrong\u003eG) \u003c/strong\u003eExpression of miR-21, miR-155 and Let-7c were analysed and the cycle threshold (Ct) values were graphed, a lower Ct value is indicative of higher gene expression (n=4).\u003cstrong\u003e \u003c/strong\u003eBiological duplicates were used for qRT-PCR and averaged. Statistical analysis was performed using a one way ANOVA for \u0026gt;2 groups and a T-test for the comparison of two groups. *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-7436803/v1/efbffa6392f3e84f96406c3e.png"},{"id":90174228,"identity":"5fdcac7c-2dfc-48a6-95d7-6ccbee21ba94","added_by":"auto","created_at":"2025-08-29 12:05:11","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":453603,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eiPSC EVs inhibit the chemotactic effects of the macrophage secretome on monocytes\u003c/strong\u003e. Primary PBMC-derived macrophages were treated with 5x10^8 iPSC-EVs for 24hr. LPS (50ng/mL) was added for the final 4hr, ATP (5mM) was added for the final hour and supernatants and RNA were then harvested. EVs were re-suspended in media thus media was used as the control. \u003cstrong\u003eA) \u003c/strong\u003eIL-1β, IL-6, IL-8, IL-10 and IL-12p70 were analysed in the cell supernatant by MSD assay and \u003cstrong\u003eB) \u003c/strong\u003e\u003cem\u003eCXCL8 \u003c/em\u003e(IL-8) was analysed by qRT-PCR using \u003cem\u003eGAPDH\u003c/em\u003e as the endogenous control. \u003cstrong\u003eC) \u003c/strong\u003eMCP-1 was analysed in the cell supernatant by ELISA and \u003cstrong\u003eD) \u003c/strong\u003e\u003cem\u003eCCL2 \u003c/em\u003e(MCP-1) was analysed by qRT-PCR using \u003cem\u003eGAPDH\u003c/em\u003e as the endogenous control. \u003cstrong\u003eE) \u003c/strong\u003eGraphical summary of transmigration experiment design. Conditioned media (CM) from macrophages treated with iPSC-EVs was used in the low chamber in the transmigration assay (TMA) to test the chemotactic potency in the migration of THP-1 monocytes. \u003cstrong\u003eF) \u003c/strong\u003eThe average number of THP-1 monocytes that migrated through the transmembrane was calculated for each condition. The three independent TMA were performed and graphed displaying biological duplicates. CM from macrophages stimulated with iPSC EVs was generated from three independent experiments. \u003cstrong\u003eG) \u003c/strong\u003eTHP-1 monocyte nuclei stained with Hoechst. Representative images of each independent experiment (12 random images per insert were obtained). Scale bar is 100µm. ‘N=’ denotes an independent experiment. Statistical analysis was performed using an ordinary one way ANOVA comparing select columns using Bonferroni’s multiple comparisons test. ns= not statistically significant, *=p\u0026lt;0.05, ****=p\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-7436803/v1/2df249eb24aa90944113b657.png"},{"id":90173940,"identity":"94eceeb8-be97-475e-98ef-39e0e212f2dd","added_by":"auto","created_at":"2025-08-29 11:57:11","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":528306,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMass-spectrometry analysis on EVs derived from iPSCs and NT-2 cells.\u003c/strong\u003eEVs were isolated from 3 different passages of iPSCs and NT-2 cells respectively and mass spectrometry based proteomic analysis was performed.\u003cstrong\u003eA) \u003c/strong\u003eTo identify the number of EV associated proteins identified in both iPSC EVs (n=3) and NT-2 EVs (n=3), the mass spectrometry datasets were intersected with proteins from the ExoCarta Mass Spectrometry database, a repository of EV reported/identified proteins. \u003cstrong\u003eB)\u003c/strong\u003e The iPSC EV proteins were analysed by PANTHER Cellular Component Analysis and graphed based on statistical significance (negative log of the false discovery rate). \u003cstrong\u003eC)\u003c/strong\u003e Relative protein quantification for the top EV-associated markers from the ExoCarta database detected in iPSC and NT-2 EVs. \u003cstrong\u003eD) \u003c/strong\u003eThe top 10 most abundant proteins unique to the iPSC EV proteome.\u003cstrong\u003e E) \u003c/strong\u003eScatter plot of the significantly enriched EV proteins in iPSC EVs and NT-2 EVs following\u003cstrong\u003e \u003c/strong\u003eunivariate analysis. \u003cstrong\u003eF) \u003c/strong\u003eIPA analysis of the unique iPSC EV proteins was used to categorise the protein type and proteins of interest for some categories were listed. \u003cstrong\u003eG) \u003c/strong\u003eProteomaps was used to analyse the unique iPSC EV proteins to categorise their known biomolecular and cellular associations and functions.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-7436803/v1/42792653b96111892d5b54d2.png"},{"id":90174230,"identity":"82c7e0d7-1999-4851-968a-b17ecec9791b","added_by":"auto","created_at":"2025-08-29 12:05:11","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":726571,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe macrophage proteome is altered by treatment with iPSC EVs.\u003c/strong\u003e Primary human PBMC derived-macrophages were left untreated (Control (CTRL)) or treated with iPSC-EVs or NT-2 EVs and mass spectrometry based proteomic analysis was performed. Three independent experiments with biological duplicates were performed which consisted of macrophages from 3 different PBMC donors and EVs from 3 different cell passages. \u003cstrong\u003eA) \u003c/strong\u003eThe significantly changed macrophage proteins altered by treatment with iPSC EVs in comparison to control (iPSC EV vs Untreated) were intersected with those altered by macrophage exposure to NT-2 EVs (NT-2 EVs vs Untreated). The \u0026nbsp;proteins identified in iPSC EVs alone were also intersected to generate a Venn Diagram. \u003cstrong\u003eB) \u003c/strong\u003eA heatmap was generated showing the significantly altered proteins in macrophages stimulated with iPSC EVs when compared to the untreated control. Those overlapping with the iPSC EV proteome were regarded as possible iPSC EV transfer while those overlapping with NT-2 EV effects were regarded as a generic EV effect. Scatter plots were used to highlight the significantly changed macrophages proteins altered by iPSC EVs in comparison to \u003cstrong\u003eC) \u003c/strong\u003econtrol (untreated) and \u003cstrong\u003eD) \u003c/strong\u003eNT-2 EV stimulation. \u003cstrong\u003eE) \u003c/strong\u003eAnalysis of the significantly altered proteins in macrophage with iPSC EVs using IPA ‘Disease \u0026amp; Biological Functions’ and \u003cstrong\u003eF) \u003c/strong\u003eUpstream Regulator Analysis. \u003cstrong\u003eG)\u003c/strong\u003e The altered proteins identified from analysis of iPSC EVs on macrophages when compared against NT-2 EVs were intersected with IPA Upstream Regulators identified via analysis of the iPSC EV proteome. TGFB1 LFQ intensity values were compared between the two groups. Statistical analysis was performed using a Man-Whitney t-test where *=p\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-7436803/v1/6eff180a1b2ab631b819350f.png"},{"id":90173944,"identity":"25d2ee01-dda1-450b-8d3a-182514a90e9f","added_by":"auto","created_at":"2025-08-29 11:57:11","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":677007,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe miR profile of iPSC EVs has greater diversity than NT-2 EVs\u003c/strong\u003e. RNA was isolated from iPSC EVs and NT-2 EVs and analysed via Taqman OpenArray Card using a QuantStudio™ 12K Flex instrument. Data was filtered to account for quantitative and qualitative data. The relative threshold (Crt) values of the top 12 most abundant miRs was graphed for \u003cstrong\u003eA)\u003c/strong\u003e iPSC EVs and \u003cstrong\u003eB)\u003c/strong\u003e NT-2 EVs. A lower Crt value is indicative of higher miR expression.\u003cstrong\u003e C) \u003c/strong\u003eVenn diagram illustrating the intersection of the top 22 miRs from iPSCs with previously published miR datasets on iPSC EVs \u003csup\u003e22\u003c/sup\u003e and MSC-EVs \u003csup\u003e34\u003c/sup\u003e.\u003cstrong\u003e D) \u003c/strong\u003eThe fold changes of the significantly altered miRs in iPSC EVs relative to NT-2 EVs. Data points are representative of 3 independent experiments with 1 technical replicate each. \u003cstrong\u003eE) \u003c/strong\u003eData was filtered to identify miRs that were detected in all iPSC EV independent experiments (3/3 or 100%) and absent in all NT-2 EV independent experiments (0/3, 0%) and tabulated as qualitative data. miRs marked with an asterisk (*) were among the top 22 miRs identified previously in iPSC EVs by Povero\u003cem\u003e et al. \u003c/em\u003e\u003csup\u003e22\u003c/sup\u003e. \u003cstrong\u003eF)\u003c/strong\u003e To identify potential miR-protein interactions, the significantly increased iPSC EV miRs were analysed with proteins downregulated in iPSC EV treated macrophages by IPA to identify the known miR-gene/protein interactions. miR-gene/protein interactions were displayed using a circos plot. Statistical analysis was performed using a t-test where *=p\u0026lt;0.05 and **=p\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-7436803/v1/1bf33c7014ca1b12f0e46268.png"},{"id":90175268,"identity":"ee357544-8d0f-416c-9ecd-03bade992412","added_by":"auto","created_at":"2025-08-29 12:21:14","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4685649,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7436803/v1/60bc073d-73d6-429f-ae98-48d5a2dd62f9.pdf"},{"id":90173956,"identity":"1a904569-8e27-4f88-970e-4baa586e985d","added_by":"auto","created_at":"2025-08-29 11:57:11","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":10712019,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformationiPSCEVEffectsonMacrophages.docx","url":"https://assets-eu.researchsquare.com/files/rs-7436803/v1/8056a3c1280e940dffca186f.docx"},{"id":90174227,"identity":"a70ea539-e33b-417d-856e-9da3c824b954","added_by":"auto","created_at":"2025-08-29 12:05:11","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":189603,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGraphical Abstract: \u003c/strong\u003eiPSC-derived EVs were characterized and the protein and microRNA (miR) content was analysed. Human PBMC derived macrophages were treated with iPSC EVs which increased anti-inflammatory associated proteins and miR-21 and decreased chemoattractant proteins. The conditioned media from these iPSC-EV treated macrophages inhibited transmigration of human THP-1 monocytes. Asterisk (*) is used to indicate a statistically significant difference in the iPSC EVs when compared to the NT-2 EVs.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7436803/v1/a322859e879daadeab50f6a9.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Extracellular vesicles derived from induced pluripotent stem cells mediate anti- inflammatory effects in primary human macrophages","fulltext":[{"header":"Introduction","content":"\u003cp\u003eInduced pluripotent stem cells (iPSCs) and mesenchymal stem cells (MSCs) have a novel therapeutic potential in multiple diseases owing to their multilineage differentiation potential, immunomodulatory, anti-inflammatory, regenerative and neuroprotective properties \u003csup\u003e\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. The therapeutic and anti-inflammatory effects of stem cells are mediated in part through paracrine effects including the secretion of extracellular vesicles (EVs) \u003csup\u003e\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. EVs are nanometre sized lipid membranous spherical particles, secreted from all cells and can contain lipids, proteins, DNA, mRNA and miRNA. EVs have the capacity to transfer biologically functional cargo to recipient cells to mediate cell signalling. Tumour derived EVs are known to function in pre-metastatic niches in target organs \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Endothelial cell derived EVs decreased proinflammatory macrophage and increased anti-inflammatory macrophages, in part through a decrease in transfer of miR-155 \u003csup\u003e8\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eIn contrast to the use of viable stem cells as a potential therapeutic, the EVs derived from such cells are acellular and have been proposed as a safer alternative for therapeutic application. For example, in a murine model of myocardial infarction (MI), injection of murine derived allogenic iPSC-EVs did not induce teratoma formation which was in contrast to the effect observed following injection of iPSCs \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Specifically, cardiac tumors developed in over 50% of the iPSC-treated group representing a substantial concern that could prevent clinical translation of direct iPSC injections. Importantly, in this model, iPSC-EVs were more effective in enhancing left ventricular (LV) ejection fraction and in reducing LV mass post MI when compared to iPSCs \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. This suggests that EVs may be a safer alternative due to the concerns of live cell therapy such as tumorigenesis \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e and embolism \u003csup\u003e\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e .\u003c/p\u003e\u003cp\u003eThe ability of stem cell-derived EVs to modulate the macrophage phenotype has been extensively reviewed \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Macrophages are phagocytic cells and EVs are up taken into cells via phagocytosis, caveolin- and clathrin- mediated endocytosis \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Some EVs, such as epithelial cell derived EVs, and their associated miRNA contents have been shown to promote a pro-inflammatory M1-like macrophage phenotype \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e, whereas stem cell derived EVs enhanced a pro resolving M2-like macrophage phenotype, associated with a reduction in inflammation \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. In a model of myocardial ischaemia reperfusion injury, mesenchymal stem cell (MSC)-derived EVs alleviated infarct size and inflammation through their ability to shift the macrophage from an M1 like phenotype to an M2 like phenotype \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. In addition, MSC-derived EVs decreased atherosclerosis development in ApoE knockout mice via promotion of an M2-like phenotype \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThere is now a growing body of evidence demonstrating the efficacy of iPSC-EVs \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e. Previous studies have shown that iPSC-EVs decreased NF-κB inflammatory signalling and markers of fibrosis in TGFbeta stimulated mouse mesangial cells \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. iPSC-EVs also decreased fibrotic markers, chemotaxis and proliferation of TGF-beta stimulated hepatic stellate cells and had an anti-fibrotic effect in a murine model of hepatic fibrosis and cholestatic liver fibrosis \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. iPSC-EVs have shown neuroprotective effects when combined with electroacupuncture in murine models of ischemic stroke \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Furthermore, EVs from iPSC derived neural stem cells decreased IL-6 secretion in LPS stimulated macrophages. In this study \u003cem\u003ein vivo\u003c/em\u003e administration also decreased MCP-1, TNF-α and IL-1β in the hippocampus after acute seizure \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eIn vivo\u003c/em\u003e, EVs from human iPSC-derived mesenchymal stomal cells decreased hepatic injury coincident with reduced inflammation, apoptosis and enhanced antioxidant markers \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eAlthough the proteome of murine iPSC EVs has previously been described \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e, in this study we analysed the proteome of human iPSC EVs and 216 human miRs within these EVs. EVs from a pluripotent carcinoma cell line were used as a control to identify the unique proteins and miRs within iPSC EVs. We also assessed the inflammatory impact of iPSC EVs on primary human macrophages and whether they have a functional relevance on monocyte transmigration, a key component in the propagation of inflammation.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eCharacterisation of iPSC EVs\u003c/h2\u003e\u003cp\u003eEVs were isolated from the supernatant of iPSCs by differential centrifugation. Several methods were employed to extensively characterise the isolated particles as recommended in the MISEV guidelines \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. These isolated EVs were analysed by TEM (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA and \u003cb\u003eSupplemental Fig.\u0026nbsp;4\u003c/b\u003e). EVs within the nanometre size range appeared to be intact with cup shaped morphology, a common artefact of the TEM preparation process. Super resolution microscopy was used to demonstrate that among the iPSC-EV population, EVs were positive for the exosome markers CD9, CD63 and CD81 and were within the nanometre size range (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Some EVs were single positive or double positive for these markers and the full diversity of the EV population can be observed in \u003cb\u003eSupplemental Fig.\u0026nbsp;5\u003c/b\u003e. To assess the size of the isolated iPSC EVs en masse, nanoparticle tracking analysis (NTA) was utilised (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). The mode size was 97.5nm\u0026thinsp;\u0026plusmn;\u0026thinsp;29.9nm with an average total number of particles of 2.6x10\u003csup\u003e10\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6x10\u003csup\u003e10\u003c/sup\u003e. NTA was also used to calculate the concentration of particles in solution and to calculate the different EV amounts to be applied to macrophages. Bodipy membrane labelling followed by flow cytometry was used to identify particles with a lipid membrane that were then gated for CD63 positivity (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). There was an average of 5.4x10\u003csup\u003e7\u003c/sup\u003e particles positive for CD63, representing approximately 72% of the overall membrane labelled population. These particles could also be visualized by the ImageStream flow cytometer (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). Collectively, the particles isolated from iPSCs have a size, surface proteins and a morphology that is indicative of an EV population.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eiPSC EVs induce upregulation of\u003c/b\u003e \u003cb\u003eMRC1\u003c/b\u003e \u003cb\u003eand miR-21 in human macrophages.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eMacrophages are phagocytotic cells and readily take up EVs, however the effects of stem cell EVs on macrophage phenotype has not been fully elucidated. Here we investigated that effect of iPSC EVs on macrophage gene and miRNA expression, using human derived macrophage which were characterized prior to treatment (\u003cb\u003eSupplemental Fig.\u0026nbsp;2\u003c/b\u003e). Primary human macrophages were treated with two concentrations of iPSC EVs (1x10\u003csup\u003e5\u003c/sup\u003e and 5x10\u003csup\u003e5\u003c/sup\u003e) for 24hr. LPS was used as a pro-inflammatory stimulus for the final 4hr and ATP was added for the final hour to ensure IL-1beta secretion. Gene expression analysis showed no changes in \u003cem\u003eTNFA\u003c/em\u003e expression however there was a dose dependent increases in \u003cem\u003eMRC1\u003c/em\u003e expression and a trend towards increased \u003cem\u003eIL10\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-C). To investigate whether the increase in \u003cem\u003eMRC1\u003c/em\u003e was iPSC EV mediated, Triton X-100 diluted in PBS, was applied to the iPSC EVs to disrupt the membrane and resulted in a decrease in the mode particle size (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). While PBS treated iPSC EV increased \u003cem\u003eMRC1\u003c/em\u003e expression in macrophages, application of Triton X100 exposed iPSC EVs did not, suggesting the induction of \u003cem\u003eMRC1\u003c/em\u003e relies on undisrupted or intact EVs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE).\u003c/p\u003e\u003cp\u003eEVs are known carriers of microRNAs, here we analysed two anti-inflammatory miRs, miR-21 and Let-7c, and a pro-inflammatory miR, miR-155. There was a significant increase in miR-21 in macrophages exposed to the iPSC EVs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). To determine if this increase was due to the presence of the EVs or transfer of their miR content, the RNA content of the EVs was analysed for the three miRs. Let-7c was undetected while miR-21 and miR-155 had similar Ct values (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG) in iPSC EVs. Thus, the miR-21 increase in macrophages exposed to EVs is likely due to upregulation of this miR within the macrophage rather than transfer from the EVs.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eiPSC EVs decrease MCP-1 secretion from macrophages and decrease the chemotactic properties of the macrophage secretome.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eNext we investigated if iPSC EVs alter inflammatory cytokine secretion in primary human macrophages. iPSC EVs decreased pro-inflammatory IL-1β secretion (Control 100% vs 5x10\u003csup\u003e8\u003c/sup\u003e EVs 87%+/-1.9%) and IL-8 secretion (Control 100% vs 5x10\u003csup\u003e8\u003c/sup\u003e EVs 68%+/-17.5%) from unstimulated macrophages (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). There was no change in the gene expression of \u003cem\u003eCXCL8\u003c/em\u003e which encodes IL-8 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-B). IL-8 and IL-1β cytokines had the highest basal levels of secretion however the effects of the iPSC-EVs on these cytokines was not observed in the presence of LPS (\u003cb\u003eSupplemental Fig.\u0026nbsp;6\u003c/b\u003e). There was a significant decrease in MCP-1 secretion (Control 132.8%+/-36.8% vs 5x10\u003csup\u003e8\u003c/sup\u003e EVs 74%+/-16.6%) from iPSC-EV treated macrophages in the presence of LPS which was mirrored by a trending decrease at the gene level in \u003cem\u003eCCL2\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC-D). Given that iPSC EVs decreased this chemoattractant in macrophages, a transmigration assay was used identify any functional significance of this reduction as further recruitment of inflammatory cells is a critical step in sustaining chronic inflammation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). Conditioned media (CM) from the human macrophages was used to establish its chemotactic effects on THP-1 monocytes. There was a statistically significant decrease in monocyte migration toward the CM from macrophages treated with iPSC EVs (CM-EV) when compared to CM from the untreated controls (CM-CTRL). The CM from LPS stimulated macrophages (CM-CTRL\u0026thinsp;+\u0026thinsp;LPS) increased monocyte migration which was significantly reduced in the presence of CM-EV\u0026thinsp;+\u0026thinsp;LPS (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF-G\u003cb\u003e).\u003c/b\u003e Representative 5X images were also obtained (\u003cb\u003eSupplemental Figs.\u0026nbsp;6\u0026ndash;7\u003c/b\u003e). In summary, iPSC-EVs decreased the macrophage secretion of MCP-1, and decreased the chemotactic properties of the macrophage secretome as demonstrated by reduced monocyte migration, suggesting that iPSC-EVs mediate an anti-inflammatory effect on monocyte/macrophage cells.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eThe iPSC EV proteome contains EV associated proteins and has greater diversity than the NT-2 proteome\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIn order to investigate the proteins in iPSC EVs that may mediate the observed anti-inflammatory effects, mass spectrometry based proteomics was performed. EVs from the pluripotent human embryonal carcinoma cell line NTERA-2 (NT-2) were isolated and characterised by TEM (\u003cb\u003eSupplemental Fig.\u0026nbsp;8\u003c/b\u003e) and NTA and used as an EV control. Here the objective was to identify iPSC EV specific proteins and effects through comparison with a pluripotent tumour cell line. Approximately 572 proteins were detected in iPSC EVs compared to 252 identified in NT-2 EVs suggesting a greater diversity and abundance of proteins within the iPSC EV proteome (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Notably, 89% of iPSC EV proteins detected had been previously identified in EVs as reported in the ExoCarta Mass Spectrometry Database. The proteins common to both iPSC EV and NT-2 EVs (220) were considered to be generic EV associated proteins. \u0026lsquo;Extracellular exosome\u0026rsquo;, \u0026lsquo;extracellular vesicle\u0026rsquo; and \u0026lsquo;extracellular organelle\u0026rsquo; were among the most significant terms identified by PANTHER Cellular Component Analysis of the iPSC- and NT-2 EV proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB and \u003cb\u003eSupplemental Fig.\u0026nbsp;9A\u003c/b\u003e). We further identified specific proteins overlapping with the top 20 EV-proteins (ExoCarta). Some of the classical EV-related markers, CD9, CD81, Alix (PDCD6IP), HSP70 and HSP90 identified here, were previously reported in iPSC-EV proteomics \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Histone proteins, actin, tubulin and ribosomal proteins were the most highly abundant proteins identified in both EV groups. Filtering the proteins unique to each group of EVs identified the sialomucin, podocalyxin (PODXL) as the most abundant in iPSC EVs followed by Insulin (INS) and SLC2A3 (GLUT3) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD \u003cb\u003eand Supplemental Fig.\u0026nbsp;9B).\u003c/b\u003e PODXL and Lin-28 Homolog A (LIN28A) were present only in the iPSC-EV group and have been previously identified as being specifically enriched in iPSC-EVs \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Independently, PODXL has been identified as a glycoprotein ligand on iPSC EVs \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Comparison of the iPSC- and NT-2 EV proteomes identified 298 proteins enriched in iPSC EVs and 76 proteins enriched in NT-2 EVs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE and \u003cb\u003eSupplemental Fig.\u0026nbsp;9C\u003c/b\u003e). Ingenuity pathway analysis (IPA) of the unique iPSC EV proteins categorised 31% of the proteins as enzymes among them were the thioredoxin and peroxiredoxin associated proteins. Ezrin (EZR), and basic fibroblast growth factor (FGF2) were highlighted given their known role in M2-like macrophage polarization \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. IPA of iPSC EV proteins also identified a list of transcription factors and upstream regulators predicted to be activated, Transforming Growth Factor Beta 1 (TGFB1) was among them (\u003cb\u003eSupplemental Fig.\u0026nbsp;9D-E\u003c/b\u003e). Proteins unique for iPSC EVs and unique for NT-2 EVs were analysed by Proteomaps to highlight their known involvement in functions and pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG and \u003cb\u003eSupplemental Fig.\u0026nbsp;10\u003c/b\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eiPSC EVs decrease chemotactic proteins in human macrophages.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eNext iPSC-EVs and NT-EVs were incubated with macrophages and mass-spectrometry based proteomics was performed. 71 proteins were significantly changed in macrophages stimulated with iPSC-EVs in comparison to the control (untreated) macrophages. Incubation with NT-2 EVs altered 205 proteins in comparison to the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). The iPSC EV proteome was intersected with these datasets to identify proteins that may be increased due to physical transfer/presence of the EVs. A heatmap was generated with annotation to show the proteins commonly regulated by both iPSC EV and NT-2 EVs in macrophages and which were considered to be \u0026lsquo;generic EV effects\u0026rsquo;. Elimination of these proteins left 40 unique proteins (as marked by asterisks) that were considered to represent an iPSC EV specific effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Analysis of these proteins identified several processes in macrophages altered by the iPSC EVs in comparison to the untreated control (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). iPSC EVs decrease macrophage secretory proteins; Azurocidin 1 (AZU1), Growth Differentiation Factor 15 (GDF15), Ribosomal Protein S19 (RPS19), Leucyl And Cystinyl Aminopeptidase (LNPEP), Complement 9 (C9) and Complement C1q B Chain (C1QB). In concert, a mediator of SNARE exocytosis, STX4, was also decreased. They also altered the mitochondrial associated proteins; Inner Membrane Mitochondrial Protein (IMMT), Translocase Of Inner Mitochondrial Membrane 50 (TIMM50), Acyl-CoA Synthetase Family Member 2 (ACSF2) and D-Glutamate Cyclase (DGLUCY). iPSC EVs also enhanced the anti-oxidant proteins: Apurinic/Apyrimidinic Endodeoxyribonuclease 1 (APEX1) and Glutathione S-Transferase Zeta 1 (GSTZ1). AZU1 proteins levels were significantly decreased in macrophages treated with iPSC-EVs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD) notably, the mRNA expression of \u003cem\u003eAZU1\u003c/em\u003e was unchanged (\u003cb\u003eSupplemental Fig.\u0026nbsp;11A\u003c/b\u003e). The effects of iPSC EV on macrophages were compared to the NT-2 EV effects. Plexin Domain Containing 2 (PLXDC2) and TGFBI was among the proteins increased by iPSC EVs and notably inflammatory associated Intercellular Adhesion Molecule 1 (ICAM1) and Interferon Induced Protein With Tetratricopeptide Repeats 1 (IFIT1) were decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). GDF15 was among the 8 proteins that was decreased in both dataset comparisons (iPSC EV vs Ctrl GDF15 FC = -6.25, p-value\u0026thinsp;=\u0026thinsp;0.0053, iPSC EV vs NT-2 EV GDF15 FC -4.59, p-value\u0026thinsp;=\u0026thinsp;0.024). Collectively, IPA analysis of iPSC-EV versus NT-2 EV treated macrophages predicted inhibition of pro-inflammatory signalling molecules including IL1A, IL1B, TNF and IFNG (\u003cb\u003eSupplemental Fig.\u0026nbsp;11B\u003c/b\u003e). IPA analysis of the effects of iPSC EVs on macrophages versus untreated macrophages identified the most significantly decreased functions as \u0026lsquo;Microtubule dynamics\u0026rsquo;, \u0026lsquo;Organization of cytoplasm\u0026rsquo; and \u0026lsquo;Organization of cytoskeleton\u0026rsquo;. Given our previous findings that the iPSC EV treated macrophage secretome decreased chemotaxis of myeloid cells it was of interest to highlight that \u0026lsquo;chemotaxis of phagocytes\u0026rsquo; were predicted to be inhibited (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). The key proteins involved in these pathways were AZU1, Mitogen-Activated Protein Kinase 1 (MAPK), Rac Family Small GTPase 2 (RAC2) and RPS19 which as a dimer has monocyte chemoattractant properties \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. In addition, \u0026lsquo;Degranulation of Phagocytes\u0026rsquo; was identified. Peroxisome Proliferator Activated Receptor Alpha (PPARA) was among the upstream molecules predicted to be activated in macrophages stimulated with iPSC EVs when compared to untreated control (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG). The upstream regulators, predicted by IPA, based on analysis of the EV proteome were intersected with the proteins that were significantly changed by iPSC EVs in macrophages. TGFBI was the only molecule predicted to be activated that was significantly increased in iPSC-EV treated macrophages (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH). In summary, iPSC EVs alter the macrophage proteome decreasing chemotactic proteins secreted by macrophages.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eiPSC EV miR content and miR targets altered in treated macrophages\u003c/h3\u003e\n\u003cp\u003eThe miR content of the iPSC EVs and NT-2 EVs was also analysed. The most abundant miR was miR-302d-5p, which was previously reported to be among the most abundant miRs in iPSC EVs \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e and MSC-EVs \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). miR-221-3p, also identified in iPSC EVs, was the most abundant in NT-2 EVs (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Intersection of the top miRs in iPSC EVs identified in this study with previously published data on the top miRs identified in iPSC EVs \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e and MSC-EVs \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e revealed that miRs in common included miR-302d-5p, miR-92b-3p, miR-191-5p, miR-99b-5p, miR-23a-3p and miR-25-3p (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). A comparison of iPSC EVs and NT-2 EVs revealed that 13 miRs were differentially expressed between the two groups. Notably, iPSC EVs had less expression of miR-Let-7c, miR-27a-3p and miR-146a-5p (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). The miR profile of the iPSC EVs was more diverse than that of the NT-2 EVs. The miRs specific to iPSC EVs were tabulated showing those detected in all 3 independent experiments and absent in NT-2 EVs (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE\u003cb\u003e).\u003c/b\u003e The miRs marked with an asterisk were previously identified in iPSC derived EVs by Povero \u003cem\u003eet al\u003c/em\u003e. \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Further experiments are required to identify if any of these miRs have an effect on the recipient macrophages. Bioinformatic analysis was used to identify miR-protein interactions using two datasets: 1) miRs increased in iPSC EVs vs NT-2 EVs and 2) proteins decreased in macrophages treated with iPSC EVs vs NT-2 EVs. The circos plot illustrates the interactions of the nine upregulated miRs and their known targets downregulated in iPSC EV treated macrophages (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study highlights that iPSC EVs decrease macrophage chemotactic proteins and secretory proteins and increase anti-inflammatory associated \u003cem\u003eMRC1\u003c/em\u003e and miR-21. In addition, iPSC EVs alter the macrophage secretome to the extent that the chemoattractant capacity to induce monocyte migration was impaired. Here we also provide insights into the iPSC EV proteome which included anti-oxidant proteins and the miRs that are detectable in iPSC EVs. Understanding of the key signalling molecules within iPSC EVs may facilitate mimicry and concentration of such molecules for therapeutic application.\u003c/p\u003e\u003cp\u003eRecruitment of inflammatory cells to the area of damage is critical for repair and resolution of inflammation. However under certain circumstances, when resolution of inflammation is impaired there is sustained inflammatory cell recruitment and activation mediated via secretion of chemoattractants and pro-inflammatory cytokines. M1 like, pro-inflammatory macrophages secrete Il-1β, TNF-α and chemoattractants and hence modulation of their secretome may reduce cyclic low grade chronic inflammation that underlies multiple diseases. We have shown in this study that MCP-1 (\u003cem\u003eCCL2\u003c/em\u003e), a potent chemoattractant, is downregulated by iPSC EVs. MCP-1 is frequently used as a chemoattractant stimulus in monocyte transmigration assays. Here, monocyte transmigration towards iPSC EV macrophage conditioned media was significantly decreased. Reduction in monocyte transmigration may be beneficial in decreasing the pro-inflammatory response in chronic inflammatory conditions.\u003c/p\u003e\u003cp\u003eIt must be noted that other known chemoattractants, Il-8, AZU1 \u003csup\u003e35\u003c/sup\u003e, GDF15 (also known as Macrophage Inhibitory Cytokine 1 (MIC-1)) and RPS19 \u003csup\u003e32,33\u003c/sup\u003e were also significantly decreased by iPSC-EV treatment. In contrast, parasite derived EVs increased IL-8 in unstimulated THP-1 macrophages \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Notably, iPSC EVs were carriers of RPS19 and RPS6 but crucially these were downregulated in the recipient macrophages, indicative of regulation rather than transfer. RPS6 (protein with the greatest fold change decrease) was previously linked with IL-8/\u003cem\u003eCXCL8\u003c/em\u003e where Ang \u003cem\u003eet al.\u003c/em\u003e showed that induction of rps6 phosphorylation at S235/236 enhanced the translation of \u003cem\u003eCXCL8\u003c/em\u003e in macrophages and that both rsp6 and \u003cem\u003eCXCL8\u003c/em\u003e could be attenuated by ERK1/2 inhibitors \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eIL-8 and AZU1, known to be stored in secretory vesicles, were decreased but unchanged at the transcript level suggesting the iPSC EVs modulate translation or affect vesicle granule release and/or degradation initiated via the endosomal pathway. This decrease in chemotactic vesicle proteins may be due to inhibition of degranulation as our proteomics analysis identified several altered proteins (STX4, RAC2, RAB14) associated with \u0026lsquo;Degranulation of Phagocytes\u0026rsquo;. EVs are frequently taken via endocytosis in an energy dependent process \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e and macrophages can phagocytose EVs \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. It could be hypothesized that the addition of iPSC-EVs and their complexity activates the endosomal lysosomal pathway within the recipient macrophages, supresses exocytosis/degranulation via STX4 inhibition and in tandem drives the degradation of endogenous vesicles containing secretory molecules such as IL-8 and AZU1. Additionally, the cell may receive signals that there is an abundance of vesicles in the immediate microenvironment resulting in a negative feedback inhibiting further vesicle production/secretion of endogenous vesicles. The mechanism by which iPSC EVs alter these proteins remains to be elucidated and was beyond the scope of this study.\u003c/p\u003e\u003cp\u003e\u003cem\u003eIn vivo\u003c/em\u003e studies have demonstrated that stem cell derived EVs alter the macrophage phenotype to an anti-inflammatory, M2-like phenotype however the mechanism and full spectrum of their effects remains to be elucidated \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. Here, iPSC EVs increased macrophage expression of \u003cem\u003eMRC1\u003c/em\u003e, PLXDC2, TGFB1 and miR-21 which are indicative of an anti-inflammatory phenotype. This is in keeping with previous studies which found that administration of MSC EVs led to increased CD206 (\u003cem\u003eMRC1\u003c/em\u003e) in heart tissue in a model of myocardial I/R injury \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Additionally, adipose-MSC derived EVs enhanced CD206 and Arginase 1 in PBMC-derived macrophages \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. Macrophage PLXDC2, induced by iPSC EVs, was previously shown to correlate with M2 macrophage associated genes \u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. Previously, PLXDC2 was identified as the lead immunoregulatory target in bone marrow derived macrophages stimulated with \u003cem\u003eHelicobacter pylori\u003c/em\u003e. In \u003cem\u003ein vivo\u003c/em\u003e models, loss of Plxdc2 in macrophages was associated with increased M1 markers (TNFa, iNOS/Arg1 and IL6), increased inflammation and disease severity \u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. Although miR-21 and miR-155 were present within the EVs, only miR-21 was significantly upregulated, suggesting activation of endogenous macrophage transcription of miR-21. MiR-21 is a key mediator of the inflammatory response and resolution in macrophages and is upregulated by pro-inflammatory stimuli to modulate inflammation \u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. Macrophage efferocytosis of apoptotic cells induces miR-21 expression which in turn supresses pro-inflammatory signalling \u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eiPSC EVs were carriers of EZR and FGF2, both of which have previously been shown to induce an M2 macrophage phenotype \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Specifically EZR in EVs induce an M2-like phenotype while knockdown induced an M1 phenotype in the context of pancreatic ductal adenocarcinoma \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Notably, in this study peroxiredoxins (\u003cem\u003ePRDX2, PRDX3, PRDX4)\u003c/em\u003e and thioredoxins (TXN, TXNDC5, TMX1) were unique to the iPSC EV proteome. These are anti-oxidant enzymes which scavenge reactive oxygen species. iPSC EVs may drive degradation or release of IRAP (\u003cem\u003eLNPEP\u003c/em\u003e) from the cell. IRAP (\u003cem\u003eLNPEP\u003c/em\u003e) is primarily found in the endosomal vesicles and can re-locate to the cell membrane acting as a receptor for angiotensin IV \u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. Notably, IRAP (\u003cem\u003eLNPEP\u003c/em\u003e) expression was enhanced in pro-inflammatory M1 like macrophages compared to M2-like macrophages \u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. Indeed, IRAP inhibitors have been proposed as an anti-inflammatory strategy.\u003c/p\u003e\u003cp\u003eThe ability of MSCs and their derivatives (apoptotic and membrane particles) to modulate macrophage phenotype through phagocytosis has been concisely reviewed by \u003cem\u003eLu et al.\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. MSCs in direct contact with macrophages enhanced CD206 (\u003cem\u003eMRC1\u003c/em\u003e) and increased phagocytotic activity \u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. MSCs phagocytosed by lung macrophages induced an M2-like phenotype \u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. Exposure of macrophages to apoptotic MSCs also altered the macrophage secretome and application of this macrophage supernatant to hypoxic cardiomyocytes enhanced viability and survival time \u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. MSC-EVs mediated macrophage anti-inflammatory effects via mitochondrial transfer \u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. MSCs package mitochondria into EVs and when transfered to macrophages resulted in enhanced bioenergetics (oxygen consumption rate) \u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. It is not just the exposure of macrophages to EVs or any spherical particles that triggers the change in phenotype as here we observed that equivalent amounts of NT-2 EVs did not have the same effect on secretory proteins as those mediated by iPSC EVs. Additionally, macrophages exposed to silica particles induced mitochondrial ROS production which was mitigated when combined with MSC-EVs \u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. It is evident that the content and/or unique membrane of the EVs is a key mediator.\u003c/p\u003e\u003cp\u003eEVs are known to shuttle functional miRNA content between cells \u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e,\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e. Whether the amount of miRs within the iPSC EVs is sufficient to illicit a biological shift remains to be determined. However, assessment of the content and use of mimics of the most efficacious miRs may be a viable future therapeutic strategy. The most abundant miR in iPSC EVs was miR-302d-3p which was not detected in NT-2 EVs. Previously, MSC-EVs loaded with miR-302d-3p mimic inhibited its target, BCL6, downregulated the NF-κB pathway and reduced inflammation and cardiac dysfunction in a murine model of acute myocardial dysfunction \u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. miR-302d-3p regulates IFN signalling via IRF9 suppression \u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e and was identified as a crucial signalling molecule in MSC-EVs that inhibits migration and lymphangiogenesis of lymphatic endothelial cells \u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. Notably miR-25-3p was another one of the most abundant miRs in iPSC-EVs. miR-25-3p mimic induces an M2-like macrophage phenotype (\u003cem\u003eARG1\u003c/em\u003e, \u003cem\u003eCD163\u003c/em\u003e, \u003cem\u003eIL10\u003c/em\u003e) while downregulating M1-like makers (\u003cem\u003eTNFA\u003c/em\u003e and \u003cem\u003eNOS2\u003c/em\u003e) \u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. Similarly, bone marrow MSC-EVs loaded with miR-25-3p enhance the M2-like macrophage phenotype (\u003cem\u003eIL10\u003c/em\u003e, \u003cem\u003eARG1\u003c/em\u003e) and decreased the M1-like phenotype (\u003cem\u003eIL1B\u003c/em\u003e and \u003cem\u003eIL6\u003c/em\u003e) \u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e. However, it is challenging to identify the specific miRs or proteins that are altering the macrophage phenotype. Future studies would require knock out or suppression of these miRs or proteins in iPSCs to elucidate the effects of the EV derivatives on recipient macrophages.\u003c/p\u003e\u003cp\u003eOur findings describe key proteins and miRs that were associated with iPSC EVs. iPSC EVs are more biologically complex than NT-2 EVs as they consist of a wider array of proteins and miRs. In addition, we have observed that these iPSC-EVs promote an anti-inflammatory macrophage phenotype and decrease the abundance of secretory proteins some of which have known chemotactic properties. Here we showed that the alterations in the secretome of macrophages treated with EVs is of functional relevance as it decreased monocyte transmigration, a key process in the propagation of inflammation. Further work will be necessary to determine the key signalling molecules within the iPSC EVs, the diversity of iPSC EVs across iPSC lines and critical EV surface proteins responsible for cell uptake. Here we have identified several potential anti-inflammatory mediators and mechanisms which may be amenable to mimicry and enhance the efforts to use stem cell derived EVs as anti-inflammatory therapeutics.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003eCell Culture of Human iPSCs\u003c/h2\u003e\u003cp\u003eThe human iPSC line WTSli028-A from the European Bank for Induced Pluripotent Stem Cells (EBISC) was used to generate EVs. These iPSCs were derived from the dermal fibroblasts of a healthy female (50\u0026ndash;54 year old) of white-British descent. Non-integrating Sendai virus to induce transient overexpression of KLF4, C-Myc, Sox2 and Oct-4 was used to derived these cells. iPSC were cultured on 6-well plates coated with Vitronectin XF (10 \u0026micro;g/mL) (StemCell Technologies, Cat No. 07180) for 1hr at room temperature before use. Essential 8\u0026trade; Flex Media (Gibco, Cat no. A28858501) was used to maintain iPSCs incubated at 37\u0026deg;C in 5% CO2. iPSCs were subcultured using a passage ratio of 1:6. Supernatant was collected from iPSCs upon reaching 70\u0026ndash;80% confluency and frozen at -80\u0026deg;C. iPSC were then passaged via ReLeSR\u0026trade; Passaging Reagent (StemCell Technologies, Cat no. 100\u0026ndash;0483). iPSCs were passaged upon reaching confluency into Essential 8\u0026trade; Flex Media supplemented with ROCK Inhibitor, Y-27632, (10 \u0026micro;M) (Tocris, Cat No. 1254) and maintained in this media for 24h. Colonies had classical morphology as observed by brightfield microscopy, had high expression of pluripotency genes \u003cem\u003eSOX2\u003c/em\u003e, \u003cem\u003eNANOG\u003c/em\u003e and \u003cem\u003eOCT4\u003c/em\u003e and were fluorescently positive for OCT4 (\u003cb\u003eSupplemental Fig.\u0026nbsp;1\u003c/b\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eCell Culture of NT-2 Cells\u003c/h2\u003e\u003cp\u003eNT-2 cells also known as NTERA-2 cells (ATCC, Cat. No. CRL-1973) are a human pluripotent embryonal carcinoma cell line from the testis. NT-2 cells were used to generate control EVs to compare the EV content to that of iPSC EVs. NT-2 cells were cultured in T75 flasks in high glucose Dulbecco's Modified Eagle Medium (DMEM) (4.5g/L glucose) supplemented with 10% (v/v) Fetal Bovine Serum (Hyclone), 100U/ml penicillin and 100U/ml streptomycin (Gibco). Cells were subcultured using 0.25% Trypsin-EDTA (Invitrogen). Confluent NT-2 cells were cultured in serum-free DMEM for 24hr prior to passaging of cells. The supernatant/conditioned media was collected and centrifuged at 1,200 rpm for 5mins and frozen at -80\u0026deg;C prior.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eExtracellular Vesicle Isolation\u003c/h3\u003e\n\u003cp\u003eExtracellular vesicles were isolated from iPSC and NT-2 cell supernatant \u003cem\u003evia\u003c/em\u003e a series of differential centrifugations (Eppendorf Centrifuge 5417R) at 4\u0026deg;C. Samples were centrifuged in 1.5mL Eppendorfs at 2,000g for 20min, the supernatant was then centrifuged at 20,000g for 90min, the EV pellet was washed in double filtered phosphate buffered saline (DF-PBS) and then the centrifugation step of 20,000g was repeated and the EVs were resuspended in i) neat M199 media (Analab, Dublin, Ireland) or ii) DF-PBS.\u003c/p\u003e\u003cp\u003e\u003col\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eFor nanoparticle tracking analysis (NTA) and RT-PCR, the supernatant was then centrifuged at 24,000g for 60min, the EV pellet was washed in PBS and then the centrifugation step was repeated and the EVs were resuspended in DF-PBS or TRIzol.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eFor flow cytometry and transmission electron microscopy (TEM), the supernatant was then centrifuged at 20,000g for 90min, the EV pellet was washed in DF-PBS and then the centrifugation step was repeated and the EVs were resuspended in PBS.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003c/ol\u003e\u003c/p\u003e\u003cp\u003eFor NTA and flow cytometry analysis 4 EV pellets were pooled and resuspended in 300\u0026micro;L of DF-PBS. For TEM 4 EV pellets were pooled and resuspended in 50\u0026micro;L of double filtered sterile DF-PBS. For RT-PCR analysis, 4 pellets were pooled in 500\u0026micro;L of TRIzol reagent.\u003c/p\u003e\u003cp\u003eFor disruption of EVs; 0.1% Triton X-100 in DF-PBS was added to 7.5x10^8 EVs and incubated at 37\u0026deg;C for 15mins. DF-PBS was used as the control. Samples were centrifuged at 20,000g for 90mins and resuspended in 100\u0026micro;L of DF-PBS with 50\u0026micro;L used for NTA and 50\u0026micro;L remaining for the treatment of macrophages. Macrophages were treated with 500 EVs per cell.\u003c/p\u003e\n\u003ch3\u003eTransmission Electron Microscopy (TEM) of EVs\u003c/h3\u003e\n\u003cp\u003eTEM was performed as previously described \u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e. In brief, formvar carbon coated copper (G200-Cu) (EMS, Pennsylvania, USA), grids were used to mount the EVs. EVs were fixed using 2.5% glutardialdehyde (Merck, Darmstadt, Germany) and then incubated with 2% uranyl acetate (Agar Scientific, Essex, UK). Grids containing EVs were treated with a solution of 1.8% methyl cellulose (Sigma-Aldrich, Dorset, UK) and 0.4% uranyl acetate followed by air drying. An FEI Tecnai 12 Transmission Electron Microscope with the acceleration voltage set at 120 kv was used to image the EVs. Images were obtained at a magnification of 43,000X and 135,000X.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eSuper Resolution Microscopy of EVs\u003c/h2\u003e\u003cp\u003eThe EV Profiler Kit (ONI, Oxford, Cat. no. EV-MAN-1.0) was used as per manufacturer\u0026rsquo;s instructions to assess EVs by super resolution microscopy. EVs were immobilized on the profiler chip and stained for the presence of the tetraspanin antibodies, CD9, CD63 and CD81. Positive control EVs (supplied by ONI, Oxford) and negative controls were also included. Profiler chips with EVs were analysed on the Nanoimager (ONI, Oxford) with temperature (32\u0026deg;C) and illumination angle (53\u0026deg;) set. 100nm TetraSpeck\u0026trade; Microspheres (Thermofisher, UK) were used for channel mapping calibration. Direct stochastic optical reconstruction microscopy (dSTORM) analysis was applied. For each field of view (FOV) acquisition 1000 images were captured using the 488nm and 561nm laser and 500 images were captured using the 640nm laser. The exposure (100ms) and the frequency (10Hz) was set. 3\u0026ndash;4 FOV were captured per sample with Z-lock applied. Images were visualised using the Collaborative Discovery (CODI) Software with drift correction applied.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eNanoparticle Tracking Analysis of EVs\u003c/h2\u003e\u003cp\u003eThe concentration and size of the EVs was measured by nanoparticle tracking analysis using the NanoSight NS300 (Malvern Panalytical Ltd, UK) which uses light scattering and Brownian motion to assess the size distribution and concentration of particles suspended in DFPBS (\u003cb\u003eSupplemental Fig.\u0026nbsp;2\u003c/b\u003e). EV samples were diluted 1 in 100 in DFPBS and delivered into the sample chamber \u003cem\u003evia\u003c/em\u003e automated injection. Particles in suspension scatter the light of a laser beam that is passed through the solution which is visualized by a 20X microscope and recorded by a camera. Fifteen videos of 1 minute duration were recorded for each sample using the appropriate camera level and focus. Videos were analysed using the Nanosight NTA 3.1 software using 10nm sized bins, a set detection threshold of 8 and then the concentration was calculated accounting for the dilution factor. Data from each sample were expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD of the fifteen recordings. Graphs were generated using R Studio and GraphPad Prism.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eHuman Peripheral Blood Monocyte Cell Isolation and Differentiation to Macrophages\u003c/h2\u003e\u003cp\u003ePeripheral venous blood was donated by healthy volunteers to the Irish Blood Transfusion Service (IBTS), National Blood Bank located in St. James\u0026rsquo;s Hospital, James\u0026rsquo;s Street, Dublin 8 for transfusion and research purposes. Written informed consent that was obtained from all volunteers. Written approval was granted by the IBTS which provides de-identified blood components, pro-bono, to academic researchers. IBTS approval number: 001-03-19. Peripheral blood monocyte cells (PBMCs) were isolated as previously described \u003csup\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e. In brief, whole blood was centrifuged at 190xg for 15min to remove the platelet rich plasma and then diluted 1:1 in PBS. This mix was layered onto Lymphoprep\u0026trade; (Alere Ltd, UK) and centrifuged at 450xg for 30min. The PBMC layer was removed and washed in PBS three times. Cells were re-suspended in M199 media (Analab, Dublin, Ireland) supplemented with 10% human serum (Sigma), L-glutamine (2mM) (Gibco) and antibiotic (100U/mL Penicillin and 100\u0026micro;g/mL Streptomycin) (Bio-sciences, Dublin, Ireland) and incubated on 14cm petri dishes for 2hr at 37\u0026deg;C, 5% CO\u003csub\u003e2\u003c/sub\u003e. Adhered cells were considered monocytes and were re-seeded at a density of 1x10\u003csup\u003e6\u003c/sup\u003e cells/mL in 12 well plates (day 0).\u003c/p\u003e\u003cp\u003eOn day 4 the 1mL of M199 media was added to each well. On day 7 the M199 media was discarded and replaced with 1mL of M199 media to allow for further macrophage differentiation (\u003cb\u003eSupplemental Fig.\u0026nbsp;3\u003c/b\u003e). On day 10, the M199 media on the macrophages was changed to serum free M199 media containing the EV treatments. EV treatments of 1x10\u003csup\u003e8\u003c/sup\u003e EVs per well (equivalent to 100 EVs per cell) and 5x10\u003csup\u003e8\u003c/sup\u003e EVs per well (equivalent to 500 EVs per cell) were added. Macrophages were treated with EVs for 24h in total. Lipopolysaccharide (LPS) (InvivoGen, Toulouse, France) (50ng/mL) was added for the final 4h and adenosine triphosphate (ATP) (5mM) was added for the final hour before harvesting for RNA and protein.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eGene/miR Expression Analysis by qRT-PCR\u003c/h2\u003e\u003cp\u003eRNA was isolated from EVs and cells using the TRIzol\u0026trade; Reagent (Thermo Fisher Scientific) protocol. Messenger RNA was reverse transcribed using SuperScript\u0026trade; III Reverse Transcriptase (Thermo Fisher Scientific, Cat no. 18080-044). Samples were analysed using SYBR\u0026trade; Green Universal Master Mix and Universal Taqman PCR Mix on the QuantStudio\u0026trade; 7 Flex Real-Time PCR System (Applied Biosystems, ThermoFisher Scientific). \u003cem\u003eGAPDH\u003c/em\u003e was used as the endogenous control. For miRNA synthesis the Applied Biosystems TaqMan microRNA reverse transcription kit (Applied Biosystems, Thermo Fisher Scientific) was used. TaqMan\u0026trade; microRNA Assays (Applied Biosystems) were used for reverse transcription and miR expression analysis was performed using Universal Taqman PCR Mix on the QuantStudio\u0026trade; 7 Flex Real-Time PCR System. U6 snRNA was used as the endogenous control for miRNA expression analysis. \u003cb\u003eSupplemental Table\u0026nbsp;1\u003c/b\u003e consists of a list of assays used for mRNA/gene and microRNA expression. RT-PCR results were analysed via the 2(-Delta Delta C(T)) method \u003csup\u003e\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eCytokine/Chemokine Analysis and MesoScale Discover Assay\u003c/h2\u003e\u003cp\u003eSupernatants from stimulated macrophages were analysed for MCP-1 via Enzyme-linked Immunosorbent Assay (ELISA). MCP-1 ELISA (ThermoFisher Scientific) was performed as per manufacturer\u0026rsquo;s instructions. A CLARIOStar Microplate Reader was used to read plate absorbance at 450nm. Supernatants from stimulated macrophages were analysed using Mesoscale Discovery Assays Multiplex V7 (Meso Scale Discovery) which included IL-8. Electrochemiluminescence signal was analysed on a Meso\u0026trade; QuickPlex SQ 120 (Meso Scale Discovery). Values outside of the standard curve range were excluded from the downstream analysis. Results were expressed as a percentage relative to the unstimulated and untreated macrophage control sample.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eMonocyte Transmigration Assay\u003c/h2\u003e\u003cp\u003eA transmigration assay was used to assess the chemotactic efficacy of the conditioned media/supernatant derived from macrophages treated with and without iPSC EVs. Pro-inflammatory macrophages frequently release chemokines and inflammatory cytokines to recruit additional monocytes and propagate the inflammatory process. Thus, human THP-1 monocytes cells (ATCC\u0026reg; TIB-202\u0026trade;, Middlesex, UK) were used as to assess the chemotactic efficacy of the macrophage conditioned media. THP-1 monocytes were maintained in in RPMI 1640 medium supplemented with GlutaMAX, 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin antibiotic. THP-1 monocytes were seeded in 125\u0026micro;L of serum free RPMI at a concentration of 1x10^5 cells per polycarbonate membrane transwell insert (5\u0026micro;M pores) (Corning, Cat. no. 3421). 250\u0026micro;L of macrophage conditioned media was added to 250\u0026micro;L of serum free media and added to the lower chamber. Transwell inserts with THP-1 monocytes were lowered into the lower chamber and incubated for 2 hr at 37\u0026deg;C, in 5% CO\u003csub\u003e2\u003c/sub\u003e. THP-1 monocytes migrate through the pores to the underside of the transwell membrane insert. The negative control consisted of THP-1 cells in the upper well and serum free media only in the lower chamber. Transwell inserts were removed and fixed in 3% formaldehyde (Fisher). Media was removed from inside the transwell chamber and the inner membrane insert was swabbed with a cotton bud. Transwells were washed in PBS and then stained with Hoechst 33342, trihydochloride, trihydrate (ThermoFisher Scientific, Cat no. H3570). The membrane insert was cut and mounted on a slide with the underside of the transwell membrane facing upwards. Fluorescence microscopy was performed using a Zeiss Axio Imager M1 Microscope (Zeiss). 12 random images at 20X were obtained per insert. The presence of Hoechst positive nuclei were considered as migrated cells and results were shown as average number of migrated cells/field of view. Three independent experiments consisting of three different passages of THP-1 monocytes and conditioned media from three independent experiments with iPSC EV treatment were performed using biological duplicates for all conditions.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eProteomics on EVs\u003c/h2\u003e\u003cp\u003eSamples were processed for mass spectrometry analysis as described by \u003cem\u003eHoward et al.\u003c/em\u003e 2022 \u003csup\u003e63\u003c/sup\u003e. In brief, EV samples in PBS (~\u0026thinsp;20uL) were diluted in a 40\u0026micro;L solution of 8M urea and 50mM Tris HCL and then sonicated. The protein samples were reduced using dithiothreitol (DTT) (8mM) (BioLabs, B7705S) followed by incubation in a thermomixer (1000 rpm) for 30mins at 30\u0026deg;C. Idoacetamide (IAA) (20mM) was added to each sample and incubated in a thermomixer (1000 rpm) for 30mins at 30\u0026deg;C (samples were protected from light). The samples were diluted in 50mM Tris HCL to decrease the concentration of urea to \u0026lt;\u0026thinsp;2M. Sequencing Grade Modified Trypsin (Promega, Cat: V511C) was added to each sample in a ratio of 1:20, trypsin enzyme to substrate. Samples were incubated overnight in a thermomixer (1000 rpm) at 37\u0026deg;C. Trypsin digestion was stopped using formic acid (diluted to 1% of the final concentration). Samples were cleaned using HyperSep\u0026trade; SpinTips C18 (Thermo Scientfic, Cat. 60109-412) and eluted in a solution of 80% acetonitrile (ACN), 0.1% trifluoroacetic acid in LC/MS grade water. Samples were evaporated in a SpeedVac Concentrator for ~\u0026thinsp;90mins at 30\u0026deg;C and peptides were resuspended in LC/MS grade water containing 0.5% acetic acid\u0026thinsp;+\u0026thinsp;2.5% ACN. Peptides from iPSC EVs were quantified and then analysed on a Q-Exactive mass spectrometer (Thermo Scientific) which was fitted with a reversed-phase NanoLC UltiMate 3000 high performance liquid chromatography (HPLC) system (Thermo Scientific) as previously described \u003csup\u003e\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e. MaxQuant \u003csup\u003e\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e,\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/sup\u003e (version 2.0.3.0) was used to process the raw data from the Q-Exactive through incorporation of the Andromeda search engine \u003csup\u003e\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e\u003c/sup\u003e. In order to identify the peptides ad proteins, the UniProt Homo Sapiens database (Version 2021_03) containing a total of 78,120 entries was used to match the mass spectrometry/mass spectrometry spectra detected. Mass spectrometry data and processing protocol have been made available at the PRIDE \u003csup\u003e\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u003c/sup\u003e Database (EMBL-EBI) with the dataset identifier PXD067567. \u003cem\u003eReviewer access details: Log in to the PRIDE website using the following details: Project accession: PXD067567 Token: 5O3Mmyynluyl. Alternatively, reviewer can access the dataset by logging in to the PRIDE website using the following account details: Username: [email protected] Password: tzg6x5Ar49vn\u003c/em\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eProteomics on Macrophages\u003c/h2\u003e\u003cp\u003eMacrophages were washed with PBS and then lysed in a 40\u0026micro;L solution of 6M urea in 50mM Ammonium Bicarbonate. 50\u0026micro;g of protein was reduced with DTT (final concentration of 4mM) and incubated for 30mins at 60\u0026deg;C. IAA (8mM) solution in 50mM Ammonium Bicarbonate was added and the samples were incubated in the dark for 40mins. The samples were diluted in 50mM Tris HCL to decrease the concentration of urea to \u0026lt;\u0026thinsp;2M. Sequencing Grade Modified Trypsin (Promega, Cat: V511C) was added to each sample in a ratio of 1:15, trypsin enzyme to substrate. Samples were incubated overnight in a thermomixer (1000 rpm) at 37\u0026deg;C. Trypsin digestion was stopped using acetic acid (diluted to 1% of the final concentration). Samples were cleaned using C18 Stage tips prepared as per Rappsilber \u003cem\u003eet al.\u003c/em\u003e [34]. Following stage tip activation, peptides were loaded, washed and eluted in a solution of 80% acetonitrile (ACN), 0.1% trifluoroacetic acid in LC/MS grade water. Peptides were evaporated in a SpeedVac Concentrator for ~\u0026thinsp;90mins at 45\u0026deg;C and peptides were resuspended in LC/MS grade water containing 0.5% acetic acid\u0026thinsp;+\u0026thinsp;2.5% ACN. 1.5mg/mL of peptide solution was loaded into mass spectrometry vials. Samples were analysed on a timsTOF Pro mass spectrometer (Bruker Daltonics, Bremen, Germany) coupled to a nanoELute (Bruker Daltonics, Bremen, Germany) ultra-high pressure nanoflow chromatography system. MaxQuant \u003csup\u003e\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e,\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/sup\u003e (version 1.6.17.0) was used to process the raw data from the timsTOF through incorporation of the Andromeda search engine \u003csup\u003e\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e\u003c/sup\u003e. In order to identify the peptides ad proteins, the UniProt Homo Sapiens database (Version 2020_09) containing a total of 75,777 entries was used to match the mass spectrometry/mass spectrometry spectra detected. Mass spectrometry data and processing protocol have been made available at the PRIDE \u003csup\u003e\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u003c/sup\u003e Database (EMBL-EBI) with the dataset identifier PXD067612. \u003cem\u003eReviewer access details: Log in to the PRIDE website using the following details: Project accession: PXD067612 Token: HXbf9mooxar4. Alternatively, reviewer can access the dataset by logging in to the PRIDE website using the following account details: Username: [email protected] Password: jpJvDE3OXvkO\u003c/em\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eProteomic Analysis\u003c/h2\u003e\u003cp\u003eHeatmaps were generated using Perseus Software \u003csup\u003e[65]\u003c/sup\u003e. Proteins detected in all 3 independent experiments were analysed using the PANTHER Classification System (PANTHER19.0) \u003csup\u003e\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e\u003c/sup\u003e. Specifically, \u0026lsquo;Cellular Component Analysis\u0026rsquo; was performed with Fisher\u0026rsquo;s Extact T-test and FDR applied. Data was filtered for the top EV markers using the Vesiclepedia database and a heatmap was generated showing the relative protein quantification. The LFQ values of the most abundant proteins unique to iPSC EVs and unique to NT-2 EVs were graphed. Univariate analysis was performed comparing iPSC EV proteins with NT-2 proteins using imputation of data in the case of absence in either group. A scatter plot showing enrichment of iPSC EV proteins and NT-2 EV proteins were generated using GraphPad Prism. All proteins identified in iPSC and NT-2 EVs and the proteins unique to each group were analysed via Proteomaps \u003csup\u003e\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e,\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e\u003c/sup\u003e. Ingenuity pathway analysis (IPA) (Qiagen) was used to identify the EV proteins that are upstream regulators and transcription regulators.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003emicroRNA Analysis by TaqMan\u0026trade; OpenArray\u0026trade; Human Advanced microRNA Panel\u003c/h2\u003e\u003cp\u003eA customised TaqMan\u0026trade; OpenArray\u0026trade; Human Advanced microRNA Panel was used to analyse the microRNAs that were isolated from the EVs. This panel contained 216 miRs as listed in \u003cb\u003eSupplemental Table\u0026nbsp;2\u003c/b\u003e. The TaqMan OpenArray microRNA Panel was analysed using a QuantStudio\u0026trade; 12K Flex instrument. The relative threshold (Crt) was determined for all miRs with lower values indicative of higher miR abundance. The results were filtered for valid values in all 3 independent experiments in at least one group. The fold change was determined for miRs that were detected in both groups (3/3 valid values) and the statistically significant results were graphed. miRs detected in one group (3/3 valid values) but not the other were tabulated. IPA software (Qiagen) was applied to identify the microRNAs upregulated in the iPSC EVs with the ability to target specific mRNA (proteins used in this case) that were downregulated in macrophages following iPSC EV exposure. The results were visualised using a circos plot \u003csup\u003e\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003eStatistical Analysis\u003c/h2\u003e\u003cp\u003eStatistical analysis was performed using GraphPad Prism Software. The normality of the data was assessed. A one-way ANOVA comparing multiple selected columns was used for comparisons of more than two groups. For comparisons of two groups, a t-test was performed. Individual dots are representative of independent experiments. Error bars are representative of \u0026plusmn;\u0026thinsp;standard error of the mean (SEM).\u003c/p\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding Declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis project was funded by the\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eUCD Strategic Support Wellcome Trust Fund (17533).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUCD Conway Microscopy Core. ONI Nanoimaging Company.\u0026nbsp;Irish Blood Transfusion Service.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eS.F.-conception and design of experiments, data collection, data analysis and interpretation, drafting of manuscript. O.B.-design of experiments, data interpretation, drafting of manuscript. S.O. and M.M.S.M.-data collection and data interpretation. J.C., S.C., C.K. and J.D.- data collection. D.A.- design of experiments. ETD- data analysis. All authors reviewed the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003cbr\u003e\u0026nbsp;The author(s) declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMass spectrometry data and processing protocol have been made available at the PRIDE \u003csup\u003e68\u003c/sup\u003e Database (EMBL‐EBI) with the dataset identifiers PXD067567 and PXD067612. Additional supporting data can be found in the supplemental tables and figures.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eReviewer access details: Log in to the PRIDE website using the following details: Project accession: PXD067612 Token: HXbf9mooxar4. Alternatively, reviewer can access the dataset by logging in to the PRIDE website using the following account details: Username:\u0026nbsp;\u003c/em\u003e\u003cem\[email protected]\u003c/em\u003e\u003cem\u003e\u0026nbsp;Password: jpJvDE3OXvkO\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eReviewer access details: Log in to the PRIDE website using the following details: Project accession: PXD067567 Token: 5O3Mmyynluyl. Alternatively, reviewer can access the dataset by logging in to the PRIDE website using the following account details: Username: [email protected] Password: tzg6x5Ar49vn\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePeripheral venous blood was donated by healthy volunteers to the Irish Blood Transfusion Service (IBTS), National Blood Bank located in St. James\u0026rsquo;s Hospital, James\u0026rsquo;s Street, Dublin 8 for transfusion and research purposes. Written informed consent that was obtained from all volunteers. Written approval was granted by the IBTS which provides de-identified blood components, pro-bono, to academic researchers. IBTS approval number: 001-03-19.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eHoang, D. M. et al. Stem cell-based therapy for human diseases. \u003cem\u003eSignal. Transduct. Target. Therapy\u003c/em\u003e. \u003cb\u003e7\u003c/b\u003e, 272. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41392-022-01134-4\u003c/span\u003e\u003cspan address=\"10.1038/s41392-022-01134-4\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2022).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWang, L. T., Liu, K. J., Sytwu, H. K., Yen, M. L. \u0026amp; Yen, B. L. 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Circos: An information aesthetic for comparative genomics. \u003cem\u003eGenome Res.\u003c/em\u003e \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1101/gr.092759.109\u003c/span\u003e\u003cspan address=\"10.1101/gr.092759.109\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2009).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Extracellular vesicles, iPSCs, macrophages, monocytes, inflammation","lastPublishedDoi":"10.21203/rs.3.rs-7436803/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7436803/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eExtracellular vesicles derived from induced pluripotent stem cells (iPSC EVs) have immunoregulatory potential with the ability to alter the macrophage phenotype. Modulating the macrophage phenotype towards an anti-inflammatory, pro-resolving state may be beneficial in the treatment of chronic inflammatory diseases. The contents of iPSC EVs and their effects on macrophages are poorly understood. Here iPSC EVs were characterized and analysed by mass-spectrometry based proteomics and a targeted microRNA (miR) panel and their immunomodulatory effects on primary human macrophages were assessed.\u003c/p\u003e\n\u003cp\u003ePodocalyxin-like protein 1 (PODXL1) and \u0026nbsp;Insulin (INS) were the most abundant proteins unique to the iPSC EVs while miR-302d-3p was the most abundant miR. Notably, thioredoxin- and peroxiredoxin-related proteins were detected. iPSC EVs increased the anti-inflammatory associated Mannose Receptor C-Type 1 (\u003cem\u003eMRC1\u003c/em\u003e)\u003cem\u003e \u003c/em\u003eand miR-21, while monocyte chemoattractant protein 1 (MCP-1) and IL-8 were decreased. Proteomics revealed that treated macrophages had decreased levels of chemoattractant proteins, Azurocidin 1 (AZU1), Growth Differentiation Factor 15 (GDF15), and Ribosomal Protein S19 (RPS19). Conditioned media from macrophages treated with iPSC EVs inhibited monocyte transmigration, a key component in the propagation of inflammation. This study provides insights into the protein and miR cargo of iPSC EVs and highlights their capacity to inhibit chemotactic proteins in macrophages.\u003c/p\u003e","manuscriptTitle":"Extracellular vesicles derived from induced pluripotent stem cells mediate anti- inflammatory effects in primary human macrophages","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-29 11:57:06","doi":"10.21203/rs.3.rs-7436803/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-09-02T14:55:51+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-08-27T12:24:10+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-08-27T12:19:16+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e55ca9ca-f1c3-493c-9b3b-c2dff9839a4e","owner":[],"postedDate":"August 29th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":53785796,"name":"Biological sciences/Cell biology"},{"id":53785797,"name":"Biological sciences/Immunology"},{"id":53785798,"name":"Biological sciences/Molecular biology"},{"id":53785799,"name":"Biological sciences/Stem cells"}],"tags":[],"updatedAt":"2026-04-15T16:10:13+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-29 11:57:06","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7436803","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7436803","identity":"rs-7436803","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}