CD146+ Endometrial-Derived Mesenchymal Stem/Stromal Cell Subpopulation Possesses Exosomal Secretomes with Strong Immunomodulatory miRNA Attributes

The perivascular localization of endometrial mesenchymal stem/stromal cells (eMSC) allows them to sense local and distant tissue damage, promoting tissue repair and healing. Our hypothesis is that eMSC therapeutic effects are largely exerted via their exosomal secretome (eMSC EXOs) by targeting the immune system and angiogenic modulation. For this purpose, EXOs isolated from Crude and CD146+ eMSC populations were compared for their miRNA therapeutic signatures and immunomodulatory functionality under inflammatory conditions. eMSC EXOs profiling revealed 121 in Crude and 88 in CD146+ miRNAs, with 82 commonly present in both populations. Reactome and KEGG analysis of miRNAs highly present in eMSC EXOs indicated their involvement among others in immune system regulation. From the commonly present miRNAs, four miRNAs (hsa-miR-320e, hsa-miR-182-3p, hsa-miR-378g, hsa-let-7e-5p) were more enriched in CD146+ eMSC EXOs. These miRNAs are involved in macrophage polarization, T cell activation, and regulation of inflammatory cytokine transcription (i.e., TNF-α, IL-1β, and IL-6). Functionally, stimulated macrophages exposed to eMSC EXOs demonstrated a switch towards an alternate M2 status and reduced phagocytic capacity compared to stimulated alone. However, eMSC EXOs did not suppress stimulated human peripheral blood mononuclear cell proliferation, but significantly reduced secretion of 13 pro-inflammatory molecules compared to stimulated alone. In parallel, two anti-inflammatory proteins, IL-10 and IL-13, showed higher secretion, especially upon CD146+ eMSC EXO exposure. Our study suggests that eMSC, and even more, the CD146+ subpopulation, possess exosomal secretomes with strong immunomodulatory miRNA attributes. The resulting evidence could serve as a foundation for eMSC EXO-based therapeutics for the resolution of detrimental aspects of tissue inflammation.


Introduction
The functional layer of the human endometrium is a highly regenerative tissue undergoing monthly cycles of growth and differentiation, and it is maintained by perivascular endometrial-derived mesenchymal stem/stromal cells (eMSCs) [1][2][3]. In general, mesenchymal stem/stromal cells (MSCs) are non-hematopoietic cells showing ease of isolation, extensive proliferation capacity, and multilineage differentiation potential in vitro [4,5]. CD146 expression in MSCs plays a key role in the perivascular niche, skeletogenesis, and hematopoietic support in vivo [6][7][8]. On this basis, our previous data showed that the CD146 signature is correlated with innately higher MSC immunomodulatory and secretory capacities, and, thus, therapeutic potency in vivo [9]. Interestingly, among the first markers used to identify eMSCs possessing higher clonogenic capacity in vitro was CD146, strongly indicating their perivascular origin in intact full-thickness endometrium [10]. Specifically, SUSD2, a single marker distinguishing perivascular eMSCs from the surrounding endometrial stromal cells, has been found to co-express CD146 and CD140b, MSC/pericytic markers which play an important role in the cyclical regeneration of this highly regenerative tissue [1,11,12]. Furthermore, we and others have demonstrated that MSCs acquiring a potent immunomodulatory phenotype actively reverse both inflammation and fibrosis linked with macrophage polarization from an M1 in disease to an M2 phenotype [13][14][15][16][17][18].
In our previous study, we demonstrated that the exposure of eMSC to an inflammatory environment upregulates their immunomodulatory transcriptional and inflammatory-/angiogenesis-related secretory profiles [12]. Consequently, MSC-derived exosomal cargos (EXOs) are a promising alternative to cellular therapeutics. In this study, EXOs isolated from Crude and CD146 + eMSC populations were characterized and compared for their miRNA therapeutic signatures. Furthermore, we assessed the capacity of eMSCs to affect macrophage and peripheral blood mononuclear cell functionality under inflammatory conditions. These types of observations could provide a rationale for further testing eMSC EXOs as a viable therapeutic modality to manufacture cell-free products for acute inflammation resolution, as well as in chronic conditions such as osteoarthritis and diabetes, where inflammation is increasingly recognized as an important component of disease progression.

Isolation, Culture, and Expansion of eMSC
Human endometrial tissue (n = 6) was collected according to our previous study [12] after participants provided written informed consent to the CryoVida stem cell bank (Guadalajara, Mexico). After eMSC expansion passage 0 (P0), cells were shipped to the University of Miami (Miami, FL, USA), then isolated, cultured, and expanded with complete Dulbecco's Modified Eagle's Medium plus 10% fetal bovine serum medium at 37 • C and 5% (v/v) CO 2 . All eMSCs were cultured by seeding 0.25 × 10 6 cells/175 cm 2 flask until 80% confluency until P3, and then detached with TrypLE Select Enzyme 1X (Gibco, Thermo Fisher Scientific, Waltham, MA, USA). We then assessed cell viability with 0.4% (w/v) Trypan Blue (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA). All tests were performed in accordance with the relevant guidelines and regulations following "not as human research" approval (based on the nature of the samples as discarded tissue).

Immunophenotype of eMSC
Flow cytometric analysis was performed on P3 eMSC (n = 3). Then, 2.0 × 10 5 cells were labeled with antibodies specific for SUSD2 (BioLegend, San Diego, CA, USA) and CD146 (Miltenyi Biotec, Auburn, CA, USA), in addition to the corresponding isotype controls. All cells were stained with eFluor 780 fixable viability dye (Invitrogen). The fluorescent signal was acquired using a CytoFLEX S (20,000 events) and analyzed with Kaluza analysis software (Beckman Coulter, Miami, FL, USA).

CD146 + eMSC Selection
The eMSC original population (designated as the Crude group) was sorted based on CD146 expression to yield the CD146 + subpopulation. Briefly, Crude eMSCs were re-suspended in staining buffer containing PBS with 0.5% bovine serum albumin (BSA) and 2 mM EDTA, and then incubated with biotinylated anti-human CD146 (Miltenyi Biotech, Inc., Auburn, CA) at 4 • C for 20 min. The Invitrogen™ CELLection Dynabeads™ Biotin Binder Kit (Thermo Fisher Scientific) was used according to manufacturer's instructions for magnetic selection.

Clonogenic Assay of Crude and CD146 + eMSCs
Crude and CD146 + eMSCs at passage three (P3) (n = 3) were seeded in 100-mm culture plates in triplicate, at a density of 10 3 cells per plate in complete medium. Colony-forming unit fibroblasts (CFU-Fs) were manually enumerated on day 15 after cytochemical staining with 0.01% Crystal Violet (Sigma, Billerica, MA, USA).

Cell Proliferation Assay of Crude and CD146 + eMSCs
Crude and CD146 + eMSCs at passage three (P3) (n = 3) were seeded in a 24-well plate at a concentration of 1 × 10 3 cells/well in triplicate, in complete medium. Growth curves were generated as percent confluency achieved on day 10 of culture, from brightfield images obtained using an IncuCyte live-cell analysis system with IncuCyte ZOOM software (Essen Bioscience, Ann Arbor, MI, USA).

Isolation and Validation of Crude and CD146 + eMSC EXOs
Crude and CD146 + eMSCs at passage three (P3) were seeded in complete medium until 70% confluency. Briefly, non-adherent cells were removed by Dulbecco's phosphate buffered saline (DPBS; Sigma Aldrich, St. Louis, MO, USA). After gentle rinsing, an exosome-depleted medium was added to each group for 48 hrs. Conditioned media from each group cultured in an exosome-depleted medium were collected and centrifuged at 2000× g for 10 min to remove debris, then at 10,000× g for 30 min, and finally ultracentrifuged at 120,000× g for 16 h.
Pre-enriched exosome populations were incubated with the Dynabeads ® -based Exosome-Human CD63 Isolation/Detection Reagent (Invitrogen) and purified according to the manufacturer's instructions for magnetic selection. CD9 (Invitrogen) expression was used to validate exosome presence in CD63 + -gated particles by flow cytometry. The specific fluorescent labeling of 20,000 events was analyzed on a CytoFLEX S with Kaluza analysis software (Beckman Coulter).
Nanoparticle tracking analysis (NTA) (NanoSight NS300, Malvern, Westborough, MA, USA) from Crude and CD146 + eMSC EXO was performed for quantity and size determination. All samples were diluted 1:50 in PBS. The following settings were set according to the manufacturer's software: detection threshold 5; room temperature; number of frames 30; and measurement time 30 s. The size distribution and particle concentration each represent the mean of five individual measurements.
The functional assessment of Crude and CD146 + eMSC EXOs was performed by macrophage polarization and immunopotency assays. For EXO tracking of eMSCs, exosomes were stained with a PKH26 red fluorescent membrane staining kit (Fluorescent Cell Linker Kits, Sigma) according to the manufacturer's instructions, and then co-cultured with target cells in functional assessments.

Quantitative Real-Time PCR (qPCR) of Crude and CD146 + eMSCs
A pre-designed 90-gene Taqman-based mesenchymal stem cell qPCR array (Stem Cell Technologies, Vancouver, Canada) was performed (n = 2) using 1000 ng cDNA per eMSC (Crude and CD146 + ) sample, and processed using a StepOne Real-time thermocycler (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA, USA). Data analysis was performed using Stem Cell Technologies' qPCR online analysis tools (Stem Cell Technologies). Sample and control Ct values were expressed as 2 −∆∆ Ct (with 38 cycles as the cut-off point). Expression levels were represented in bar plots ranked by transcript expression levels on a log-transformed scale of the sample cohort compared to the control. Bar plots were colorcoded by the functional class of genes (namely Stemness, MSC, MSC-related/Angiogenic, Chondrogenic/Osteogenic, Chondrogenic, Osteogenic, and Adipogenic). A t-test (unpaired, two-tailed test with equal variance) was used in all statistical analysis, and p-values were corrected for multiple comparisons by the Benjamini-Hochberg procedure.

miRNA Profile of Crude and CD146 + eMSC EXOs
A total Exosome RNA and Protein Isolation Kit (Thermo Fisher Scientific) was used to extract miRNA from Crude and CD146 + eMSC EXOs, according to the manufacturer's instructions. Total exosome miRNA (1 µg) was used for first-strand cDNA synthesis with the All-in-One miRNA First-Strand cDNA Synthesis Kit (GeneCopoeia, Rockville, MD, USA).
Pre-designed qPCR arrays covering 166 miRNA primers related to human MSC exosomes (GeneCopoeia) were performed using 1000 ng cDNA per Crude and CD146 + eMSC EXOs (n = 2), and then processed using a StepOne Real-time thermocycler (Applied Biosystems, LLC). The analysis was performed using GeneCopoeia's online Analysis System (http://www.genecopoeia.com/product/qpcr/analyse/ (accessed on 20 May 2022)). Mean values were normalized with SNORD47 (a small nucleolar RNA) and expression levels were calculated using the 2 −∆Ct method.
A miRNet centric network visual analytics platform (https://www.mirnet.ca/ (accessed on 10 June 2022)) was used to created miRNA interactomes. The miRNA target gene data were collected from the well-annotated database miRTarBase v8.0. miRNA-gene interactome network refining was performed with 2.0 betweenness cut-off. Values (with a 34-cycle cut-off point) were represented in a topology miRNA-gene interactome network using a force atlas layout and hypergeometric test algorithm.
RNA extraction from THP-1 cultures was performed using the RNeasy Mini Kit (Qiagen, Frederick, MD, USA) according to the manufacturer's instructions. Total RNA (1 µg) was used for reverse transcription with a SuperScript™ VILO™ cDNA synthesis kit (Invitrogen). A pre-designed 40-gene Human Macrophage Polarization array (GeneQuery™ Human Macrophage Polarization Marker qPCR Array Kit, ScienCell) was performed using 1000 ng cDNA per culture and processed using a StepOne Real-time thermocycler (Applied Biosystems). Mean values were normalized to GAPDH, and expression levels were calculated using the 2 −∆Ct method. Values were represented in a stacked bar plot for M0, M1, and M2 polarization as the relative fold change of the PMA/IO + THP-1/eMSC EXOs to PMA/IO + THP-1 (reference sample, 2 −∆Ct = X sample/X reference sample).
The miRDB online database (http://mirdb.org (accessed on 20 June 2022)) for prediction of functional microRNA targets has been used to correlate highly expressed target genes in macrophages with specific miRNAs identified by eMSC EXO miRNA profiling. MirTarget prediction scores are in the range of 0-100% probability, and candidate transcripts with scores ≥ 50% are presented as predicted miRNA targets in miRDB [27].

Phagocytosis Assay
For this step, 9.0 × 10 3 PMA/IO-stimulated THP-1 (macrophages) were mixed with Crude or CD146 + eMSC EXOs (n = 2 for each) per well of a 96-well plate, and cultured in M1-macrophage generation medium for 2 days. Parallel wells were designed as negative (M1 medium only) and positive (PMA/IO-stimulated THP-1 only) controls. On day 2, all cultures were incubated with 1.0 mg/mL fluorescein-labeled Escherichia coli K-12 bioparticles at 37 • C and 5% CO 2 for 2 h, then cytochemical stained with 1.25 mg/mL Trypan Blue according to the manufacturer's instructions (Thermo Fisher Scientific). Levels of phagocytosed fluorescent bioparticles were determined at 480 nm and 520 nm (SpectraMax M5 spectrophotometer, Molecular Devices, San Jose, CA, USA) and quantified using the following formula: %fluorescence = (net experimental reading/net positive control reading) * 100.

Protein Profile of Cells/eMSC EXOs Co-Cultures Secretome
Multiplex protein arrays of 60 cytokines (RayBio ® C-Series, RayBiotech, Peachtree Corners, GA, USA) were used to determine THP-1/eMSC EXO and PBMC/eMSC EXO co-culture secretomes (n = 2). For each assay, 1 mL of co-culture supernatant was used, following the manufacturer's instructions. The data shown represent 40 s of exposure in the FluorChem E chemiluminescence imaging system (ProteinSimple, San Jose, CA, USA). Results were generated by quantifying the mean spot pixel density of each array using a protein array analyzer plugin coupled with ImageJ software (Fiji/ImageJ, NIH website). Signal intensities were normalized with the background, and separate signal intensity results represent the average pixel density of two spots per protein. The signal intensity for each protein spot is proportional to the relative concentration of the antigen in the sample.

Statistical Analysis
A normal distribution of values was assessed by the Kolmogorov-Smirnov normality test. Statistical analysis was performed using paired and unpaired Student's t-test for normally distributed data with Wilcoxon (for paired data) or Mann-Whitney (for unpaired data) tests. One-way ANOVA was used for multiple comparisons. All tests were performed with GraphPad Prism v7.03 (GraphPad Software, San Diego, CA, USA). The level of significance was set at p < 0.05.
Molecular profiling of CD146 + eMSCs versus Crude eMSCs revealed that 55 out of 90 genes tested were more highly expressed in CD146 + eMSCs, with 21 genes being more than two-fold higher (VCAM1, PPARG, CSPG4, IL6, VEGFA, MCAM, BDNF, LIF, CD200, DLX2, ACAN, HIC1, FGF2, PDGFRB, COL1A1, TWIST2, NOTCH1, EGF, KITLG, GDF15, FUT4) ( Figure 1D). Interestingly, the tested genes were grouped into phenotype/functionrelated cohorts, with the MSC-associated, stemness, and MSC cohorts showing the most prominent fold expression change overall between CD146 + eMSC and Crude eMSC cultures ( Figure 1D). The VCAM-1 gene, whose expression was 16-fold higher in CD146 + eMSCs, is directly related to robust pro-angiogenic and immunosuppressive MSC actions. From the highly expressed genes (more than three-fold), PPARG, IL6, and BDNF have important roles in immunoregulation and cell apoptosis. PPARG regulates the expression of genes involved in the DNA damage response of inflamed endometrium [28]. In addition, IL6 has an intracellular role in MSC immunosuppression and proliferation, as its high expression is related to the increased capacity of MSCs to suppress activated T-cell proliferation [29]. In the same context, BDNF gene expression has pro-survival effects, and regulates intracellular signaling molecules in order to inhibit inflammatory cytokine expression in MSC [30].
In contrast, Crude eMSC molecular profiles showed higher expression of genes involved in MSC differentiation programs towards adipogenic, chondrogenic, and osteogenic lineages, with nine genes (FGF10, CEBPA, TERT, FGF18, SP7, ALPL, KDR, BMP6, VWF) being more highly expressed by over two-fold compared to CD146 + eMSCs. Specifically, the FGF10, CEBPA, and TERT genes seem to be a characteristic molecular signature for Crude eMSCs, as they are expressed 225-, 112-, and 95-fold more highly, respectively. These genes are involved in the proliferation and differentiation of MSC signaling. Specifically, studies showed that FGF10 expression and protein paracrine secretion control epithelial proliferation and ligand-receptor signaling in the endometrium [31]. Additionally, CEBPA and TERT gene expressions increase MSCs' stem-like properties and proliferation potential [32,33].
Reactome and KEGG analysis of miRNAs highly present in Crude and CD146 + eMSC EXOs indicated their involvement in the regulation of gene expression, immune system, cell cycle, cellular responses to stress, cytokine signaling, and MAPK signaling pathways (Figures 3 and 4). Furthermore, miRNA-gene interactome network analysis revealed four miRNAs (hsa-mir-21-5p, hsa-mir-32-5p, hsa-mir-98-5p, and hsa-let-7e-5p) for Crude eMSC EXOs and three miRNAs (hsa-mir-27b-3p, hsa-mir-98-5p, and hsa-let-7e-5p) for CD146 + eMSC EXOs with higher node degrees that act as hubs in the gene network. Even though the levels of these miRNAs as cargo within the eMSC EXOs are variable, they regulate multiple genes related to important signaling pathways.   . (A,B) Four miRNAs (hsa-mir-21-5p, hsa-mir-32-5p, hsa-mir-98-5p, and hsa-let-7e-5p) for Crude eMSC EXOs with higher node degrees act as hubs in the gene network. Crude eMSC EXOs showed their involvement in the regulation of six gene groups related to gene expression, immune system, cell cycle, cellular responses to stress, MAPK signaling, and WNT signaling pathways. (A,B) Three miRNAs (hsa-mir-27b-3p, hsa-mir-98-5p, and hsa-let-7e-5p) for CD146 + eMSC EXOs with higher node degrees act as hubs in the gene network. CD146 + eMSC EXOs showed their involvement in the regulation of six gene groups related to gene expression, immune system, cell cycle, cellular responses to stress, cytokine signaling in immune system, and MAPK signaling pathways.
Functionally, PMA/IO-stimulated THP-1 exposed to Crude or CD146 + eMSC EXOs for 2 days showed reduced capacity to phagocytize fluorescent bioparticles ( Figure 5E). In contrast, PMA/IO-stimulated THP-1 monocultures showed increased phagocytosis capacity in vitro. According to previous studies, high levels of phagocytic activity are directly related to strong polarization of macrophages towards the M1 pro-inflammatory phenotype, especially during acute inflammation [36]. In contrast, M2-like macrophage polarization resulted in their reduced phagocytosis capacity. In pathogen-free inflammation, this phenomenon can be related to removal of the remaining apoptotic cells at the final stages of inflammation, when macrophages have already polarized towards the M2 phenotype [37].
In parallel, two anti-inflammatory proteins, IL-10 and IL-13, showed higher secretion, especially in CD146 + eMSC EXOs + stimulated PBMC compared to stimulated PBMC alone cultures. Importantly, both IL-10 and IL-13 play crucial roles in macrophage polarization towards the M2 phenotype by upregulating the expression of arginase 1 (Arg1) and CD206 M2-polarization macrophage markers [38,39]. Collectively, from these data as well as those from the macrophage polarization assay, we can conclude that both Crude and CD146 + eMSC EXOs show strong M2 macrophage polarization effects in vitro.

Discussion
The human endometrium has emerged as an attractive source of mesenchymal stem/stromal cells (eMSC) that are easily isolated by non-invasive procedures, and show increased immunomodulatory and pro-angiogenic properties [12]. Furthermore, we have demonstrated that the CD146 signature is correlated with innately higher MSC immunomodulatory and secretory capacity, and, thus, better therapeutic potency in vivo [9]. Interestingly, at the extracellular vesicle level, our previous studies clearly demonstrated that infrapatellar fat pad-derived (IFP) MSCs show a potent miRNA immunomodulatory exosomal (EXOs) profile. Functionally, IFP-MSC EXOs can significantly affect macrophage polarization under inflammatory conditions both in vitro and in vivo by inducing macrophages towards an anti-inflammatory therapeutic M2 phenotype [22]. Herein, for the first time, we elucidated the miRNA exosomal profile of Crude and CD146 + eMSCs. Our findings provide critical information on the immunomodulatory effects of eMSC EXOs on macrophages' and peripheral blood mononuclear cells' functionality. These types of investigations could provide a rationale for further testing of eMSC EXOs as a viable therapeutic modality to manufacture cell-free products for inflammatory conditions, including osteoarthritis and diabetes. Specifically, macrophage infiltration and pro-inflammatory activation has been associated with synovitis/fat pad fibrosis severity in the knee joints of osteoarthritis patients [40,41], as well as pancreatic islet viability/insulin secretion capacity in diabetes patients [42,43].
SUSD2 was identified as a single marker capable of purifying eMSCs possessing MSC properties, and confirmed that these cells reside in a perivascular niche [3]. Consistent with previous studies [3,10], herein, we showed that SUSD2 + eMSC partially co-express the CD146 pericytic marker. According to our findings, the CD146 + eMSC subpopulation shows a distinct molecular profile that is directly related to immunosuppressive, proangiogenic, and anti-apoptotic MSC actions. Specifically, increased VCAM-1 expression in CD146 + eMSC is a potent mechanism by which MSCs exert their immunosuppressive effects via increased cell-cell adhesion with T cells [44]. Studies have shown that VCAM-1 + MSCs possess a favorable angiogenic paracrine activity, and display therapeutic potential in vascular ischemia animal models [45]. In contrast, the Crude eMSC molecular profile is mainly characterized by high expression of genes involved in MSC differentiation programs. Together, our data indicate that CD146 + eMSCs possess a superior immunomodulatory, pro-angiogenic, pro-survival molecular profile compared to Crude eMSCs.
Upon eMSC EXOs purification from Crude and CD146 + eMSC populations, hsa-miR-107, hsa-miR-125a, and hsa-miR-301a-3p miRNAs involved in immune system regulation were highly present in their exosomes. Specifically, the expression levels of hsa-miR-107 have been demonstrated to be related to TLR4 activation, whereas decreased expression of hsa-miR-107 may be a regulative feedback effect to limit insulin resistance in inflammation [46]. In addition, hsa-miR-125a stabilizes Treg-mediated immune homeostasis [47], whereas hsa-miR-301a-3p induces the M2 polarization of macrophages via activation of the PTEN/PI3Kγ signaling pathway [48]. Furthermore, we demonstrated that these immunomodulatory exosomal signatures can effectively induce PMA/IO-stimulated macrophages to polarize towards an anti-inflammatory therapeutic M2 phenotype. Notably, we observed that exposure of stimulated macrophages to CD146 + eMSC EXOs resulted in a more robust polarization towards M2-like macrophages by upregulation of the MRC1, TGFB1, and CCL2 genes. Studies have shown that TGF-β induces M2-like macrophage polarization via SNAIL-mediated suppression of a pro-inflammatory phenotype [49]. CCL2 is associated with monocyte recruitment in inflamed tissues via the CCR2 chemokine receptor after pro-inflammatory cytokine activation. Importantly, CCL2 and CCR2 determine the extent of M2 macrophage polarization by enhancing the production of the anti-inflammatory IL-10 cytokine [50].
In silico prediction analysis for MRC1, TGFB1, and CCL2 gene interaction with identified miRNA cargos from miRNA EXOs profiling revealed four miRNAs (hsa-let-7e-5p, hsa-miR-125a, hsa-miR-1255a, and hsa-miR-3065-5p) that strongly modulate their expression. Previous studies have demonstrated that the let family miRNAs may regulate M2 polarization through the SOCS1/STAT pathway [51]. Additionally, hsa-miR-125a affects monocyte adhesion and chemotaxis by direct targeting of the chemotaxis-mediating chemokine receptor CCR2 [52], whereas hsa-miR-1255a can regulate SMAD4 to participate in the TGF-β signaling pathway [53]. However, little is known about the biological function of hsa-miR-3065-5p on macrophages, and, therefore, it requires further investigation. Functionally, the pronounced M2-like macrophage polarization action by eMSC EXOs has been clearly demonstrated by the reduced phagocytic activity of macrophages. This may suggest that macrophage exposure to eMSC EXOs not only alters their molecular profile and phenotype, but may also significantly affect their functionality during inflammatory conditions in vivo.
At the cellular level, we have previously demonstrated that the immunomodulatory potential of eMSC is specifically related to their strong inhibitory effect on PBMC proliferation [12]. Along the same lines, Queckbörner et al. reported that eMSC co-culturing with PBMC can effectively suppress the proliferation and activation of CD4 + T cells [54]. In the present study, we investigated the effect of Crude and CD146 + eMSC EXOs on PBMC proliferation and pro-inflammatory secretory activity. Importantly, our data indicate that even though eMSC EXOs show a privileged immunomodulatory miRNA profile, they cannot significantly affect the proliferation of stimulated PBMC. To the best of our knowledge, this is the first time that such an effect has been reported. This finding may be attributed to the mechanisms used by MSC to suppress activation and proliferation of PBMC in vivo. In general, MSCs' immunoregulatory function requires their preliminary activation by immune cells through local secretion and stimulation by pro-inflammatory molecules, such as IFNγ, TNFα, IL-1α, and IL-1β [55]. In turn, MSCs activate their immunosuppressive and anti-inflammatory responses, mediated by several soluble factors, including IDO, PGE2, transforming growth factor β (TGFβ), insulin-like growth factor (IGF), and interleukin 10 (IL-10) [56][57][58]. Therefore, an absence of these important immunomodulatory soluble factors from eMSC EXOs can justify their limited capacity to suppress PBMC proliferation. However, the strong immunomodulatory attributes of eMSC EXOs are evident by the acquisition of a reduced pro-inflammatory secretory profile by stimulated PBMC. Specifically, we confirmed that 13 (GM-CSF, ICAM-1, IFN-γ, IL-2, IL-6 IL-6 sR, IP-10, MCP-1, MIP-1α, MIP-1β, RANTES, sTNF RII, and TIMP-2) inflammation-related cytokines had significantly lower secretion in eMSC EXOs + stimulated PBMC cultures compared to stimulated PBMC alone. In parallel, PBMC exposure to eMSC EXOs induces their higher secretion of the major anti-inflammatory proteins IL-10 and IL-13. IL-10 is a potent anti-inflammatory cytokine that inhibits MHC class II and costimulatory molecule B7-1/B7-2 expression on monocytes and macrophages, and limits the production of pro-inflammatory cytokines (including IL-1α and β, IL-6 and TNF-α) and chemokines (IP-10, MCP-1, and RANTES) [59]. Similarly, IL-13 is a strong anti-inflammatory cytokine, produced by T helper 2 cells and naïve or memory CD4 + /CD8 + T cells, that can inhibit the secretion of pro-inflammatory mediators, including nitric oxide (NO), IL-1β, IL-6, IL-12, and TNF-α [38]. Overall, both Crude and CD146 + eMSC EXOs induce a strong suppression of stimulated PBMC pro-inflammatory secretory activity.

Conclusions
In summary, eMSCs possess a potent miRNA immunomodulatory exosomal profile. Specifically, the CD146 + eMSC subpopulation demonstrates a significantly reinforced antiinflammatory molecular profile compared to that of Crude eMSC, an effect that is reflected in their differential miRNA EXOs signatures. Functionally, eMSC EXOs, and foremost, CD146 + eMSC EXOs, significantly affect macrophage and peripheral blood mononuclear cell functionality under inflammatory conditions in vitro. On this basis, our results help to elucidate the various local therapeutic anti-inflammatory activities of eMSC EXOs.