Released Exosomes Contribute to the Immune Modulation of Cord Blood-Derived Stem Cells

Background: Clinical studies demonstrated the immune modulation of cord blood-derived stem cells (CB-SC) for the treatment of type 1 diabetes and other autoimmune diseases, with long-lasting clinical efficacy. To determine the molecular mechanisms underlying the immune modulation of CB-SC, the actions of exosomes released from CB-SC were explored in this study. Methods: Exosomes were isolated from CB-SC cultures using ultracentrifugation and confirmed with different markers. The activated T cells and purified monocytes from peripheral blood mononuclear cells (PBMC) were treated with CB-SC in the presence or absence of the purified exosomes, followed by functional and flow cytometry analysis of phenotypic changes with different immune cell markers. Results: CB-SC-derived exosomes displayed the exosome-specific markers including CD9, CD63, and Alix, at the size of 85.95 ± 22.57 nm. In comparison with the treatment of CB-SC, functional analysis demonstrated that the CB-SC-derived exosomes inhibited the proliferation of activated PBMC, reduced the production of inflammatory cytokines, downregulated the percentage of activated CD4+ T and CD8+ T cells, and increased the percentage of naive CD4+ T and CD8+ T cells. Using the fluorescence dye DiO-labeled exosomes, flow cytometry revealed that exosomes preferably bound to the monocytes in the PBMC, leading to an improvement of mitochondrial membrane potential of treated monocytes. Further study indicated that the purified monocytes gave rise to spindle-like macrophages displaying type 2 macrophage (M2) surface markers and upregulating an expression of immune tolerance-related cytokines after the treatment with exosomes. Conclusions: CB-SC-derived exosomes display multiple immune modulations and primarily on monocytes, contributing to the immune education of CB-SC in the clinical treatment of autoimmune diseases.


INTRODUCTION
Autoimmunity, leading to the destruction of pancreatic islet β cells in type 1 diabetes (T1D), is mediated by abnormalities in multiple types of immune cells, including T cells, B cells, regulatory T cells (Treg), monocytes/macrophages (Mo/Mϕ), and dendritic cells (DC) (1). While insulin therapy allows T1D patients to manage blood sugar, it does not address the underlying immune dysfunction. Pancreatic and islet transplantations can overcome the shortage of insulin-producing islet β cells. However, donor scarcity and risk of immune rejection severely limit the use of these treatment modalities. Insulin-producing cells have been generated from embryonic stem (ES) cells and induced pluripotent stem cells (iPSC) through ex vivo inductions (2). However, transplanting these stem cells can also cause immune rejection (3), raising ethical and safety concerns. Alternative approaches are needed to correct the underlying autoimmunity of T1D.
We developed the Stem Cell Educator (SCE) therapy, which harnesses the unique therapeutic potential of cord blood-derived stem cells (CB-SC) to treat the multiple immune dysfunctions in diabetes (4)(5)(6)(7). SCE therapy circulates patient's blood through a blood cell separator, cocultures the patient's lymphocytes with adherent CB-SC in vitro, and returns "educated" lymphocytes to the patient's circulation (4,5,7). In contrast to conventional immune therapies, SCE therapy modifies rather than destroys the cells responsible for autoimmunity. Our clinical phase 1/2 trials indicate that SCE therapy reverses autoimmunity, promotes regeneration of islet β cells, and improves metabolic control for the treatment of T1D (4-7) and T2D (6,7). SCE therapy elicits immune tolerance by altering autoimmune T cells and pathogenic Mo/Mϕs through the autoimmune regulator (AIRE) and other molecular pathways (8). Exosomes are the smallest extracellular vesicles (EVs) (30-150 nm) that are produced by a variety of cells, and existing in all biological fluids, with diverse biological functions in the maintenance of homeostasis (9)(10)(11) To understand the molecular mechanisms underlying the immune education of SCE therapy to improve its clinical efficacy for the treatment of T1D and other autoimmune diseases, we explored the action of CB-SC-derived exosomes in this study. The data demonstrated multiple immune modulations of CB-SC-derived exosomes on immune cells.

Cell Culture for CB-SC
The culture of CB-SC was performed as previously described (4). In brief, human umbilical cord blood units (50-100 ml/U) were collected from healthy donors and purchased from Cryo-Cell International blood bank (Oldsmar, FL). Cryo-Cell has received all accreditations for cord blood collections and distributions, with hospital institutional review board (IRB) approval and signed consent forms from donors. Mononuclear cells were isolated with Ficoll-Hypaque (γ = 1.077, GE Health), and red blood cells were removed using ammonium-chloride-potassium (ACK) lysis buffer (Lonza). The remaining mononuclear cells were washed three times with phosphate-buffered saline (PBS) and seeded in 150 × 15 mm style non-tissue culture-treated Petri dishes (Becton Dickinson Labware) at 1 × 10 6 cells/ml. Cells were cultured in X-Vivo 15 chemical-defined serum-free culture medium (Lonza) and incubated at 37 • C with 8% CO 2 for 10-14 days. To characterize the phenotype and purity of CB-SC, the detached CB-SC were performed by flow cytometry with associated markers, including leukocyte common antigen CD45, ES cell markers OCT3/4 and SOX2, hematopoietic stem cell marker CD34, and the immune tolerance-related markers CD270 and CD274. Isotype-matched immunoglobulin G (IgGs) served as control.

Isolation of Exosomes From CB-SC Culture
At 80-90% of confluence, CB-SC were washed with PBS three times to remove all cellular debris, supplied with 25 ml fresh serum-free medium, and continued culturing. After 4 days, the conditioned medium was harvested for purifying exosomes by ultracentrifugation in an Optima L-100XP ultracentrifuge (Beckman Coulter) as previously described (12,13). Initially, the conditioned medium was centrifuged at 2,000 g for 20 min to remove cellular debris and other components, and followed by centrifugation at 10,000 g for 30 min to remove mitochondria. Consequently, the supernatant was collected and transferred to a new tube with a 10-kDa Amicon R Ultra-15 Centrifugal Filter Unit (Millipore Sigma) and centrifuged at 4,000 g for 30 min to concentrate the supernatant, followed by centrifugation at 100,000 g for 70 min. Finally, the pellets were rewashed once with PBS at 100,000 g for 70 min. The purified exosomes were resuspended in 100 µl PBS and transferred to a new centrifuge tube with a 0.22-µm filter and centrifuged at 2,000 g for 2 min. All centrifugation was performed at 4 • C. The purified exosomes were kept at −80 • C for later applications.

Electron Microscopy
The concentrated exosomes were loaded onto the electron microscope grids coated with Formvar. After being contrasted with uranyl-acetate solution and embedded in methylcellulose, exosomes were observed and photographed with a FEI Titan Themis 200 kV scanning transmission electron microscope (Thermo Fisher Scientific).

Dynamic Light Scattering
The size of the exosomes was determined using a dynamic light scattering (DLS) method, performed with a Nano-ZS Zetasizer Analyzer (Malvern Instruments Ltd, Malvern, united Kingdom) with a refractive index (RI) at 1.39.

Western Blotting
Purified exosomes or cells were treated with radioimmunoprecipitation assay (RIPA) buffer; protein concentration was determined by a bicinchoninic acid (BCA) protein assay. Proteins were separated by 10% Tris-HCl gel (Bio-Rad) and transferred to the polyvinylidene fluoride (PVDF) membrane, blotted overnight with anti-human Calnexin (Biolegend) and anti-human Alix monoclonal antibodies (mAb) (Biolegend), followed by anti-rat or anti-mouse horseradish peroxidase (HRP)-conjugated secondary mAb (Thermo Fisher Scientific). Membranes were incubated with chemiluminescent substrate (Millipore Sigma), and chemiluminescent signal was detected upon exposure to autoradiographic films.

FACS Assessment of Exosomes Characterization
To further confirm the release of exosomes from CB-SC, we performed the flow cytometry analysis after being captured with Exosome-Human CD63 Isolation/Detection Beads (Thermo Fisher, Waltham, MA). Owing to the small size of CB-SCderived exosomes and the limitation (>200 nm) of particle size detected by the Gallios Flow Cytometer (Beckman Coulter), the human CD63 isolation/detection beads (4.5-µm size) were utilized to isolate the exosomes from the ultracentrifugeconcentrated supernatant of CB-SC cultures for flow cytometry. Exosomes (10-20 µg protein) were incubated with 20 µl of 4.5-µm size anti-human CD63 beads in 100 µl volume and incubated overnight at 4 • C under 500 rpm agitation. Consequently, exosomes bound with CD63-capturing beads were place in the DynaMag TM -2 Magnet Stand (Thermo Fisher) and washed twice with PBS, and followed by the preparation for flow cytometry. Exosomes captured by anti-CD63 beads were aliquoted and incubated with phycoerythrin (PE)-conjugated anti-human CD63 (BD Bioscience), fluorescein isothiocyanate (FITC)-conjugated anti-human CD81 (BD Bioscience), and PEconjugated anti-human CD9 mAb (BD Bioscience), respectively, for 45 min at room temperature and then washed twice with PBS in the DynaMag TM -2 Magnet Stand, followed by flow cytometry.

PBMC Isolation and Proliferation Assay
Human buffy coat blood units were purchased from the New York Blood Center (New York, NY). Human peripheral bloodderived mononuclear cells (PBMC) were harvested as previously described (5). PBMC were stained with carboxyfluorescein succinimidyl ester (CFSE) (Life Technologies) according to the manufacturer protocol, then stimulated with Dynabeads coupled with anti-CD3 and anti-CD28 antibodies (Life Technologies) for 72 h in the presence of treatment with exosomes at 10 µg/ml, 20 µg/ml, and 40 µg/ml in duplicate, respectively, and were incubated at 37 • C in 5% CO 2 in the tissue culture-treated 96-well plate. Exosome-untreated cells served as control.

Flow Cytometry
Flow cytometric analyses of surface and intracellular markers were performed as previously described (6).

BCA Assay
Exosome concentration was measured by protein quantification using BCA assay (14), using the Pierce TM BCA Protein Assay Kit (Thermo Fisher Scientific). For protein concentration quantification, 10 µl of exosome samples were incubated with 200 µl BCA reagent (Thermo Fisher Scientific) at 37 • C for 30 min. Absorbance was read at 562 nm within 10 min. The protein concentration was determined by interpolating test sample concentrations relative to the standard concentration curve using bovine serum albumin.

Action of CB-SC Derived Exosomes on Monocytes
Monocytes were purified from PBMC using CD14 + microbeads (Miltenyi Biotec) according to the manufacturer's instruction, with purity of CD14 + cells >95%. The purified CD14 + monocytes were initially seeded in the tissue culture-treated six-well plate at 5 × 10 5 cells/well and cultured in X-Vivo 15 serum-free medium, at 37 • C, 5% CO 2 conditions. After adhering for 2 h, the attached monocytes were washed twice with PBS to remove all floating cells and cell debris, followed by treatment with or without CB-SC-derived exosomes (40 µg/ml) in X-Vivo 15 serum-free medium, at 37 • C, 5% CO 2 conditions. After treatment for 3 days, both the supernatant and detached cells were collected for ELISA and flow cytometry, respectively. The morphological change was photographed by phase-contrast microscope before cells were detached with 0.25% trypsin/ethylenediaminetetraacetic acid (EDTA) (Corning, New York) or an 18-cm cell scraper (BD Falcon). The exosome-treated and untreated cells were immunostained with a combination of mAbs, including Krome Orange-conjugated anti-human CD14 (Beckman Coulter), APC-conjugated antihuman CD80 (Beckman Coulter), AF750-conjugated antihuman CD86 (Beckman Coulter), PE-conjugated anti-human CD163, FITC-conjugated anti-human CD206, and BV421conjugated anti-human CD209 (BD Bioscience) for 30 min at room temperature and then washed with PBS at 2,000 rpm for 5 min before flow analysis. Isotype-matched mouse anti-human IgG antibodies (Beckman Coulter) served as a negative control.
To examine the effects of CB-SC-derived exosomes on mitochondrial function of monocytes, 3 × 10 5 fresh PBMC were plated on non-tissue-treated hydrophobic 24-well plates (avoiding the attachment of monocytes); PBMC were cultured with or without CB-SC-derived exosomes. After incubation for 3 h, the PBMC were harvested to be stained with anti-human CD14-Krome Orange-conjugated antibody and cytoplasmic Ca 2+ dye (Fluo-4) (Thermo Fisher Scientific) and mitochondrial Ca 2+ dye (Rhod-2) (Thermo Fisher Scientific) and tetramethylrhodamine, ethyl ester (TMRE) (Abcam) for the detection of mitochondrial membrane potential.

ELISA Assay
To detect the cytokine production by PBMC, 1 × 10 5 PBMC were stimulated with anti-CD3/anti-CD28 beads in the presence or absence of CB-SC-derived exosomes at 10, 20, and 40 µg/ml in triplicate in a 96-well plate with a total of 200 µl X-Vivo 15 serum-free culture medium (Lonza) per well. After the treatment for 72 h, the supernatants were collected to examine the inflammatory cytokines tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ) using human TNF-α and IFNγ enzyme-linked immunosorbent assay (ELISA) kits (Biolegend) according to the manufacturer protocols, respectively.
To examine the cytokine production by monocytes, the purified CD14 + monocytes were initially seeded in the tissue culture-treated six-well plate at 5 × 10 5 cells/well and cultured in X-Vivo 15 serum-free medium, at 37 • C, 5% CO 2 conditions. After adhering for 2 h, the attached monocytes were washed twice with PBS to remove all floating cells and cell debris, followed by treatment with or without CB-SC-derived exosomes (40 µg/ml) in X-Vivo 15 serum-free medium, at 37 • C, 5% CO 2 conditions. After treatment for 3 days, supernatants were collected to test the transforming growth factor (TGF)-β1 and interleukin (IL)-10 using ELISA kits (Biolegend) according to manufacturer's recommended protocol, respectively.

Statistical Analysis
Statistical analyses were performed with GraphPad Prism 8 (version 8.0.1) software. The normality test of samples was performed by the Shapiro-Wilk test. Statistical analyses of data were performed by the two-tailed Student's t-test to determine statistical significance for parametric data. Mann-Whitney Utest was utilized for non-parametric data. Values were given as mean ± standard deviation (SD). Statistical significance was defined as P < 0.05, with two sided.
Next, exosomes were purified from CB-SC cultures using serial centrifugations ( Figure 1B). Phenotypic characterization confirmed the expression of exosome-specific markers such as CD9 and CD81 on the CB-SC-derived exosomes by flow cytometry, which were analyzed following the purification by conjugation with anti-CD63 beads ( Figure 1C). The presence of exosomes was demonstrated by transmission electron microscopy ( Figure 1D), with the size of 85.95 ± 22.57 nm ( Figure 1E). Western blot further proved the expression of the exosome-associated universal marker Alix, but failed to exhibit the endoplasmic reticulum (ER)-associated marker calnexin ( Figure 1F). The data indicated that CB-SC release exosomes.

Suppression of PBMC Proliferation by CB-SC-Derived Exosomes
To explore the immune modulation of CB-SC-derived exosomes, the anti-CD3/CD28 bead-activated PBMC were initially treated with different dosages of CB-SC-derived exosomes ranging from 10 to 40 µg/ml. The PBMC proliferation was evaluated by carboxyfluorescein succinimidyl ester (CFSE) staining and flow cytometry analysis. The data demonstrated that the proliferation of PBMC was markedly declined after the treatment with CB-SC, with a percentage reduction about of 61.55 ± 6.43% (Figures 2A,B). In comparison, treatment with different dosages of exosomes declined the percentage of PBMC proliferation at 5.54% for the dosage of 10 µg/ml exosomes, 10.99% for 20 µg/ml, and 15.37% for 40 µg/ml, respectively (Figure 2A). There were significant differences for the dose groups at 20 µg/ml (P < 0.01) and 40 µg/ml (P < 0.005) relative to the group of anti-CD3/CD28-activated PBMC (Figure 2B).
In addition, we tested the inhibitory effect of CB-SC-derived exosomes at high doses such as 80 and 160 µg/ml on the anti-CD3/CD28 bead-activated PBMC. The inhibitory effect of exosomes on the proliferation of anti-CD3/CD28 bead-activated PBMC was markedly improved at these high dosages (Figure S1). In consideration of the clinical design (4, 7), the 9-or 12-layer Stem Cell Educators are utilized for the treatment of patients. Based on the current protocol, a maximum of 80 µg exosomes was normally purified from 240 ml supernatants of CB-SC cultures (20 ml per layer for 12 layers). Therefore, the optimal dose of exosomes at 40 µg/ml was utilized for the following experiments. Meanwhile, inflammatory cytokines were examined by ELISA assay. We found that the levels of TNF-α and IFNγ were markedly decreased following treatment with CB-SCderived exosomes at 20 µg/ml (P < 0.05) and 40 µg/ml (P < 0.01 and p < 0.005 respectively) (Figures 2C,D). In addition, the CD4/CD8 ratio was analyzed, and it failed to display a marked change after the treatment with exosomes, with CD4/CD8 ratios at 2.49 ± 0.97 for the PBMC group, 2.53 ± 0.94 for the PBMC + beads group, and 2.49 ± 0.96 for the PBMC + beads + exosomes group (P > 0.05, N = 5).

CB-SC-Derived Exosomes Primarily Target Monocytes in PBMC
To further explore the action of CB-SC-derived exosomes on other immune cells, PBMC were treated with CB-SC-derived exosomes labeled with DiO dye. Different blood lineage cells were characterized and gated with cell-specific markers such as CD3 for T cells, CD4 for CD3 + CD4 + T cells, CD8 for CD3 + CD8 + T cells, CD11c for myeloid dendritic cells (DC), CD14 for monocytes, CD19 for B cells, and CD56 for NK cells (Figure 4A). After an incubation for 4 h, flow cytometry demonstrated that different blood cell compartments displayed at different levels of DiO-positive exosomes ( Figure 4B). Notably, monocytes exhibited higher fluorescence intensity of DiO-positive exosomes than those of other immune cells, about six times higher than that of DC ( Figure 4C). It suggested that CB-SC-derived exosomes were mainly found in monocytes.

Differentiation of Monocytes Into Type 2 Macrophage (M2) After ex vivo Treatment With CB-SC-Derived Exosomes
To determine the direct effects of CB-SC-derived exosomes on monocytes, the purified CD14 + monocytes from PBMC were treated with CB-SC-derived exosomes. Phase contrast microscopy showed that about 50.80 ± 1.70% of exosometreated monocytes turned into spindle-like morphologies after the treatment for 3 days (Figures 5A,B), which was similar to previously characterized fibroblast-like macrophages (16). In contrast, most of the untreated monocytes were round and spread with a few elongated cells ( Figure 5A). Next, we tested their phenotype through gating the viable (PI − ) monocytes. In comparison with untreated monocytes (Figure 5C, green histogram and Figure 5D), expressions of M2-associated markers including CD163, CD206, and CD209 were markedly increased after the treatment with CB-SC-derived exosomes, specifically for the level of CD206 expression (Figure 5C, red histogram and Figure 5D). There were no substantial changes in the expression levels of CD14, CD80, and CD86 (Figures 5C,D).
In addition, the ELISA assay further indicated the increase in the production of immune tolerance-associated cytokines such as TGF-β1 and IL-10 in the supernatant of exosome-treated monocytes ( Figure 5E). Thus, these data imply that monocytes gave rise to macrophages with M2 phenotype after the treatment with CB-SC-derived exosomes.
Owing to the metabolic status of mitochondria contributing to the polarization of M1 and M2 macrophages (17,18), we examined mitochondrial mass and associated markers following the treatment with CB-SC-derived exosomes. Labeling with MitoTracker deep red for the detection of mitochondrial mass failed to show marked difference between exosome-treated and untreated groups ( Figure 5F). There were no significant differences in levels of cytoplasmic Ca 2+ ( Figure 5G) and mitochondrial Ca 2+ (Figure 5H). However, the mitochondrial membrane potential ( ψm) was considerably upregulated after the treatment with CB-SC-derived exosomes (Figure 5I). This may contribute to polarizing the differentiation of M2 macrophages after the treatment with CB-SC-derived exosomes.

DISCUSSION
Exosomes are becoming one of the hot topics for pharmacology and translational medicine. The current studies demonstrated the releasing of exosomes from CB-SC cultures, with specific exosome markers. Ex vivo data confirmed the direct modulation of CB-SC-derived exosomes on activated PBMC and monocytes. In comparison with the substantial effects of CB-SC, the data demonstrated that CB-SC-derived exosomes inhibited the proliferation of activated PBMC, reduced the production of inflammatory cytokines, and could be effective on specific subsets of T cells such as in downregulating the percentage of activated CD4 + and CD8 + T cells and increasing the percentage of naive CD4 + and CD8 + T cells. Notably, the differentiation of purified monocytes into M2 macrophages following the treatment with CB-SC-derived exosomes provide additional molecular mechanisms underlying the immune education and induction of immune tolerance observed in the clinical use of SCE therapy for the treatment of T1D (4-6) and other autoimmune diseases like alopecia areata (19).
Previous studies have demonstrated the long-lasting clinical efficacy of SCE therapy for the treatment of T1D and T2D patients, with a complete recovery of islet β-cell functions in some subjects after a single treatment with SCE therapy sustained for 4 years (6), as well as in the treatment of alopecia areata (19). These observations suggest that SCE therapy has the potential to fundamentally correct the immune dysfunction of these subjects with autoimmune diseases. Several possible molecular mechanisms underlying the immune education of SCE therapy were reported elsewhere (8), such as an expression of autoimmune regulator (AIRE) transcription factor in CB-SC, displaying high levels of the programmed death-ligand 1 (PD-L1) and CD270 on the cell surface of CB-SC, and the release of TGF-β1 and nitric oxide (NO) (20). Current data confirmed that the released CB-SC-derived exosomes contributed to the immune down-modulation of activated CD4 and CD8 T cells, along with a corresponding increase in naive CD4 and CD8 T cells. In the process of clinical treatment, SCE therapy circulates a patient's blood through a blood cell separator, cocultures the patient's T cells and monocytes with adherent CB-SC in vitro, and returns "educated" autologous cells to the patient's circulation (4,7). During the cocultures, CB-SC may release exosomes that target dysfunctional monocytes and/or T cells, leading to the tolerization of these cells and expanding the therapeutic potential of SCE therapy after infusing the cells back to the subjects. CB-SC-derived exosomes would also be transferred back to the subjects along with the CB-SC-treated patient cells, leading to the expansion of the therapeutic potential of SCE therapy in patients. Owing to their small size, the circulating CB-SCderived exosomes may penetrate into the damaged tissues such as pancreatic islets and target the pathogenic T cells and/or macrophages, contributing to the induction of tolerance and the clearance of residential autoimmune memory T cells (21). In this respect, previous clinical study established the modulation of autoimmune T-cell memory (e.g., CD4 + T CM and CD8 + T EM ) by SCE therapy in T1D subjects (5). Current ex vivo study demonstrated the marked downregulation of percentages of CD4 + T CM , CD4 + T EM , and CD8 + T CM after the treatment with CB-SC, but only slight reductions in the presence of CB-SC-derived exosomes. To overcome this limitation and improve the clinical efficacy of SCE therapy, it is necessary to further explore by which additional CB-SC-derived signals (8) contribute to the synergistic effect with exosomes in the modulation of autoimmune memory T cells. To mimic the clinical setting, it will be better to utilize T1D patient-derived PBMC, instead of using the anti-CD3/CD28-activated PBMC.
Monocytes/macrophages (Mo/Mϕ) are frontline immune cells defending against viral and bacterial infections and maintaining homeostasis, with diverse functions and  heterogeneity. Based on their profiles, macrophages are simply characterized with two subpopulations: type 1 macrophages (M1, proinflammation) and type 2 macrophages (M2, antiinflammation) (22). Increasing clinical evidence and animal studies demonstrate the dysfunction of Mo/Mϕ causing the pathogenesis of diabetes and other autoimmune diseases (17,(23)(24)(25)(26). Notably, this study established that purified monocytes gave rise to cells with an M2 phenotype after the treatment with CB-SC-derived exosomes, displaying the elongated morphology and the expression of M2-associated markers (e.g., CD163, CD206, and CD209). However, the level of costimulating molecules CD80 and CD86 expressions failed to show the marked changes after the treatment with CB-SC-derived exosomes. This may be associated with our current protocol in which the purified CD14 + monocytes were treated with CB-SC-derived exosomes in the presence of serum-free culture medium X-Vivo 15, without adding any other growth factors such as granulocyte-macrophage colony-stimulating factor (GM-CSF) or macrophage colony-stimulating factor (M-CSF). This was different from the conventional protocol that utilized cytokines such as M-CSF, IL-4, and/or IL-10 during the M2 differentiation (27). Both CD80 (28) and CD86 expressions are normally upregulated on the activated macrophages and served as a marker for M1 (28,29). Owing to the plasticity of monocytes/macrophages, the phenotypes of M1/M2 were significantly affected by ex vivo culture conditions such as the serum-containing or serum-free culture media and cytokine stimulations (27). For instance, the expression of CD80 was substantially increased on the M1 in the presence of 10% fetal bovine serum (FBS) Roswell Park Memorial Institute (RPMI) compared to the serum-free X-Vivo 10 media, but without significant difference in the level of CD86 expression between M1 and M2 (27). Therefore, culture conditions need to be optimized and standardized for the M1/M2 study. To elucidate the mechanisms by which signaling pathway contributed to the M2 differentiation, flow cytometry revealed that the mitochondrial membrane potential ( ψm) of monocytes was markedly increased after the treatment with CB-SC-derived exosomes, without affecting both intracellular and mitochondrial calcium concentrations. Since the differentiation to a M2 macrophage is strongly favored by an increase in oxidative phosphorylation (17), the upregulation of mitochondrial membrane potential by CB-SC-derived exosomes may thus lead to the differentiation to M2 macrophages. From this perspective, Calabria and colleagues reported that the mitochondrial membrane potential of neuronal cells was recovered by the treatment with exosomes isolated from adipose stem cells (30). The reason causing the upregulation of ψm by the interaction between exosomes and mitochondria need to be clarified in future studies. It is important to note that the differentiation of CB-SC-derived exosome-treated monocytes may be affected by patient's internal environment after being infused back during the SCE therapy. To this end, previous clinical data demonstrated that the percentage of CD86 + CD14 + of monocytes was markedly reduced 4 weeks after the treatment with SCE therapy in type 2 diabetic subjects (7), even no change in the level of CD86 expression after in vitro treatment.
Based on the above-mentioned immune modulation potentials of CB-SC-derived exosomes, quantification of released exosomes from CB-SC cultures may provide a valuable biomarker for the Quality Control (QC) analysis of good manufacturing practice (GMP)-manufactured Stem Cell Educators before their clinical applications. However, the size of CB-SC-derived exosomes was too small at ∼100 nm, which cannot be directly detected by flow cytometry and visualized by optical microscopes. It will be challenging to perform the absolute quantification with current limited technologies. The conventional approach for the isolation of exosomes was achieved by ultracentrifugation, ultrafiltration, and/or precipitation, with a duration of 4-5 h. These isolated exosomes can be quantified using current commercial techniques such as asymmetrical-flow field flow fractionation (AF4) coupled with multidetection, nanoparticle tracking analysis (NTA), DLS, and surface plasmon resonance (SPR) (31). However, due to their sensitivity, specificity, cost, and time-consuming process, the isolation and quantification of exosomes would need to be standardized with the optimized protocol. It should be noted that we utilized the conventional approach for the isolation of CB-SC-derived exosomes in the current work that might have other vesicles in different proportions. In addition, different cord blood donor-derived CB-SC may release exosomes with variable contents of bioactive molecules, and ex vivo functional analysis will be necessary to parallel the exosome quantification for the QC analysis of SCE products.
Previous works demonstrated that CB-SC could give rise to three germ-layer-derived cells in the presence of different inducers (15,20), and differentiate into functional insulinproducing cells after transplantation into the chemical streptozotocin (STZ)-induced diabetic mice, leading to the reduction in hyperglycemia (15). In addition, CB-SC expressed the ES cell markers such as the self-renewalassociated transcription factors OCT3/4 and NANOG (15). Exosomes released from CB-SC might carry the capability of tissue regeneration of parent cells in addition to the immune modulation capacity. To further optimize the clinical treatment protocol and improve the clinical efficacy of SCE therapy, detailed mechanisms need to be further investigated through ex vivo studies such as docking of exosomes on monocytes, trafficking of exosomes in the cytoplasm of monocytes, and polarizing the M2 differentiation by the exosome treatment, along with in vivo studies in the autoimmune-caused diabetic mouse models (32,33). Specifically, the proteomic analysis through mass spectrometry-based proteome profiling and exosome RNA sequencing will be necessary to dissect the detailed molecular mechanisms underlying the long-lasting clinical efficacy of SCE therapy for the treatment of T1D and other autoimmune diseases.
The current ex vivo study provides a better understanding of how SCE therapy results in the anti-inflammatory clinical effects. CB-SC-derived exosomes preferably and quickly bounded to monocytes in 2-3 h. As mentioned above, during the coculture of CB-SC with patient's immune cells for clinical treatment during 8-9 h (4,5,7,19), the SCE-treated monocytes may carry the CB-SC-derived exosomes back into the body, probably resulting in further M2 differentiation and induction of tolerance. Therefore, SCE therapy has the potential to revolutionize the treatment of diabetes and multiple autoimmune diseases through CB-SCmediated immune modulation, without the safety and ethical concerns associated with conventional immune and/or stem-cellbased approaches.

DATA AVAILABILITY STATEMENT
The datasets generated for this study are available on request to the corresponding author.

AUTHOR CONTRIBUTIONS
YZ supervised experiments and contributed to concepts, experimental design, data analysis, and interpretation, manuscript writing, and final approval of manuscript. WH performed most experiments and data analysis. XS, HY, and JS performed stem cell culture, flow cytometry, and TEM for exosomes.