Unique and shared miRNAs of the three exosome types

Next, we investigated the role of the unique miRNAs from the three exosome types in signal regulation. The Venn diagram revealed 16, 27, and 61 unique miRNAs in hESC-Exos, hiPSC-Exos, and hUC-MSC-Exos, respectively, and 70 shared miRNAs (Fig. 6A). Te regulatory network of miRNA-protein interactions were evaluated by targeting the unique miRNAs. In hESC-Exos, the  16 unique miRNAs were found to regulate autophagy,  PI3K-AKT, Foxo, HIF-1, ErbB, mTOR, longevity, AMPK  pathway, etc. (Fig. 6B). For the 27 unique miRNAs in hiPSC-Exos, KEGG analysis revealed multiple significant ontologies, including the metabolism of xenobiotics,  AGE-RAGE signaling, mTOR signaling, retinol metabolism, cellular senescence, MAPK signaling, etc. (Fig. 6C).  In hUC-MSC-Exos, the 61 unique miRNAs were found to participate in the regulation of PI3K-AKT signaling,   human papillomavirus infection, cGMP-PKG signaling,   cellular senescence, Ras signaling, mTOR signaling, JAK-STAT signaling, NF-κB signaling, etc. (Fig. 6D).

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The miRNA components indirectly contribute greatly to multiple signaling pathways. To investigate the interaction between the unique miRNA clusters and canonical pathways, we performed a regulatory network analysis using the IPA database. IPA results revealed that the top read count miRNAs (P<0.05, abundance>1000) in the regulatory network were mapped to multiple canonical signaling pathways. miRNAs and their corresponding pathways are listed in Additional File 5. Using hESC-Exos miRNAs as examples, multiple high-abundance miRNAs,   including has-miR-95-3p, has-miR-30a-3p, has-miR- 181-5p, has-miR-183-3p, and has-miR-301a-3p, were involved in AMPK, autophagy, ErbB, longevity regulation, and the FOXO signaling pathway, among others.  Cytoscape was used to draw the network of unique miRNAs from the three exosome types and their regulated signaling pathways (Additional file 1: Fig. S11).

miRNA profiles related to pluripotency regulation

To investigate the regulatory effect of exosome-derived miRNAs on the pluripotency of stem cells, we predicted the target genes and screened the miRNAs involved in regulating the above process. Pluripotency-related miRNAs (abundance>1000) in the three exosome types are listed (Fig.  7A–C). Te top three miRNAs in the three   exosome types were has-miR-302b-3p, has-miR-302a-5p,   and has-miR-302d-3p (hESC-Exos), has-miR-372-3p,   has-miR-371a-5p, and has-miR-221-3p (hiPSC-Exos),   and has-miR-21-5p, has-miR-146a-5p, and has-miR- 320a-3p (hUC-MSC-Exos). Furthermore, we described the regulatory network of the top ten miRNAs from exosomes and their target genes involved in pluripotency regulation (Fig.  7D–F). Venn diagram analysis revealed  12 overlapping miRNAs, which indicated that they might be crucial miRNA sets in regulating the pluripotency of stem cells (Fig. 7G–H).

Specific proteins and miRNAs in the three exosome types

To further verify the actual expression of specific proteins in the three exosomes, we performed western blotting to detect candidate proteins in different pathways (Fig.  8A and B). For the cell cycle, three crucial regulatory factors, MCM5, PCNA1, and CDK1, were more highly expressed in hESC-Exos and hiPSCExos than in hUC-MSC-Exos. PRKAA1, belonging to the ser/thr protein kinase family, is a cellular energy,   conserved factor in all eukaryotic cells and showed a similar load in the three exosomes. SYK is widely expressed in hematopoietic cells and is involved in the coupling of activated immunoreceptors to downstream signaling events. BTK plays a crucial role in B cell development and immunoregulation. The protein loads of SYK and BTK were elevated in hUC-MSCExos compared to those in hESC-Exos or hiPSC-Exos. Wnt5 is a member of the Wnt gene family, which has been implicated in developmental processes, including the regulation of cell fate and patterning during embryogenesis. hESC-Exos were the most enriched in  Wnt5, followed by hiPSC-Exos and hUC-Exos. RHEB   is vital in regulating growth and cell cycle progression given its role in the mTOR/S6K signaling pathway.  Western blotting indicated that the three exosome types carried a similar RHEB load. EGFR is a cell surface protein that binds the epidermal growth factor, thus inducing receptor dimerization and tyrosine autophosphorylation, leading to cell proliferation.  Both hESC-Exos and hUC-MSC-Exos had higher  EGFR levels than hiPSC-Exos did. ICAM2 is a member of the intercellular adhesion molecule (ICAM) family and mediates adhesive interactions important for antigen-specific immune response, NK cell-mediated clearance, lymphocyte recirculation, and other cellular interactions important for immune response and surveillance. Among the three exosomes, hUC-MSC-Exos had the highest ICAM2 level. In addition, the top five miRNAs in the three exosome types were evaluated by  RT-qPCR, and their expression was consistent with the  RNA sequencing results (Fig. 8C).

Discussion

EVs are rich in various biologically active substances and are mainly composed of proteins, nucleotides, and lipids  [3, 17]. In recent decades the research community has ushered in the golden age of analytical techniques for protein, nucleotide, and lipid omics. Among these techniques, mass spectrometry [14, 20] and high-throughput sequencing [6, 54] enable the large-scale screening and identification of EV components. Two databases, Vesiclepedia (https://microvesicles.org) [28] and Expedia (https://evpedia.info) [29], are continuously updated and provide a summary of the components of mammalian and non-mammalian EVs, respectively. As EVs,   exosomes differ from MVs in terms of their biological origin and physical dimensions [46]. With continuous advances in the field of EVs, new methods are constantly being optimized to facilitate the isolation and purification of exosomes, meeting the rigor needed for comparative analysis. To replenish the exosome databases and expand the scope of their clinical applications, in this study, we describe the component framework of exosomes from hESCs and hiPSCs, which has not yet been reported. We also compared their composition and biological functions with those of hUC-MSC-derived exosomes. To this end,   we arrested exosomes from EVs at recognized sizes using ultracentrifugation combined with filtration, excluding  MVs and apoptotic bodies, which facilitated the refinement of exosome components. During the logarithmic growth phase of the cells, the collected CMs were ready for exosome preparation. The particle concentration of hESC-Exos was found to be significantly higher than that of hUC-MSC-Exos, but similar to that of hiPSC-Exos.  Similar results were obtained when comparing the protein concentrations. In contrast, total RNAs exhibited a significant decrease in gradients from hESC-Exos and hiPSC-Exos to hUC-MSC-Exos. These results indicate that hESCs (H9) may play a central role in the replication activity and have a stronger ability to secrete exosomes among the three stem cells.

In previous studies, researchers have focused on the accessibility of exosomes [1, 11, 58] while being confined to their research field. As a result, less attention has been paid to difference analysis between exosomes derived from different sources. Although some EV proteomics of the three stem cells has been performed, researchers have not yet refined the dimension of exosomes [19, 32, 59],   and the assay method is generally based on a single mass spectrometry technique. In the present study, both TMT   and LFQ methods were used to measure and quantify the protein components of the isolated exosomes. Bioinformatics analysis revealed that highly loaded proteins could coordinate the processes of development, damage repair,   and metabolism via intervening Wnt, AMPK, VEGF, and cell cycle signaling pathways, which was consistent with a previous report [19]. Similarly, hiPSC-Exos could also participate in the above biological processes by affecting the collateral signals. However, the EV proteomics of hiPSCs induced from HFFs showed a different gene ontology overview, focusing more on the processes of DNA replication and RNA catabolism [45]. Therefore, we deduced that exosomes secreted from hiPSCs derived from different tissues may have different protein profiles. In terms of hUC-MSCs-Exos, its components blended into many immunomodulatory activities by regulating complement system, microbial infection, NF-κB signaling, and so on,   and affected multiple metabolic signals, such as cholesterol metabolism, phospholipase D metabolism, and purine metabolism, providing results similar to those of previous reports [1, 59].

In the present study, we compared the proteomes of three types of exosomes pairwise and further analyzed their exclusive proteins regarding functional pathways and regulatory networks. The hiPSCs are a type of pluripotent stem cells that can be generated by reprogramming somatic cells to mimic the pluripotency of hESCs [48], which shows that these cells are similar to a certain extent. Although their exosome proteins were highly overlapping, those of hESC-Exos were enriched in developmental regulation and cell cycle functions,   indicating that hESC-Exos might have a stronger ability to regulate pluripotency than hiPSC-Exos in our study.  The pluripotency of hUC-MSCs is inferior to that of hiPSCs and hESCs, as reflected by their notably different protein profiles. The proteome of hUC-MSC-Exos differed from that of hESC-Exos and hiPSC-Exos in immunoregulation, being enriched in proteins regulating the activities of natural killer cells and the complement system. Furthermore, bioinformatics of the proteins shared by the three exosome types revealed that the upregulation of hESC-Exos or hiPSC-Exos proteins focused more on metabolism, development, and cell proliferation functions by interfering with classical signaling pathways such as the AMPK, Wnt, mTOR, and the cell cycle. In comparison, the upregulated proteins of hUC-MSC-Exos were not only enriched in immune-related signaling, including the complement system, microbial infection, NF-κB signaling, and B cell receptor signaling, but also in metabolic processes such as PPAR signaling, cholesterol metabolism, and MAPK signaling.

Exosome cargos are of unique tissue and cellular origins and contain miRNAs [16]. The present study also showed that exosomes isolated from CMs produced in vitro by hESCs, hiPSCs, and hUC-MSCs contained distinctive and specific miRNA signatures. We found that each exosomal miRNA had a unique landscape.   miRNA expression and interaction with the 3’ or 5’ UTR   of their target genes are involved in complex physiological and pathophysiological activities[18]. The top-loaded miRNAs of the three exosome types regulate a series of biological processes, including the cell cycle and Hippo,  Wnt, AMPK, and TGF-β signaling. However, the miRNA profiles of hUC-MSC-Exos had a stronger immunomodulatory ability than that of hESC-Exos or hiPSC-Exos regarding the behaviors of B and T cells, as well as TNF,  JAK-STAT, and NF-κB signaling. In particular, most unique miRNA profiles were linked to the regulation of autophagy, longevity, PI3K-Akt, mTOR, AMPK,   and p53 signaling, indicating that these miRNA clusters may modulate aging, aging-related diseases, tissue repair after injury, and metabolism. The unique miRNAs of hiPSC-Exos regulate mTOR signaling, cellular senescence, retinol metabolism, and TNF signaling, which also contributes to senescence and metabolism regulation. In addition to mTOR signaling and cell senescence,   the unique miRNAs of hUC-MSC-Exos regulate Foxo,  Jak-STAT, and NF-κB signaling, which may contribute to remodeling the metabolic and immune microenvironment. The analysis of shared miRNAs among the three exosome types further supported these differences in signaling regulation. Overall, the miRNA clusters in hESC-Exos or hiPSC-Exos coordinated the occurrence of multiple events, such as development, cell cycle, and cell differentiation. The miRNA set of hiPSC-Exos seems to play a less important role in these functions than that of hESC-Exos but a more important role than that of hUCMSC-Exos. As for hUC-MSC-Exos, the miRNA profiles found indicate their superior ability in regulating the immune environment, particularly in wound and infection healing.

In developmental biology, exosomes derived from pluripotent stem cells promote the maintenance of the pluripotent state. Therefore, miRNAs entering the cell microenvironment contribute significantly to the maintenance of stemness [4, 32]. The three exosome types in the present study contained different miRNA clusters that regulate pluripotent signaling. Nevertheless, the 12   miRNAs shared among the three exosome types may be crucial for pluripotency regulation, including miR- 21-5p, miR-92a-3p, and miR-221-3p. This hypothesis remains to be tested, and more stem cell types need to be investigated. In addition, the overlapping miRNAs between hESC-Exos and hiPSC-Exos may be involved in cell differentiation and reprogramming, as indicated by the results of more detailed analyses. For example, the   miR-302 family was highly enriched in and exclusive to hESC-Exos and hiPSC-Exos and contributed greatly to influencing stem cell behavior by modulating reprogramming [33, 50]. This point indirectly matches previous research regarding the uniqueness of miR-302 in human and mouse ESCs [33, 50]. Expression analysis of miRNA clusters that were highly expressed in ESCs during the initial phase of reprogramming revealed the induction of miR-17 [41] and miR-106a/106b [37]. Overexpression of miR-93 promoted an increase in the colony number of iPSCs [35].

The signaling network established by exosome cargos drives intracellular events, further intervening in a   series of pathological and physiological processes [3].  The exosomes of hESCs contain numerous loaded proteins and miRNAs predicted to regulate the landscape of development, metabolism, and anti-aging via blending into the AMPK, mTOR, and Wnt signaling pathways and regulating autophagy, longevity, and the cell cycle.  Multiple studies have highlighted the functions of hESCEVs in rejuvenating the aging hippocampus [25, 26], bone marrow [19], and endothelial cells [10], as well as alleviating recurring osteoarthritis by delivering specific proteins or miRNAs [58]. Our findings also support previous research, in which ESC-derived EVs were found to have positive implications in restoring impaired cardiovascular function [5]. ESC-derived EVs enabled maintaining the stemness of ESCs, thus being capable of reprogramming [4], which is in line with our results for hESC-Exos cargos. In our study, hiPSCs reprogrammed from umbilical cord cells had biological functions similar to those of hESC-Exos; however, no reports have specifically supported this theory. In comparison, hUC-MSC-Exos have received more attention, and various preclinical and clinical trials have clarified their therapeutic effect on multiple diseases [51]. Although hUC-MSC-Exos contribute to tissue regeneration and tissue remodeling, these abilities are inferior to that of hESC-Exos and hiPSC-Exos. Our analysis revealed that hUC-MSC-Exos exhibited excellent immune regulation ability. These findings provide a   basis for further research on inflammation-related diseases such as COVID-19 [2].

Conclusions

Overall, hESC-Exos is outstanding in regulating development, metabolism, and anti-aging, hiPSC-Exos has similar biological functions but is inferior to hESC-Exos.  In comparison, hUC-MSC-Exos contribute more to immune regulation. Our analysis broadens the application scope of hESC-Exos, hiPSCs, and hUC-MSC-Exos and highlights their respective advantages in the intervention of disease-related signaling. To the best of our knowledge, this study is the first to report a systematic and comprehensive analysis of exosome proteomics and miRNA profiles of hESCs, hiPSCs, and hUC-MSCs. Our study further enriches the current EV databases, facilitating the mining of more valuable data for the identification of appropriate acellular therapies in clinical settings.  These exosomes also cater to drug development as an alternative delivery system to replace virus delivery systems like adenovirus [7]. Although current predictions lack substantial validation, these findings could reveal further individual or joint applications of the three exosomes in preclinical or clinical research. Moreover,   future research could also be conducted via the integrated differential analysis of exosome cargos with EVs,   as well as of the components of the cells themselves.

Abbreviations

MSC: Mesenchymal stem cell; hESCs: Human embryonic stem cells; hiPSCs:  Human-induced pluripotent stem cells; hUC-MSCs: Human umbilical cord mesenchymal stem cells; TMT: Tandem mass tag; LFQ: Label-free relative peptide quantification; hESC-Exos: Human embryonic stem cell-derived exosomes; hiPSC-Exos: Human embryonic stem cell-derived exosomes;   hUC-MSC-Exos: Human umbilical cord mesenchymal stem cell-derived exosomes; EVs: Extracellular vesicles; MVs: Microvesicles; Alix: Apoptosis-linked gene 2-interacting protein X; TSG101: Tumor susceptibility gene 101; HFFs:  Human foreskin fibroblasts; CAMs: Cell adhesion molecules; TEM: Transmission electron microscopy; PDI: Polydispersity; TEAB: Tetraethylammonium bromide;  DEPs: Differentially expressed proteins; FDR: False discovery rates.

Acknowledgments

We are grateful to Xueke Tan, Zhongshuang Lv, and Xixia Li for helping with electron microscopy sample preparation and taking TEM images at the Center for Biological Imaging (CBI), Institute of Biophysics, Chinese Academy of  Science. The authors also thank Dr. Baohua Zou from the Institute of Biophysics Chinese Academy of Sciences for her kind help in crucial reading and suggestions.

Author contributions

YB, YZ, and JG conceived and designed the experiments. YB and XQ performed the exosome isolation and identification. YB, XQ, QL, SS, KZ, XQ, XZ, CJ,  HW, and ZY contributed to the bioinformatics analysis. YB, YZ, and JG wrote the paper. All authors have read and approved the manuscript.

Funding

This work was supported by grants from the National Key Research and Development Project (2019YFA0110400 to GJ), the National Foundation of Sciences and Technology (31971051, 31771562 to GJ), and Dalian municipal Dengfeng  Clinical Medicine Grant Support (2021024 to YZ).

Availability of data and materials

All datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request. All authors have confirmed that a citation for available data in the references section was included.

Declarations

Ethics approval and consent to participate 

The authors declare no potential conflict of interest.

Consent for publication 

Not applicable.

Author Details 

1 Key Laboratory of Interdisciplinary Research, Institute of Biophysics, Chinese  Academy of Sciences, Beijing 100101, China. 2 University of Chinese Academy of Sciences, Beijing 100049, China. 3 Department of Medical Oncology, The  Second Affiliated Hospital of Dalian Medical University, Dalian 116023, China. 4 Sixth Department of Liver Disease, Dalian Public Health Clinical Center, Dalian  Medical University, Dalian 116023, China.

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