What is the transcriptomic response of human blastocysts following internalization of extracellular vesicles (EVs) secreted by the human endometrium?

EVs secreted by the maternal endometrium induce a transcriptomic response in human embryos that modulates molecular mechanisms related to embryo development and implantation.

EVs mediate intercellular communication by transporting various molecules, and endometrial EVs have been postulated to be involved in the molecular regulation of embryo implantation. Our previous studies showed that endometrial EVs carry miRNAs and proteins associated with implantation events that can be taken up by human blastocysts; however, no studies have yet investigated the transcriptomic response of human embryos to this EV uptake, which is crucial to demonstrate the functional significance of this communication system.

A prospective descriptive study was performed. Primary human endometrial epithelial cells (pHEECs), derived from endometrial biopsies collected from fertile oocyte donors (n = 20), were cultured in vitro to isolate secreted EVs. Following EV characterization, Day 5 human blastocysts (n = 24) were cultured in the presence or absence of the EVs for 24 h and evaluated by RNA-sequencing.

EVs were isolated from the conditioned culture media using ultracentrifugation, and characterization was performed using western blot, nanoparticle tracking analysis, and transmission electron microscopy. Human blastocysts were devitrified, divided into two groups (n = 12/group), and cultured in vitro for 24 h with or without previously isolated EVs. RNA-sequencing analysis was performed, and DESeq2 was used to identify differentially expressed genes (DEGs) (FDR < 0.05). QIAGEN Ingenuity Pathway Analysis was used to perform the functional enrichment analysis and integration with our recently published data from the pHEECs' EV-miRNA cargo.

Characterization confirmed the isolation of EVs from pHEECs' conditioned culture media. Among the DEGs in blastocysts co-cultured with EVs, we found 519 were significantly upregulated and 395 were significantly downregulated. These DEGs were significantly enriched in upregulated functions related to embryonic development, cellular invasion and migration, cell cycle, cellular organization and assembly, gene expression, and cell viability; and downregulated functions related to cell death and DNA fragmentation. Further, the intracellular signaling pathways regulated by the internalization of endometrial EVs were previously related to early embryo development and implantation potential, for their role in pluripotency, cellular homeostasis, early embryogenesis, and implantation-related processes. Finally, integrating data from miRNA cargo of EVs, we found that the miRNAs carried by endometrial EVs targeted nearly 80% of the DEGs in human blastocysts.

This is an in vitro study in which conditions of endometrial cell culture could not mimic the intrauterine environment.

This study provides novel insights into the functional relevance of EVs secreted by the human endometrium, and particularly the role of EV-miRNA regulation on global transcriptome behavior of human blastocysts during early embryogenesis and embryo implantation. It provides potential biomarkers that could become useful diagnostic targets for predicting implantation success.

This study was supported by the Spanish Ministry of Education through FPU awarded to M.S.-B. (FPU18/03735), Generalitat Valenciana through VALi+d Programme awarded to M.C.C.-G. (ACIF/2019/139), and Instituto de Salud Carlos III and cofounded by the European Social Fund (ESF) "Investing in your future" through the Miguel Servet Program (CP20/00120 [H.F.]; CP19/00149 [I.C.]). The authors have no conflicts of interest to disclose.

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Extracellular vesicles (EVs), including exosomes and microvesicles, have emerged as pivotal mediators of intercellular communication. Embryo implantation is a critical process in early pregnancy and requires communication between the embryo and maternal uterus. EVs are important in coordinating the communication between the embryo and maternal uterus. This review explores EV biogenesis, molecular composition, and functional roles during implantation. It emphasizes the dynamic role of EVs in modulating the maternal-embryo dialogue, which is critical for establishing a receptive endometrium and facilitating successful implantation. EVs secreted by the embryo and endometrial cells have been shown to carry a diverse cargo of proteins, lipids, and miRNAs, which collectively influence key physiological processes, including immune tolerance, endometrial receptivity, and trophoblast invasion. EVs can be potential candidates as non-invasive biomarkers to assess the quality of embryos and uterine receptivity to enhance reproductive success. By providing a comprehensive overview of the current understanding of EVs in implantation, this chapter aims to highlight the significance of EVs in reproductive biology and their potential applications in improving fertility rates.

Uterine dendritic cells (uDCs) are critical for endometrial function, yet their origin, molecular characteristics, and specific roles during the pre- and post-implantation periods in the human endometrium remain largely unknown. The complexity of the endometrial environment makes defining the contributions of uDCs subtypes challenging. We hypothesize that distinct uDC subsets carry out specialized functions, and that resident progenitor DCs generate these subtypes. Employing single-cell RNA sequencing on uterine tissues collected across different menstrual phases and during early pregnancy, we identify several uDCs subtypes, including resident progenitor DCs. CITE-seq was performed on endometrial single-cell suspensions to link surface protein expression with key genes identified by the RNAseq analysis. Our analysis revealed the developmental trajectory of the uDCs along with the distinct functional roles of each uDC subtype, including immune regulation, antigen presentation, and creating a conducive environment for embryo implantation. This study provides a comprehensive characterization of uDCs, serving as a foundational reference for future studies for better understanding female reproductive disorders such as infertility and pregnancy complications.

Successful embryo implantation relies on synchronized dialog between the embryo and endometrium, and the role of extracellular vesicles (EVs) in facilitating this cross-talk has been recently established. In our previous study, milk fat globule-EGF factor 8 protein (MFGE8) was identified as increasing in receptive endometrial epithelial cells (EECs) in response to trophoblastic EVs. However, the dynamics of MFGE8 protein in this context are not completely understood. Therefore, we examined its expression and secretion in EECs exposed to estrogen, progesterone, and trophoblastic EVs to gain deeper insights into its potential as an indicator of EV-mediated embryo-maternal dialogue. Our findings revealed that MFGE8 secretion is sensitive to estrogen and progesterone, and that trophoblastic EVs stimulate their release in both receptive and non-receptive EECs. Furthermore, trophoblast EV function was dose and time-dependent. Notably, the secretion of MFGE8 increased within a short timeframe of 30 min after addition of EVs, suggesting the possibility of rapid processes such as binding, fusion or internalization of trophoblastic EVs within EECs. Interestingly, MFGE8 released from EECs was associated with EVs, suggesting increased EV secretion from EECs in response to embryonic signals. In conclusion, increased MFGE8 secretion in this embryo implantation model can serve as an indicator of EV-mediated embryo-maternal dialogue.

Proper action of the female sex steroids 17β-estradiol (E2) and progesterone (P4) on the endometrium is essential for fertility. Beyond its role in regulating the cell cycle, cyclin A2 (CCNA2) also mediates E2 and P4 signaling in vitro, but a potential role in modulating steroid action for proper endometrial tissue development and function is unknown. To fill this gap in our knowledge, we examined human endometrial tissue from fertile and infertile cisgender women for CCNA2 expression and correlated this with pregnancy outcome. Functional assessment of CCNA2 was validated in vivo using a conditional Ccna2 uterine-deficient mouse model, while in vitro function was assessed using human cell culture models. We found that CCNA2 expression was significantly reduced in endometrial tissue, specifically the stromal cells, from women undergoing in vitro fertilization who failed to achieve pregnancy. Conditional deletion of Ccna2 from mouse uterine tissue resulted in an inability to achieve pregnancy, which appeared to be due to alterations in the process of decidualization, which was confirmed using in vitro models. From these studies, we conclude that CCNA2 expression during the proliferative/regenerative stage of the menstrual cycle allows for proper steroid responsiveness, decidualization, and pregnancy. When CCNA2 expression levels are insufficient, there is impaired endometrial responsiveness, aberrant decidualization, and loss of pregnancy.

Extracellular vesicles (EVs) serve as messengers for intercellular communication, yet the precise mechanisms by which recipient cells interpret EV messages remain incompletely understood. In this study, we explored how the origin of EVs, their protein cargo, and the recipient cell type influence the cellular response to EVs within an embryo implantation model. We treated two types of EVs to 6 different recipient cell types and expression of zinc finger protein 81 (ZNF81) gene expression in the recipient cells were quantified using quantitative polymerase chain reaction (qPCR). The proteomic contents of the EV cargos were also analyzed. The results showed that downregulation of the ZNF81 gene was a specific cellular response of receptive endometrial epithelial cells to trophoblast derived EVs. Protein cargo analysis revealed that the proteomic profile of EVs depends on their cell of origin and therefore may affect the recipient cell response to EVs. Furthermore, trophoblastic EVs were found to be specifically enriched with transcription factors such as CTNNB1 (catenin beta-1), HDAC2 (histone deacetylase 2), and NOTCH1 (neurogenic locus notch homolog protein 1), which are known regulators of ZNF81 gene expression. The current study provided compelling evidence supporting the existence of EV specificity, where the characteristics of both the EVs and the recipient cell type collectively contribute to regulating EV target specificity. Additionally, EV protein cargo analysis suggested a potential association between transcription factors and the specific functionality of trophoblastic EVs. This in vitro embryo implantation model and ZNF81 read-out provides a unique platform to study EV specific functionality in natural cell-cell communication.

Extracellular vesicles (EVs) are cell-derived nanosized membrane-bound vesicles that are important intercellular signalling regulators in local cell-to-cell and distant cell-to-tissue communication. Their inherent capacity to transverse cell membranes and transfer complex bioactive cargo reflective of their cell source, as well as their ability to be modified through various engineering and modification strategies, have attracted significant therapeutic interest. Molecular bioengineering strategies are providing a new frontier for EV-based therapy, including novel mRNA vaccines, antigen cross-presentation and immunotherapy, organ delivery and repair, and cancer immune surveillance and targeted therapeutics. The revolution of EVs, their diversity as biocarriers and their potential to contribute to intercellular communication, is well understood and appreciated but is ultimately dependent on the development of methods and techniques for their isolation, characterization and enhanced targeting. As single-stranded oligonucleotides, aptamers, also known as chemical antibodies, offer significant biological, chemical, economic, and therapeutic advantages in terms of their size, selectivity, versatility, and multifunctional programming. Their integration into the field of EVs has been contributing to the development of isolation, detection, and analysis pipelines associated with bioengineering strategies for nano-meets-molecular biology, thus translating their use for therapeutic and diagnostic utility.

Arterial thrombosis manifesting as heart attack and stroke is the leading cause of death worldwide. Platelets are central mediators of thrombosis that can be activated through multiple activation pathways. Platelet-derived extracellular vesicles (pEVs), also known as platelet-derived microparticles, are granular mixtures of membrane structures produced by platelets in response to various activating stimuli. Initial studies have attracted interest on how platelet agonists influence the composition of the pEV proteome. In the current study, we used physiological platelet agonists of varying potencies which reflect the microenvironments that platelets experience during thrombus formation: adenosine diphosphate, collagen, thrombin as well as a combination of thrombin/collagen to induce platelet activation and pEV generation. Proteomic profiling revealed that pEVs have an agonist-dependent altered proteome in comparison to their cells of origin, activated platelets. Furthermore, we found that various protein classes including those related to coagulation and complement (prothrombin, antithrombin, and plasminogen) and platelet activation (fibrinogen) are attributed to platelet EVs following agonist stimulation. This agonist-dependent altered proteome suggests that protein packaging is an active process that appears to occur without de novo protein synthesis. This study provides new information on the influence of physiological agonist stimuli on the biogenesis and proteome landscape of pEVs.

The ability of trophectodermal cells (outer layer of the embryo) to attach to the endometrial cells and subsequently invade the underlying matrix are critical stages of embryo implantation during successful pregnancy establishment. Extracellular vesicles (EVs) have been implicated in embryo-maternal crosstalk, capable of reprogramming endometrial cells towards a pro-implantation signature and phenotype. However, challenges associated with EV yield and direct loading of biomolecules limit their therapeutic potential. We have previously established generation of cell-derived nanovesicles (NVs) from human trophectodermal cells (hTSCs) and their capacity to reprogram endometrial cells to enhance adhesion and blastocyst outgrowth. Here, we employed a rapid NV loading strategy to encapsulate potent implantation molecules such as HB-EGF (NVHBEGF). We show these loaded NVs elicit EGFR-mediated effects in recipient endometrial cells, activating kinase phosphorylation sites that modulate their activity (AKT S124/129, MAPK1 T185/Y187), and downstream signalling pathways and processes (AKT signal transduction, GTPase activity). Importantly, they enhanced target cell attachment and invasion. The phosphoproteomics and proteomics approach highlight NVHBEGF-mediated short-term signalling patterns and long-term reprogramming capabilities on endometrial cells which functionally enhance trophectodermal-endometrial interactions. This proof-of-concept study demonstrates feasibility in enhancing the functional potency of NVs in the context of embryo implantation.

The integration of robust single-pot, solid-phase-enhanced sample preparation with powerful liquid chromatography-tandem mass spectrometry (LC-MS/MS) is routinely used to define the extracellular vesicle (EV) proteome landscape and underlying biology. However, EV proteome studies are often limited by sample availability, requiring upscaling cell cultures or larger volumes of biofluids to generate sufficient materials. Here, we have refined data independent acquisition (DIA)-based MS analysis of EV proteome by optimizing both protein enzymatic digestion and chromatography gradient length (ranging from 15 to 44 min). Our short 15 min gradient length can reproducibly quantify 1168 (from as little as 500 pg of EV peptides) to 3882 proteins groups (from 50 ng peptides), including robust quantification of 22 core EV marker proteins. Compared to data-dependent acquisition, DIA achieved significantly greater EV proteome coverage and quantification of low abundant protein species. Moreover, we have achieved optimal magnetic bead-based sample preparation tailored to low quantities of EVs (0.5 to 1 µg protein) to obtain sufficient peptides for MS quantification of 1908-2340 protein groups. We demonstrate the power and robustness of our pipeline in obtaining sufficient EV proteomes granularity of different cell sources to ascertain known EV biology. This underscores the capacity of our optimised workflow to capture precise and comprehensive proteome of EVs, especially from ultra-low sample quantities (sub-nanogram), an important challenge in the field where obtaining in-depth proteome information is essential.

Extracellular vesicles (EVs) are important mediators of embryo attachment and outgrowth critical for successful implantation. While EVs have garnered immense interest in their therapeutic potential in assisted reproductive technology by improving implantation success, their large-scale generation remains a major challenge. Here, we report a rapid and scalable production of nanovesicles (NVs) directly from human trophectoderm cells (hTSCs) via serial mechanical extrusion of cells; these NVs can be generated in approximately 6 h with a 20-fold higher yield than EVs isolated from culture medium of the same number of cells. NVs display similar biophysical traits (morphologically intact, spherical, 90-130 nm) to EVs, and are laden with hallmark players of implantation that include cell-matrix adhesion and extracellular matrix organisation proteins (ITGA2/V, ITGB1, MFGE8) and antioxidative regulators (PRDX1, SOD2). Functionally, NVs are readily taken up by low-receptive endometrial HEC1A cells and reprogram their proteome towards a receptive phenotype that support hTSC spheroid attachment. Moreover, a single dose treatment with NVs significantly enhanced adhesion and spreading of mouse embryo trophoblast on fibronectin matrix. Thus, we demonstrate the functional potential of NVs in enhancing embryo implantation and highlight their rapid and scalable generation, amenable to clinical utility.

The growing understanding of the role of extracellular vesicles (EVs) in embryo-maternal communication has sparked considerable interest in their therapeutic potential within assisted reproductive technology, particularly in enhancing implantation success. However, the major obstacle remains the large-scale production of EVs, and there is still a gap in understanding how different culture systems affect the characteristics of the EVs. In the current study, trophoblast analogue human chorionic carcinoma cell line was cultivated in both conventional monolayer culture (2D) and as spheroids in suspension culture (3D) and how the cell growth environment affects the physical, biochemical and cellular signalling properties of EVs produced by them was studied. Interestingly, the 3D system was more active in secreting EVs compared to the 2D system, while no significant differences were observed in terms of morphology, size, and classical EV protein marker expression between EVs derived from the two culture systems. There were substantial differences in the proteomic cargo profile and cellular signalling potency of EVs derived from the two culture systems. Notably, 2D EVs were more potent in inducing a cellular response in endometrial epithelial cells (EECs) compared to 3D EVs. Therefore, it is essential to recognize that the biological activity of EVs depends not only on the cell of origin but also on the cellular microenvironment of the parent cell. In conclusion, caution is warranted when selecting an EV production platform, especially for assessing the functional and therapeutic potential of EVs through in vitro studies.

Synchronized crosstalk between the embryo and endometrium during the periconception period is integral to pregnancy establishment. Increasing evidence suggests that the exchange of extracellular vesicles (EVs) of both embryonic and endometrial origin is a critical component of embryo-maternal communication during peri-implantation. Here, we investigated whether embryonic signals in the form of EVs can modulate the endometrial epithelial cell secretome. Receptive endometrial analog RL95-2 cells were supplemented with trophoblast analog JAr cell-derived EVs, and the secretory protein changes occurring in the RL95-2 cells were analyzed using mass spectrometry. EVs of non-trophoblastic origin (HEK 293 cells) were used as the control EV source to supplement endometrial cells. Trophoblast cell-derived EVs enriched endometrial epithelial cell secretions with proteins that support embryo development, attachment, or implantation, whereas control EVs were unable to induce the same effect. The present study suggests that embryonic signals in the form of EVs may prime receptive endometrial epithelial cells to enrich their secretory proteome with critical proteomic molecules with functional importance for periconception milieu formation.

Mating induces large changes in the female genital tract, warranting female homeostasis and immune preparation for pregnancy, including the preservation of crucial oxidative status among its pathways. Being highly susceptible to oxidative stress, sperm survival and preserved function depend on the seminal plasma, a protection that is removed during sperm handling but also after mating when spermatozoa enter the oviduct. Therefore, it is pertinent to consider that the female sperm reservoir takes up this protection, providing a suitable environment for sperm viability. These aspects have not been explored despite the increasing strategies in modulating the female status through diet control and nutritional supplementation.

To test the hypothesis that mating modifies the expression of crucial oxidative-reductive transcripts across the entire pig female genital tract (cervix to infundibulum) and, particularly in the sperm reservoir at the utero-tubal junction, before ovulation, a period dominated by estrogen stimulation of ovarian as well as of seminal origin.

The differential expression of estrogen (ER) and progesterone (PR) receptors and of 59 oxidative-reductive transcripts were studied using a species-specific microarray platform, in specific segments of the peri-ovulatory sow reproductive tract in response to mating.

Mating induced changes along the entire tract, with a conspicuous downregulation of both ER and PR and an upregulation of superoxide dismutase 1 (SOD1), glutaredoxin (GLRX3), and peroxiredoxin 1 and 3 (PRDX1, PRDX3), among other NADH Dehydrogenase Ubiquinone Flavoproteins, in the distal uterus segment. These changes perhaps helped prevent oxidative stress in the area adjacent to the sperm reservoir at the utero-tubal junction. Concomitantly, there were a downregulation of catalase (CAT) and NADH dehydrogenase (ubiquinone) oxidoreductases 1 beta subcomplex, subunit 1 (NDUFB1) in the utero-tubal junction alongside an overall downregulation of CAT, SOD1, and PRDX3 in the ampullar and infundibulum segments.

Natural mating is an inducer of changes in the expression of female genes commanding antioxidant enzymes relevant for sperm survival during sperm transport, under predominant estrogen influence through the bloodstream and semen. The findings could contribute to the design of new therapeutics for the female to improve oxidative-reductive balance.

Extracellular vesicles (EVs) function as a mode of intercellular communication and molecular transfer to elicit diverse biological/functional response. Accumulating evidence has highlighted that EVs from immune, tumour, stromal cells and even bacteria and parasites mediate the communication of various immune cell types to dynamically regulate host immune response. EVs have an innate capacity to evade recognition, transport and transfer functional components to target cells, with subsequent removal by the immune system, where the immunological activities of EVs impact immunoregulation including modulation of antigen presentation and cross-dressing, immune activation, immune suppression, and immune surveillance, impacting the tumour immune microenvironment. In this review, we outline the recent progress of EVs in immunorecognition and therapeutic intervention in cancer, including vaccine and targeted drug delivery and summarise their utility towards clinical translation. We highlight the strategies where EVs (natural and engineered) are being employed as a therapeutic approach for immunogenicity, tumoricidal function, and vaccine development, termed immuno-EVs. With seminal studies providing significant progress in the sequential development of engineered EVs as therapeutic anti-tumour platforms, we now require direct assessment to tune and improve the efficacy of resulting immune responses - essential in their translation into the clinic. We believe such a review could strengthen our understanding of the progress in EV immunobiology and facilitate advances in engineering EVs for the development of novel EV-based immunotherapeutics as a platform for cancer treatment.

Dysregulation of estrogen receptor alpha (ERα) has been linked with increased metabolic and cardiovascular disease risk. Here, we generate and characterize cardiomyocyte-specific ERα knockout (ERαHKO) mice to assess the role of ERα in the heart. The most striking phenotype was obesity in female ERαHKO but not male ERαHKO mice. Female ERαHKO mice showed cardiac dysfunction, mild glucose and insulin intolerance and reduced ERα gene expression in skeletal muscle and white adipose tissue. Transcriptomic, proteomic, lipidomic and metabolomic analyses revealed evidence of contractile and/or metabolic dysregulation in heart, skeletal muscle and white adipose tissue. We show that heart-derived extracellular vesicles from female ERαHKO mice contain a distinct proteome associated with lipid and metabolic regulation, and have the capacity to metabolically reprogram the target skeletal myocyte proteome with functional impacts on glycolytic capacity and reserve. This multi-omics study uncovers a cardiac-initiated and sex-specific cardiometabolic phenotype regulated by ERα and provides insights into extracellular vesicle-mediated interorgan communication.

Implantation success relies on intricate interplay between the developing embryo and the maternal endometrium. Extracellular vesicles (EVs) represent an important player of this intercellular signalling through delivery of functional cargo (proteins and RNAs) that reprogram the target cells protein and RNA landscape. Functionally, the signalling reciprocity of endometrial and embryo EVs regulates the site of implantation, preimplantation embryo development and hatching, antioxidative activity, embryo attachment, trophoblast invasion, arterial remodelling, and immune tolerance. Omics technologies including mass spectrometry have been instrumental in dissecting EV cargo that regulate these processes as well as molecular changes in embryo and endometrium to facilitate implantation. This has also led to discovery of potential cargo in EVs in human uterine fluid (UF) and embryo spent media (ESM) of diagnostic and therapeutic value in implantation success, fertility, and pregnancy outcome. This review discusses the contribution of EVs in functional hallmarks of embryo implantation, and how the integration of various omics technologies is enabling design of EV-based diagnostic and therapeutic platforms in reproductive medicine.

Embryo-maternal cross-talk has emerged as a vitally important process for embryo development and implantation, which is driven by secreted factors and extracellular vesicles (EVs). The EV cargo of bioactive molecules significantly influences target cells and primes them for critical stages of reproductive biology, including embryo development, adhesion, and implantation. Recent research has suggested that EVs and their cargo represent a powerful non-invasive tool that can be leveraged to assess embryo and maternal tissue quality during assisted reproduction treatments. Here, we review the current scientific literature regarding the intercellular cross-talk between embryos and maternal tissues from fertilization to implantation, focusing on human biology and signaling mechanisms identified in animal models.

Cartography of the plasma proteome remains technically challenging, primarily due to the abundance and dynamic range of plasma proteins and their concentrations, exceeding ten orders of magnitude, including low-abundant tissue-derived proteins in the pg/mL range. Data-independent acquisition mass spectrometry (DIA-MS) has seen advances in unbiased mass spectrometry-based proteomic analysis of the plasma proteome. Here, we describe a comprehensive proteomic workflow of human plasma from clinically relevant sample (10 μL) that includes anti-protein immunodepletion and highly sensitive sample preparation workflow, with optimized scheduled isolation DIA-MS and deep learning analysis. This approach results in over 960 proteins quantified from a single-shot analysis of broad dynamic range, across 8 orders of magnitude (8.2 ng/L to 0.67 g/L). We further compare data-dependent acquisition (DDA) MS to highlight the advantage in protein quantification and inter-sample variation. These developments have provided streamlined identification of the human plasma proteome, including low-abundant tissue-enriched proteins, and applications toward understanding the plasma proteome.

A series of cyclical events within the uterus are crucial for pregnancy establishment. These include endometrial regeneration following menses, under the influence of estrogen (proliferative phase), then endometrial differentiation driven by estrogen/progesterone (secretory phase), to provide a microenvironment enabling attachment of embryo (as a hatched blastocyst) to the endometrial epithelium. This is followed by invasion of trophectodermal cells (the outer layer of the blastocyst) into the endometrium tissue to facilitate intrauterine development. Small extracellular vesicles (sEVs) released by endometrial epithelial cells during the secretory phase have been shown to facilitate trophoblast invasion; however, the molecular mechanisms that underline this process remain poorly understood. Here, we show that density gradient purified sEVs (1.06-1.11 g/ml, Alix⁺ and TSG101⁺, ∼180 nm) from human endometrial epithelial cells (hormonally primed with estrogen and progesterone vs. estrogen alone) are readily internalized by a human trophectodermal stem cell line and promote their invasion into Matrigel matrix. Mass spectrometry-based proteome analysis revealed that sEVs reprogrammed trophectoderm cell proteome and their cell surface proteome (surfaceome) to support this invasive phenotype through upregulation of pro-invasive regulators associated with focal adhesions (NRP1, PTPRK, ROCK2, TEK), embryo implantation (FBLN1, NIBAN2, BSG), and kinase receptors (EPHB4/B2, ERBB2, STRAP). Kinase substrate prediction highlighted a central role of MAPK3 as an upstream kinase regulating target cell proteome reprogramming. Phosphoproteome analysis pinpointed upregulation of MAPK3 T204/T202 phosphosites in hTSCs following sEV delivery, and that their pharmacological inhibition significantly abrogated invasion. This study provides novel molecular insights into endometrial sEVs orchestrating trophoblast invasion, highlighting the microenvironmental regulation of hTSCs during embryo implantation.

Extracellular vesicles (EVs) from stem cells have shown significant therapeutic potential to repair injured cardiac tissues and regulate pathological fibrosis. However, scalable generation of stem cells and derived EVs for clinical utility remains a huge technical challenge. Here, we report a rapid size-based extrusion strategy to generate EV-like membranous nanovesicles (NVs) from easily sourced human iPSCs in large quantities (yield 900× natural EVs). NVs isolated using density-gradient separation (buoyant density 1.13 g/mL) are spherical in shape and morphologically intact and readily internalised by human cardiomyocytes, primary cardiac fibroblasts, and endothelial cells. NVs captured the dynamic proteome of parental cells and include pluripotency markers (LIN28A, OCT4) and regulators of cardiac repair processes, including tissue repair (GJA1, HSP20/27/70, HMGB1), wound healing (FLNA, MYH9, ACTC1, ILK), stress response/translation initiation (eIF2S1/S2/S3/B4), hypoxia response (HMOX2, HSP90, GNB1), and extracellular matrix organization (ITGA6, MFGE8, ITGB1). Functionally, NVs significantly promoted tubule formation of endothelial cells (angiogenesis) (p < 0.05) and survival of cardiomyocytes exposed to low oxygen conditions (hypoxia) (p < 0.0001), as well as attenuated TGF-β mediated activation of cardiac fibroblasts (p < 0.0001). Quantitative proteome profiling of target cell proteome following NV treatments revealed upregulation of angiogenic proteins (MFGE8, MYH10, VDAC2) in endothelial cells and pro-survival proteins (CNN2, THBS1, IGF2R) in cardiomyocytes. In contrast, NVs attenuated TGF-β-driven extracellular matrix remodelling capacity in cardiac fibroblasts (ACTN1, COL1A1/2/4A2/12A1, ITGA1/11, THBS1). This study presents a scalable approach to generating functional NVs for cardiac repair.

The extracellular vesicle (EV) surface proteome (surfaceome) acts as a fundamental signalling gateway by bridging intra- and extracellular signalling networks, dictates EVs' capacity to communicate and interact with their environment, and is a source of potential disease biomarkers and therapeutic targets. However, our understanding of surface protein composition of large EVs (L-EVs, 100-800 nm, mean 310 nm, ATP5F1A, ATP5F1B, DHX9, GOT2, HSPA5, HSPD1, MDH2, STOML2), a major EV-subtype that are distinct from small EVs (S-EVs, 30-150 nm, mean 110 nm, CD44, CD63, CD81, CD82, CD9, PDCD6IP, SDCBP, TSG101) remains limited. Using a membrane impermeant derivative of biotin to capture surface proteins coupled to mass spectrometry analysis, we show that out of 4143 proteins identified in density-gradient purified L-EVs (1.07-1.11 g/mL, from multiple cancer cell lines), 961 proteins are surface accessible. The surface molecular diversity of L-EVs include (i) bona fide plasma membrane anchored proteins (cluster of differentiation, transporters, receptors and GPI anchored proteins implicated in cell-cell and cell-ECM interactions); and (ii) membrane surface-associated proteins (that are released by divalent ion chelator EDTA) implicated in actin cytoskeleton regulation, junction organization, glycolysis and platelet activation. Ligand-receptor analysis of L-EV surfaceome (e.g., ITGAV/ITGB1) uncovered interactome spanning 172 experimentally verified cognate binding partners (e.g., ANGPTL3, PLG, and VTN) with highest tissue enrichment for liver. Assessment of biotin inaccessible L-EV proteome revealed enrichment for proteins belonging to COPI/II-coated ER/Golgi-derived vesicles and mitochondria. Additionally, despite common surface proteins identified in L-EVs and S-EVs, our data reveals surfaceome heterogeneity between the two EV-subtype. Collectively, our study provides critical insights into diverse proteins operating at the interactive platform of L-EVs and molecular leads for future studies seeking to decipher L-EV heterogeneity and function.

Adequate endometrial stromal cell (ESC) decidualization is vital for endometrial health. Given the importance of extracellular vesicles (EVs) in intercellular communication, we investigated how their protein landscape is reprogrammed and dysregulated during decidual response. Small EVs (sEVs) from human ESC-conditioned media at Day-2 and -14 following decidual stimuli were grouped as well- (WD) or poorly decidualized (PD) based on their prolactin secretion and subjected to mass spectrometry-based quantitative proteomics. On Day 2, in PD- versus WD-ESC-sEVs, 17 sEV- proteins were down-regulated (C5, C6; complement/coagulation cascades, and SERPING1, HRG; platelet degranulation and fibrinolysis) and 39 up-regulated (FLNA, COL1A1; focal adhesion, ENO1, PKM; glycolysis/gluconeogenesis, and RAP1B, MSN; leukocyte transendothelial migration). On Day 14, in PD- versus WD-ESC-sEVs, FLNA was down-regulated while 21 proteins were up-regulated involved in complement/coagulation cascades (C3, C6), platelet degranulation (SERPINA4, ITIH4), B-cell receptor signalling and innate immune response (immunoglobulins). Changes from Days 2 to 14 suggested a subsequent response in PD-ESC-sEVs with 89 differentially expressed proteins mostly involved in complement and coagulation cascades (C3, C6, C5), but no change in WD-ESC-sEVs ESC. Poor decidualization was also associated with loss of crucial sEV-proteins for cell adhesion and invasion (ITGA5, PFN1), glycolysis (ALDOA, PGK1) and cytoskeletal reorganization (VCL, RAC1). Overall, this study indicates varied ESC response even prior to decidualization and provides insight into sEVs-proteomes as a benchmark of well-decidualized ESC. It shows distinct variation in sEV-protein composition depending on the ESC decidual response that is critical for embryo implantation, enabling and limiting trophoblast invasion during placentation and sensing a healthy embryo.

The therapeutic properties of cell derived extracellular vesicles (EVs) make them promising cell-free alternative to regenerative medicine. However, clinical translation of this technology relies on the ability to manufacture EVs in a scalable, reproducible, and cGMP-compliant manner. To generate EVs in sufficient quantity, a critical step is the selection and development of culture media, where differences in formulation may influence the EV manufacturing process. In this study, we used human amniotic epithelial cells (hAECs) as a model system to explore the effect of different formulations of chemically defined, commercially sourced media on EV production. Here, we determined that cell viability and proliferation rate are not reliable quality indicators for EV manufacturing. The levels of tetraspanins and epitope makers of EVs were significantly impacted by culture media formulations. Mass spectrometry-based proteomic profiling revealed proteome composition of hAEC-EVs and the influence of media formulations on composition of EV proteome. This study has revealed critical aspects including cell viability and proliferation rate, EV yield, and tetraspanins, surface epitopes and proteome composition of EVs influenced by media formulations, and further insight into standardised EV production culture media that should be considered in clinical-grade scalable EV manufacture for generation of therapeutic EVs.