Urinary Extracellular Vesicles for Non-Invasive Quantification of Principal Cell Damage in Kidney Transplant Recipients.

The objective of the present study was to compare principal cell-specific aquaporin-2 (AQP2) abundances in urinary extracellular vesicles (uEVs) on the first postoperative day in deceased-donor kidney transplant recipients without and with acute kidney injury. We measured uEV markers (CD9 and CD63) and the abundances of proximal tubular sodium-glucose transporter 2, distal tubular sodium/chloride cotransporter, and principal cell-specific aquaporin-2 using Western blotting of urine. uEV-AQP2 levels were normalized to living donor controls. The validation cohort consisted of 82 deceased-donor kidney transplant recipients who had a median age of 50 years (IQR 43 to 57 years). A total of 32% of recipients had acute kidney injury. The median uEV-AQP2 was significantly higher in recipients with acute kidney injury compared to immediate allograft function (2.05; IQR 0.87 to 2.83; vs. 0.81; IQR 0.44 to 1.78; p < 0.01). The Youden index indicated a uEV-AQP2 threshold of 2.00. Stratifying uEV-AQP2 into quartiles showed that recipients with higher uEV-AQP2 levels had higher rates of acute kidney injury (Cochran-Armitage, p = 0.001). The discovery cohort showed elevated CD9, CD63, and uEV-AQP2 levels in urine from recipients with acute kidney injury compared to immediate allograft function. We were able to quantify the damage of principal cells after kidney transplant to predict acute kidney injury using uEV-AQP2.

Aged fibroblast-derived extracellular vesicles promote angiogenesis in melanoma.

Advancing age is a negative prognostic factor for cutaneous melanoma. However, the role of extracellular vesicles (EVs) within the melanoma tumor microenvironment (TME) has remained unexplored in the context of aging. While the size and morphology of the EVs isolated from young vs. aged fibroblasts remained unaltered, the contents of the protein cargo were changed. Aging reduced the expression of the tetraspanin CD9 in both the dermal fibroblasts and released EVs. CD9 is a crucial regulator of EV cargo sorting. Modulating the CD9 expression in fibroblasts was sufficient to alter its levels in EVs. Mass spectrometry analysis of EVs released by CD9 knockdown (KD) vs. control cells revealed a significant increase in angiopoietin-like protein 2 (ANGPTL2), an angiogenesis promoter. Analysis of primary endothelial cells confirmed increased sprouting under CD9 KD conditions. Together, our data indicate that aged EVs play an important role in promoting a tumor-permissive microenvironment.

CD9 promotes TßR2-TßR1 association driving the transition of human dermal fibroblasts to myofibroblast under hypoxia.

BACKGROUND: During wound healing, fibroblast to myofibroblast transition is required for wound contraction and remodeling. While hypoxia is an important biophysical factor in wound microenvironment, the exact regulatory mechanism underlying hypoxia and fibroblast-to-myofibroblast transition remains unclear. We previously found that tetraspanin CD9 plays an important role in oxygen sensing and wound healing. Herein, we investigated the effects of physiological hypoxia on fibroblast-to-myofibroblast transition and the biological function and mechanism of CD9 in it. METHODS: Human skin fibroblasts (HSF) and mouse dermis wounds model were established under physiological hypoxia (2% O2). The cell viability and contractility of HSF under hypoxia were evaluated by CCK8 and collagen gel retraction, respectively. The expression and distribution of fibroblast-to-myofibroblast transition markers and CD9 in HSF were detected by Western blotting and immunofluorescence. CD9 slicing and overexpressing HSFs were constructed to determine the role of CD9 by small interfering RNA and recombinant adenovirus vector. The association of TßR2 and TßR1 was measured by immunoprecipitation to explore the regulatory mechanism. Additionally, further validation was conducted on mouse dermis wounds model through histological analysis. RESULTS: Enhanced fibroblast-to-myofibroblast transition and upregulated CD9 expression was observed under hypoxia in vitro and in vivo. Besides, reversal of fibroblast-to-myofibroblast transition under hypoxia was observed when silencing CD9, suggesting that CD9 played a key role in this hypoxia-induced transition. Moreover, hypoxia increased fibroblast-to-myofibroblast transition by activating TGF-ß1/Smad2/3 signaling, especially increased interaction of TßR2 and TßR1. Ultimately, CD9 was determined to directly affect TßR1-TßR2 association in hypoxic fibroblast. CONCLUSION: Collectively, these findings suggest that CD9 promotes TßR2-TßR1 association, thus driving the transition of human dermal fibroblasts to myofibroblast under hypoxia.

Human tetraspanin CD81 facilitates invasion of Salmonella enterica into human epithelial cells.

Human CD81 and CD9 are members of the tetraspanin family of proteins characterized by a canonical structure of four transmembrane domains and two extracellular loop domains. Tetraspanins are known as molecular facilitators, which assemble and organize cell surface receptors and partner molecules forming clusters known as tetraspanin-enriched microdomains. They have been implicated to play various biological roles including an involvement in infections with microbial pathogens. Here, we demonstrate an important role of CD81 for the invasion of epithelial cells by Salmonella enterica. We show that the overexpression of CD81 in HepG2 cells enhances invasion of various typhoidal and non-typhoidal Salmonella serovars. Deletion of CD81 by CRISPR/Cas9 in intestinal epithelial cells (C2BBe1 and HT29-MTX-E12) reduces S. Typhimurium invasion. In addition, the effect of human CD81 is species-specific as only human but not rat CD81 facilitates Salmonella invasion. Finally, immunofluorescence microscopy and proximity ligation assay revealed that both human tetraspanins CD81 and CD9 are recruited to the entry site of S. Typhimurium during invasion but not during adhesion to the host cell surface. Overall, we demonstrate that the human tetraspanin CD81 facilitates Salmonella invasion into epithelial host cells.

Multiparametric profiling of HER2-enriched extracellular vesicles in breast cancer using Single Extracellular VEsicle Nanoscopy.

BACKGROUND: Patients with HER2-positive breast cancer can significantly benefit from HER2-directed therapy - such as the monoclonal antibody trastuzumab. However, some patients can develop therapy resistance or change HER2 status. Thus, we urgently need new, noninvasive strategies to monitor patients frequently. Extracellular vesicles (EVs) secreted from tumor cells are emerging as potential biomarker candidates. These membrane-delimited nanoparticles harbor molecular signatures of their origin cells; report rapidly on changes to cellular status; and can be frequently sampled from accessible biofluids. RESULTS: Using Single Extracellular VEsicle Nanoscopy (SEVEN) platform that combines affinity isolation of EVs with super-resolution microscopy, here we provide multiparametric characterization of EVs with ~ 8 nm precision and molecular sensitivity. We first interrogated cell culture EVs affinity-enriched in tetraspanins CD9, CD63, and CD81; these transmembrane proteins are commonly found on EV membranes. SEVEN robustly provided critical parameters of individual, tetraspanin-enriched EVs: concentration, size, shape, molecular cargo content, and heterogeneity. Trastuzumab-resistant cells (vs. trastuzumab-sensitive) secreted more EVs. Additionally, EVs from trastuzumab-resistant cells had lower tetraspanin density and higher HER2 density. We also evaluated EVs affinity-enriched in HER2; we found that these EVs (vs. tetraspanin-enriched) were larger and more elongated. We further optimized analytical sample processing to assess a rare population of HER2-enriched EVs from patient plasma. In breast cancer patients with elevated HER2 protein expression (vs. controls), HER2-enriched EVs had distinct characteristics including typically increased number of tetraspanin molecules and larger size. Importantly, these EVs were on average 25-fold more abundant compared to no cancer controls. CONCLUSIONS: SEVEN revealed unique characteristics of HER2-enriched EVs in cultured cells and complex biological fluid. In combination with current clinical approaches, this method is well poised to support precise therapeutic decisions.

Proteomic landscape of tunneling nanotubes reveals CD9 and CD81 tetraspanins as key regulators.

Tunneling nanotubes (TNTs) are open actin- and membrane-based channels, connecting remote cells and allowing direct transfer of cellular material (e.g. vesicles, mRNAs, protein aggregates) from the cytoplasm to the cytoplasm. Although they are important especially, in pathological conditions (e.g. cancers, neurodegenerative diseases), their precise composition and their regulation were still poorly described. Here, using a biochemical approach allowing to separate TNTs from cell bodies and from extracellular vesicles and particles (EVPs), we obtained the full composition of TNTs compared to EVPs. We then focused on two major components of our proteomic data, the CD9 and CD81 tetraspanins, and further investigated their specific roles in TNT formation and function. We show that these two tetraspanins have distinct non-redundant functions: CD9 participates in stabilizing TNTs, whereas CD81 expression is required to allow the functional transfer of vesicles in the newly formed TNTs, possibly by regulating docking to or fusion with the opposing cell.

Three-dimensional cell culture using CD9-positive cells isolated from marginal cell layer of intermediate lobe of rats sustains in vivo-like primary niche environment.

The adenohypophysis is composed of the anterior and intermediate lobes (AL and IL, respectively), and secretes hormones that play an important role in reproduction. CD9- and SOX2-double (CD9/SOX2) positive cells located in the marginal cell layer (MCL) facing the Rathke's cleft in the AL and IL form the primary stem cell niche in the adult adenohypophysis of rats. In this study, we successfully obtained 3-dimensional (3D) cell aggregates that closely resembled the primary niche of MCL in vivo. After incubation in a Matrigel containing several growth factors, approximately 20% of the cells in the CD9/SOX2-positive cell aggregates were differentiated into hormone-producing cells. The cell aggregates generated in this study may provide insight into the regulation of the pituitary stem/progenitor cell niche and the turnover of hormone-producing cells.

Immunohistochemical analysis of CD9, CD29 and epithelial to mesenchymal transition in triple-negative breast cancer.

BACKGROUND: Triple-negative breast carcinomas (TNBC) are a heterogeneous group of tumors with mostly aggressive behaviour and poor prognosis. In association with their aggressive behavior and chemoresistance to treatment, the concept of epithelial-mesenchymal transition (EMT) has come to the fore. CD9 and CD29 proteins are associated with EMT and may play a role in TNBC progression. Our aim was to investigate association of these markers with the lymph node metastasis, tumor grade, proliferative activity, and patient survival. PATIENTS AND METHODS: Our cohort consisted of 66 TNBC patients without neoadjuvant therapy, aged 26-81 years. The pathological tumor stages ranged from pT1b to pT3 and histological grades ranged from II to III, according to the Bloom-Richardson system. Immunohistochemical evaluation of CD9, CD29, E-cadherin, vimentin, androgen receptor and Ki-67 expression was performed semiquantitatively using the H-score. Expression of the proteins was statistically evaluated in relation to the clinicopathological parameters and survival of the patients. RESULTS: We observed lower expression of CD9 in lymph node metastases compared to the primary tumor (P = 0.021). The CD29 expression in primary tumor was significantly lower in patients with lymph node metastases compared to patients without cancer dissemination (P = 0.03). Neither CD9 nor CD29 protein expression was associated with breast cancer-specific survival (BCSS). Lower expression of E-cadherin at the periphery of the primary tumor was associated with worse BCSS (P = 0.038). Neither grade nor the presence of lymph node metastases reached significant association with the BCSS. Lower expression of E-cadherin at the periphery was also associated with higher Ki67 (Rs -0.26) and vimentin (Rs -0.33). CONCLUSION: Decreased protein expression of CD9 and CD29 were associated with lymph node metastasis growth, however, their association with survival was not proved. Lower expression of E-cadherin at the periphery of the primary tumor was associated with high proliferation and poor breast cancer-specific survival.

Transfer of cardiomyocyte-derived extracellular vesicles to neighboring cardiac cells requires tunneling nanotubes during heart development.

Rationale: Extracellular vesicles (EVs) are thought to mediate intercellular communication during development and disease. Yet, biological insight to intercellular EV transfer remains elusive, also in the heart, and is technically challenging to demonstrate. Here, we aimed to investigate biological transfer of cardiomyocyte-derived EVs in the neonatal heart. Methods: We exploited CD9 as a marker of EVs, and generated two lines of cardiomyocyte specific EV reporter mice: Tnnt2-Cre; double-floxed inverted CD9/EGFP and αMHC-MerCreMer; double-floxed inverted CD9/EGFP. The two mouse lines were utilized to determine whether developing cardiomyocytes transfer EVs to other cardiac cells (non-myocytes and cardiomyocytes) in vitro and in vivo and investigate the intercellular transport pathway of cardiomyocyte-derived EVs. Results: Genetic tagging of cardiomyocytes was confirmed in both reporter mouse lines and proof of concept in the postnatal heart showed that, a fraction of EGFP+/MYH1- non-myocytes exist firmly demonstrating in vivo cardiomyocyte-derived EV transfer. However, two sets of direct and indirect EGFP +/- cardiac cell co-cultures showed that cardiomyocyte-derived EGFP+ EV transfer requires cell-cell contact and that uptake of EGFP+ EVs from the medium is limited. The same was observed when co-cultiring with mouse macrophages. Further mechanistic insight showed that cardiomyocyte EV transfer occurs through type I tunneling nanotubes. Conclusion: While the current notion assumes that EVs are transferred through secretion to the surroundings, our data show that cardiomyocyte-derived EV transfer in the developing heart occurs through nanotubes between neighboring cells. Whether these data are fundamental and relate to adult hearts and other organs remains to be determined, but they imply that the normal developmental process of EV transfer goes through cell-cell contact rather than through the extracellular compartment.

Immune interactions and regulation with CD39+ extracellular vesicles from platelet concentrates.

Introduction: CD39 plays an important role in the immunoregulation and inhibition of effector cells. It is expressed on immune cells, including Tregs, and on extracellular vesicles (EVs) budding from the plasma membrane. Platelet transfusion may induce alloimmunization against HLA-I antigens, leading to refractoriness to platelet transfusion with severe consequences for patients. Tregs may play a key role in determining whether alloimmunization occurs in patients with hematologic disorders. We hypothesized that CD39+ EVs might play an immunoregulatory role, particularly in the context of platelet transfusions in patients with hematologic disorders. Such alloimmunization leads to the production of alloantibodies and is sensitive to the regulatory action of CD39. Methods: We characterized CD39+ EVs in platelet concentrates by flow cytometry. The absolute numbers and cellular origins of CD39+ EVs were evaluated. We also performed functional tests to evaluate interactions with immune cells and their functions. Results: We found that CD39+ EVs from platelet concentrates had an inhibitory phenotype that could be transferred to the immune cells with which they interacted: CD4+ and CD8+ T lymphocytes (TLs), dendritic cells, monocytes, and B lymphocytes (BLs). Moreover, the concentration of CD39+ EVs in platelet concentrates varied and was very high in 10% of concentrates. The number of these EVs present was determinant for EV-cell interactions. Finally, functional interactions were observed with BLs, CD4+ TLs and CD39+ EVs for immunoglobulin production and lymphoproliferation, with potential implications for the immunological management of patients.

Quality control and validation of extracellular vesicles isolated from cultured human breast cancer cells.

OBJECTIVE: Extracellular vesicles (EVs) have been shown to play a critical role in promoting tumorigenesis. As EV research grows, it is of importance to have standardization of isolation, quality control, characterization and validation methods across studies along with reliable references to explore troubleshooting solutions. Therefore, our objective with this Research Note was to isolate EVs from multiple breast cancer cell lines and to describe and perform protocols for validation as outlined by the list of minimal information for studies of EVs (MISEV) from the International Society for Extracellular Vesicles. RESULTS: To isolate EVs, two techniques were employed: ultracentrifugation and size exclusion chromatography. Ultracentrifugation yielded better recovery of EVs in our hands and was therefore used for further validation. In order to satisfy the MISEV requirements, protein quantification, immunoblotting of positive (CD9, CD63, TSG101) and negative (TGFß1, ß-tubulin) markers, nanoflow cytometry and electron microscopy was performed. With these experiments, we demonstrate that yield of validated EVs varied between different breast cancer cell lines. Protocols were optimized to accommodate for low levels of EVs, and various technical and troubleshooting suggestions are included for potential application to other cell types that may provide benefit to investigators interested in future EV studies.

Effect of primary osteoblast-derived extracellular vesicles on osteoclast differentiation. / åŽŸä»£æˆéª¨ç»†èƒžæ¥æºèƒžå¤–å›Šæ³¡ä¿ƒè¿›ç ´éª¨ç»†èƒžåˆ†åŒ–.

OBJECTIVES: To investigate the effect of osteoblast-derived extracellular vesicles (OB-EVs) on the proliferation and differentiation of osteoclasts, and to explore the possible molecular mechanism of extracellular vesicles involved in the communication between osteoblasts and osteoclasts. METHODS: Primary osteoblasts were isolated from newborn mouse calvarial bone and induced by ß-glycero phosphate, ascorbic acid and dexamethasone. Osteogenic feature was tested by alkaline phosphatase (ALP) and alizarin red S staining. Extracellular vesicles were isolated by ultracentrifugation from the cell culture supernatant. Vesicle morphology was observed by transmission electron microscopy, and the characteristic markers of tumor susceptibility gene 101 (TSG101), ALG-2 interacting protein X (Alix) and cluster of differentiation 9 (CD9) on the surface of extracellular vesicles were identified by Western blotting. Cell counting kit 8 (CCK-8) assay was used to determine the proliferation effect of OB-EVs on mouse mononuclear macrophage RAW264.7 cells. Furthermore, the expression level of specific markers of osteoclast differentiation in RAW264.7 cells was detected by Western blotting after the combined effect of OB-EVs and receptor activator for nuclear factor κB ligand (RANKL). The number of osteoclasts was observed and compared with OB-EVs-treated mouse bone marrow-derived macrophages (BMMs) by tartrate-resistant acid phosphatase (TRAP) staining, and the effect of OB-EVs on osteoclast differentiation was determined. RESULTS: The extracted OB-EVs showed a double-layer cup-like structure with a diameter of 30-150 nm, and TSG101, Alix and CD9 were expressed. RAW264.7 cells were stimulated with OB-EVs, and the results of CCK-8 assay showed that high concentration of OB-EVs (more than 20 µg/mL) inhibited cell proliferation (P<0.05). Western blotting analysis showed that the expression of osteoclast differentiation marker proteins such as c-Fos, activated T cell nuclear factor (NFATc1) and c-Jun N-terminal kinase (JNK) in RAW264.7 cells were significantly increased, and the promoting effect was enhanced with increasing of OB-EVs concentration (P<0.05). In addition, the combination of OB-EVs and RANKL on BMMs showed that the number of TRAP-positive cells was significantly higher than that of the RANKL induction group alone (P<0.05). CONCLUSIONS: OB-EVs can promote the differentiation of osteoclast precursor cells into osteoclasts, but high concentration of OB-EVs can inhibit proliferation of RAW264.7 cells.

CD9 Counteracts Liver Steatosis and Mediates GCGR Agonist Hepatic Effects.

Glucagon receptor (GCGR) agonism offers potentially greater effects on the mitigation of hepatic steatosis. However, its underlying mechanism is not fully understood. Here, it screened tetraspanin CD9 might medicate hepatic effects of GCGR agonist. CD9 is decreased in the fatty livers of patients and upregulated upon GCGR activation. Deficiency of CD9 in the liver exacerbated diet-induced hepatic steatosis via complement factor D (CFD) regulated fatty acid metabolism. Specifically, CD9 modulated hepatic fatty acid synthesis and oxidation genes through regulating CFD expression via the ubiquitination-proteasomal degradation of FLI1. In addition, CD9 influenced body weight by modulating lipogenesis and thermogenesis of adipose tissue through CFD. Moreover, CD9 reinforcement in the liver alleviated hepatic steatosis, and blockage of CD9 abolished the remission of hepatic steatosis induced by cotadutide treatment. Thus, CD9 medicates the hepatic beneficial effects of GCGR signaling, and may server as a promising therapeutic target for hepatic steatosis.

The impact of exosomes derived from B-cell acute lymphoblastic leukemia as a growth factor on bone marrow mesenchymal stromal cells.

Background The incidence of various types of cancers, including leukemia, is on the rise and many challenges in both drug resistance and complications related to chemotherapy appeared. Recently, the development and application of extracellular vesicles (EV) such as exosomes in the management of cancers, especially leukemia, holds great significance. In this article, we extracted exosomes from NALM6 cells and assessed their regulatory effects on proliferation and apoptosis in mesenchymal stem cells (MSCs). Method and result We first verified the exosomes using various techniques, including flow cytometry, transient electron microscopy, dynamic light scattering (DLS), and BCA protein assay. Then MTT analysis and flowcytometry (apoptosis and cell cycle assay) besides gene expressions were employed to determine the state of MSC proliferations. The results indicated that exosome-specific pan markers like CD9, CD63, and CD81 were present. Through DLS, we found out that the mean size of the exosomes was 89.68 nm. The protein content was determined to be 956.292 µg/ml. Analysis of MTT, flow cytometry (cell cycle and apoptosis assay), and RT-qPCR showed that in the dose of 50 µg/ml the proliferation of MSCs was increased significantly (p-value < 0.05). Conclusion All these data showed that exosomes use several signaling pathways to increase the MSCs' proliferation and drug resistance, ultimately leading to high mortalities and morbidities of acute lymphoblastic leukemia.

Endocytosis blocks the vesicular secretion of exosome marker proteins.

Exosomes are secreted vesicles of ~30 to 150 nm diameter that play important roles in human health and disease. To better understand how cells release these vesicles, we examined the biogenesis of the most highly enriched human exosome marker proteins, the exosomal tetraspanins CD81, CD9, and CD63. We show here that endocytosis inhibits their vesicular secretion and, in the case of CD9 and CD81, triggers their destruction. Furthermore, we show that syntenin, a previously described exosome biogenesis factor, drives the vesicular secretion of CD63 by blocking CD63 endocytosis and that other endocytosis inhibitors also induce the plasma membrane accumulation and vesicular secretion of CD63. Finally, we show that CD63 is an expression-dependent inhibitor of endocytosis that triggers the vesicular secretion of lysosomal proteins and the clathrin adaptor AP-2 mu2. These results suggest that the vesicular secretion of exosome marker proteins in exosome-sized vesicles occurs primarily by an endocytosis-independent pathway.

CD9 shapes glucocorticoid sensitivity in pediatric B-cell precursor acute lymphoblastic leukemia.

Resistance to glucocorticoids (GC), the common agents for remission induction in pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL), poses a significant therapeutic hurdle. Therefore, dissecting the mechanisms shaping GC resistance could lead to new treatment modalities. Here, we showed that CD9- BCP-ALL cells were preferentially resistant to prednisone and dexamethasone over other standard cytotoxic agents. Concordantly, we identified significantly more poor responders to the prednisone prephase among BCP-ALL patients with a CD9- phenotype, especially for those with adverse presenting features including older age, higher white cell count and BCR-ABL1. Furthermore, gain- and loss-offunction experiments dictated a definitive functional linkage between CD9 expression and GC susceptibility, as demonstrated by the reversal and acquisition of relative GC resistance in CD9low and CD9high BCP-ALL cells, respectively. Despite physical binding to the GC receptor NR3C1, CD9 did not alter its expression, phosphorylation or nuclear translocation but potentiated the induction of GC-responsive genes in GC-resistant cells. Importantly, the MEK inhibitor trametinib exhibited higher synergy with GC against CD9- than CD9+ lymphoblasts to reverse drug resistance in vitro and in vivo. Collectively, our results elucidate a previously unrecognized regulatory function of CD9 in GC sensitivity, and inform new strategies for management of children with resistant BCP-ALL.

Detecting and exploring kidney-derived extracellular vesicles in plasma.

BACKGROUND: Extracellular vesicles (EVs) have received considerable attention as ideal biomarkers for kidney diseases. Most reports have focused on urinary EVs, that are mainly derived from the cells in the urinary tract. However, the detection and the application of kidney-derived EVs in plasma remains uncertain. METHODS: We examined the kidney-derived small EVs (sEVs) in plasma that were supposedly released from renal mesangial and glomerular endothelial cells, using clinical samples from healthy controls and patients with kidney transplants. Plasma from healthy controls underwent ultracentrifugation, followed by on-bead flow cytometry, targeting α8 integrin, an antigen-specific to mesangial cells. To confirm the presence of kidney-derived sEVs in peripheral blood, plasma from ABO-incompatible kidney transplant recipients was ultracentrifuged, followed by western blotting for donor blood type antigens. RESULTS: Immunohistochemistry and immunoelectron microscopy confirmed α8 integrin expression in kidney mesangial cells and their sEVs. The CD9-α8 integrin double-positive sEVs were successfully detected using on-bead flow cytometry. Western blot analysis further revealed transplanted kidney-derived sEVs containing blood type B antigens in non-blood type B recipients, who had received kidneys from blood type B donors. Notably, a patient experiencing graft kidney loss exhibited diminished signals of sEVs containing donor blood type antigens. CONCLUSION: Our findings demonstrate the potential usefulness of kidney-derived sEVs in plasma in future research for kidney diseases.

Defining the activity of pro-reparative extracellular vesicles in wound healing based on miRNA payloads and cell type-specific lineage mapping.

Small extracellular vesicles (EVs) are released by cells and deliver biologically active payloads to coordinate the response of multiple cell types in cutaneous wound healing. Here we used a cutaneous injury model as a donor of pro-reparative EVs to treat recipient diabetic obese mice, a model of impaired wound healing. We established a functional screen for microRNAs (miRNAs) that increased the pro-reparative activity of EVs and identified a down-regulation of miR-425-5p in EVs in vivo and in vitro associated with the regulation of adiponectin. We tested a cell type-specific reporter of a tetraspanin CD9 fusion with GFP to lineage map the release of EVs from macrophages in the wound bed, based on the expression of miR-425-5p in macrophage-derived EVs and the abundance of macrophages in EV donor sites. Analysis of different promoters demonstrated that EV release under the control of a macrophage-specific promoter was most abundant and that these EVs were internalized by dermal fibroblasts. These findings suggested that pro-reparative EVs deliver miRNAs, such as miR-425-5p, that stimulate the expression of adiponectin that has insulin-sensitizing properties. We propose that EVs promote intercellular signaling between cell layers in the skin to resolve inflammation, induce proliferation of basal keratinocytes, and accelerate wound closure.

Fluctuation of CD9/SOX2-positive cell populations during the turnover of GH- and TSH-producing cells in the adult anterior pituitary gland.

The adenohypophysis is comprised of the anterior and intermediate lobes (AL and IL, respectively). Cluster of differentiation 9 (CD9)- and sex-determining region Y-box 2 (SOX2)-positive cells are stem/progenitor hormone-producing cells in the AL. They are located in the marginal cell layer (MCL) facing Rathke's cleft between the AL and IL (primary niche) and the parenchyma of the AL (secondary niche). We previously showed that, in rats, CD9/SOX2-positive cells in the IL side of the MCL (IL-side MCL) migrate to the AL side (AL-side MCL) and differentiate into prolactin-producing cells (PRL cells) in the AL parenchyma during pregnancy, lactation, and diethylstilbestrol treatment, all of which increase PRL cell turnover. This study examined the changes in CD9/SOX2-positive stem/progenitor cell niches and their proportions by manipulating the turnover of growth hormone (GH)- and thyroid-stimulating hormone (TSH)-producing cells (GH and TSH cells, respectively), which are Pit1 lineage cells, as well as PRL cells. After induction, the isolated CD9/SOX2-positive cells from the IL-side MCL formed spheres and differentiated into GH and TSH cells. We also observed an increased GH cell proportion upon treatment with GH-releasing hormone and recovery from continuous stress and an increased TSH cell proportion upon propylthiouracil treatment, concomitant with alterations in the proportion of CD9/SOX2-positive cells in the primary and secondary niches. These findings suggest that CD9/SOX2-positive cells have the potential to supply GH and TSH when an increase in GH and TSH cell populations is required in the adult pituitary gland.

Extended-Gate Field-Effect Transistor Consisted of a CD9 Aptamer and MXene for Exosome Detection in Human Serum.

Cancer progresses silently to the terminal stage of the impossible operable condition. There are many limitations in the treatment options of cancer, but diagnosis in an early stage can improve survival rates and low recurrence. Exosomes are the biomolecules released from cancer cells and are promising candidates for clinical diagnosis. Among them, the cluster of differentiation 9 (CD9) protein is an important exosomal biomarker that can be used for exosome determination. Therefore, here, a CD9 aptamer was first synthesized and applied to an extended-gate field-effect transistor (EGFET)-type biosensor containing a disposable sensing membrane to suggest the possibility of detecting exosomes in a clinical environment. Systematically evaluating ligands using the exponential enrichment (SELEX) technique was performed to select nucleic acid sequences that can specifically target the CD9 protein. Exosomes were detected according to the electrical signal changes on a membrane, which is an extended gate using an Au microelectrode. The fabricated biosensor showed a limit of detection (LOD) of 10.64 pM for CD9 proteins, and the detection range was determined from 10 pM to 1 µM in the buffer. In the case of the clinical test, the LOD and detection ranges of exosomes in human serum samples were 6.41 × 102 exosomes/mL and 1 × 103 to 1 × 107 exosomes/mL, respectively, showing highly reliable results with low error rates. These findings suggest that the proposed aptasensor can be a powerful tool for a simple and early diagnosis of exosomes.