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Immunomodulation is an essential consideration for cell replacement procedures. Unfortunately, lifelong exposure to nonspecific systemic immunosuppression results in immunodeficiency and has toxic effects on nonimmune cells. Here, we engineered hybrid spheroids of mesenchymal stem cells (MSCs) with rapamycin-releasing poly(lactic- co -glycolic acid) microparticles (RAP-MPs) to prevent immune rejection of islet xenografts in diabetic C57BL/6 mice. Hybrid spheroids were rapidly formed by incubating cell-particle mixture in methylcellulose solution while maintaining high cell viability. RAP-MPs were uniformly distributed in hybrid spheroids and sustainably released RAP for ~3 weeks. Locoregional transplantation of hybrid spheroids containing low doses of RAP-MPs (200- to 4000-ng RAP per recipient) significantly prolonged islet survival times and promoted the generation of regional regulatory T cells. Enhanced programmed death-ligand 1 expression by MSCs was found to be responsible for the immunomodulatory performance of hybrid spheroids. Our results suggest that these hybrid spheroids offer a promising platform for the efficient use of MSCs in the transplantation field.

The development of hydrogels and their application in various fields, such as in medical dilators and scaffolds for cartridge regeneration, are restricted by their low modulus because of the trade-...

CD9, a 24 kDa tetraspanin membrane protein, is known to regulate cell adhesion and migration, cancer progression and metastasis, immune and allergic responses, and viral infection. CD9 is upregulated in senescent endothelial cells, neointima hyperplasia, and atherosclerotic plaques. However, its role in cellular senescence and atherosclerosis remains undefined. We investigated the potential mechanism for CD9-mediated cellular senescence and its role in atherosclerotic plaque formation. CD9 knockdown in senescent human umbilical vein endothelial cells significantly rescued senescence phenotypes, while CD9 upregulation in young cells accelerated senescence. CD9 regulated cellular senescence through a phosphatidylinositide 3 kinase-AKT-mTOR-p53 signal pathway. CD9 expression increased in arterial tissues from humans and rats with age, and in atherosclerotic plaques in humans and mice. Anti-mouse CD9 antibody noticeably prevented the formation of atherosclerotic lesions in ApoE(−/−) mice and Ldlr(−/−) mice. Furthermore, CD9 ablation in ApoE(−/−) mice decreased atherosclerotic lesions in aorta and aortic sinus. These results suggest that CD9 plays critical roles in endothelial cell senescence and consequently the pathogenesis of atherosclerosis, implying that CD9 is a novel target for prevention and treatment of vascular aging and atherosclerosis.

Extracellular vesicles (EVs) that circulate in body fluids possess significant potential for disease diagnosis. Their use in clinical settings, however, has been limited owing to lack of simple and robust isolation methods. To rectify this problem, a centrifugal device for automatic, fast, and efficient isolation of EVs from whole-blood, called Exodisc-B is presented in this paper. Methods: The device comprises a built-in chamber to facilitate plasma separation and two nanoporous filters—one for removing larger particles and the other for enriching EVs. The performance of the device in comparison to ultracentrifugation (UC) was evaluated by analyzing the yield, purity, protein and RNA content of the isolated EVs. Additionally, the EV protein marker expressions were measured by ELISA and statistically analyzed to differentiate prostate cancer patients from healthy donors. Results: Compared with the UC technique, the proposed device is capable of isolating at least an order of magnitude higher number of EVs with about 30-fold higher mRNA count within 40 min. Sandwich ELISA of EV-specific membrane proteins—CD9-CD81—confirmed that Exodisc-B can isolate EVs from a volume of whole blood as low as 30 µL with a capture efficiency exceeding 75%. The device also facilitates temporal monitoring of tumor progression within live mouse xenograft models over a period of 13 weeks while using minimal volumes of weekly collected blood samples. Further, in ELISA analyses of multiple cancer-related proteins, such as prostate-specific antigen (PSA), prostate-specific membrane antigen (PSMA), epithelial cell adhesion molecule (EpCAM), epidermal growth factor receptor 1 (EGFR1), and heat shock protein 90 (HSP90), extracted from EVs isolated from human plasma, 43 patients were differentiated from 30 healthy donors. Conclusion: The results demonstrated the ability of Exodisc-B to provide a rapid, sensitive, and point-of-care-type method for extracting intact EVs from small volumes of clinical blood samples for disease diagnosis and monitoring.

AIMS CD9, a tetraspanin transmembrane protein, modulates cell motility, migration, and proliferation. The aim of this study was to investigate the prognostic significance of CD9 expression in patients with invasive breast carcinoma (IBC). METHODS AND RESULTS CD9 expression was evaluated in tissue microarrays of 1349 IBC samples via immunohistochemistry. CD9 expression in tumour cells (T-CD9 expression) and CD9 expression in stromal immune cells (S-CD9 expression) were analysed separately. T-CD9 expression was observed in 732 (54.3%) cases, and was associated with lymph node metastasis, histological type, lymphovascular invasion, high histological grade, HER2 positivity, a high Ki67 labelling index, and distant metastasis. S-CD9 expression was observed in 833 (61.7%) cases, and was associated with large tumour size, histological type, high histological grade, negative hormone receptors, HER2 positivity, a high Ki67 labelling index, and tumour-infiltrating lymphocytes. Patients with T-CD9 expression had shorter disease-free survival (DFS) than those without T-CD9 expression in the univariate and multivariate analyses. However, S-CD9 expression correlated significantly with a favourable DFS in the univariate and multivariate analyses. In the subgroup analysis, T-CD9 expression and S-CD9 expression were independent markers for DFS in luminal A and luminal B (HER2-negative) subgroups, respectively. CONCLUSIONS T-CD9 expression could be a biomarker for poor prognosis in luminal A IBC, whereas S-CD9 expression could be a marker of good prognosis in luminal B (HER2-negative) IBC. Therefore, tumour compartment-specific analyses considering molecular subtypes are necessary to study the prognostic significance of CD9 expression in IBC.

Metastasis of cancers accounts for almost all cancer-related deaths. In this study, we report a PEGylated nanostructured platform for coadministration of doxorubicin (DOX) and imatinib (IMT) intended to effectively inhibit metastatic tumors. The DOX and IMT coloaded nanostructured system (DOX/IMT-N) is characterized by an excellent encapsulation potential for both drugs and shows sequential and sustained drug release in vitro. DOX/IMT-N significantly inhibited the in vitro proliferation of MDA-MB-231 and SK-MEL-28 cells. The inhibitory effect on in vitro proliferation of the cells was significantly greater than the effect of free DOX, DOX/IMT cocktail, or the nanostructured system housing DOX only (DOX-N). DOX/IMT-N remarkably enhanced cellular drug uptake, resulting in enhanced apoptosis, caused by significant increases in the expression levels of apoptotic marker proteins. Intravenous administration of DOX/IMT-N to MBA-MB-231 xenograft tumor-bearing mice resulted in significantly improved inhibition of tumor progression compared to that with DOX, DOX/IMT, or DOX-N. Therefore, the nanostructured DOX/IMT-N system could potentially aid in overcoming drug resistance in metastatic tumors and improve the effectiveness of metastatic tumor therapeutics.

Senescent cells accumulate in various tissues over time and contribute to tissue dysfunction and aging-associated phenotypes. Accumulating evidence suggests that cellular senescence can be inhibited through pharmacological intervention, as well as through treatment with soluble factors derived from embryonic stem cells (ESCs). In an attempt to investigate the anti-senescence factors secreted by ESCs, we analyzed mouse ESC-derived extracellular microRNAs in conditioned medium via microRNA array analysis. We selected mmu-miR-291a-3p as a putative anti-senescence factor via bioinformatics analysis. We validated its inhibitory effects on replicative, Adriamycin-induced, and ionizing radiation–induced senescence in human dermal fibroblasts. Treatment of senescent cells with mmu-miR-291a-3p decreased senescence-associated β-galactosidase activity, enhanced proliferative potential, and reduced mRNA and protein expression of TGF-β receptor 2, p53, and p21. mmu-miR-291a-3p in conditioned medium was enclosed in ESC-derived exosomes and exosomes purified from ESC conditioned medium inhibited cellular senescence. The inhibitory effects of mmu-miR-291a-3p were mediated through the TGF-β receptor 2 signaling pathway. Hsa-miR-371a-3p and hsa-miR-520e, the human homologs of mmu-miR-291a-3p, showed similar anti-senescence activity. Furthermore, mmu-miR-291a-3p accelerated the excisional skin wound healing process in aged mice. Our results indicate that the ESC-derived mmu-miR-291a-3p is a novel candidate agent that can be utilized for cell-free therapeutic intervention against aging and aging-related diseases.