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Cell culture‐conditioned medium (CCM) is a valuable source of extracellular vesicles (EVs) for basic scientific, therapeutic and diagnostic applications. Cell culturing parameters affect the biochemical composition, release and possibly the function of CCM‐derived EVs (CCM‐EV). The CCM‐EV task force of the Rigor and Standardization Subcommittee of the International Society for Extracellular Vesicles aims to identify relevant cell culturing parameters, describe their effects based on current knowledge, recommend reporting parameters and identify outstanding questions. While some recommendations are valid for all cell types, cell‐specific recommendations may need to be established for non‐mammalian sources, such as bacteria, yeast and plant cells. Current progress towards these goals is summarized in this perspective paper, along with a checklist to facilitate transparent reporting of cell culturing parameters to improve the reproducibility of CCM‐EV research.

Induced pluripotent stem cell‐derived conditioned medium (iPS‐CM) could improve cell viability in many types of cells and may be a better alternative for the treatment of myocardial infarction. This study aimed to examine the influence of iPS‐CM on anti‐apoptosis and the proliferation of H9C2 cardiomyocytes and investigate the underlying mechanisms. H9C2 cardiomyocytes were exposed to 200 μmol/L hydrogen peroxide (H2O2) for 24 hours with or without pre‐treatment with iPS‐CM. The ratio of apoptotic cells, the loss of mitochondrial membrane potential (△Ψm) and the levels of intracellular reactive oxygen species were analysed by flow cytometric analysis. The expression levels of BCL‐2 and BAX proteins were analysed by Western blot. Cell proliferation was assessed using cell cycle and EdU staining assays. To study cell senescence, senescence‐associated β‐galactosidase (SA‐β‐gal) staining was conducted. The levels of malondialdehyde, superoxide dismutase and glutathione were also quantified using commercially available enzymatic kits. The results showed that iPS‐CM containing basic fibroblast growth factor significantly reduced H2O2‐induced H9C2 cardiomyocyte apoptosis by activating the autophagy flux pathway, promoted cardiomyocyte proliferation by up‐regulating the Wnt/β‐catenin pathway and inhibited oxidative stress and cell senescence. In conclusion, iPS‐CM effectively enhanced the cell viability of H9C2 cardiomyocytes and could potentially be used to inhibit cardiomyocytes apoptosis to treat myocardial infarction in the future.

Extracellular vesicles (EVs) contain cell‐derived lipids, proteins and RNAs; however, determining the tissue‐ and cell‐type‐specific EV abundances in body fluids remains a significant hurdle for our understanding of EV biology. While tissue‐ and cell‐type‐specific EV abundances can be estimated by matching the EV's transcriptome to a tissue's/cell type's expression signature using deconvolutional methods, a comparative assessment of deconvolution methods' performance on EV transcriptome data is currently lacking. We benchmarked 11 deconvolution methods using data from four cell lines and their EVs, in silico mixtures, 118 human plasma and 88 urine EVs. We identified deconvolution methods that estimated cell type‐specific abundances of pure and in silico mixed cell line‐derived EV samples with high accuracy. Using data from two urine EV cohorts with different EV isolation procedures, four deconvolution methods produced highly similar results. The three methods were also concordant in their tissue‐ and cell‐type‐specific plasma EV abundance estimates. We identified driving factors for deconvolution accuracy and highlighted the importance of implementing biological knowledge in creating the tissue/cell type signature. Overall, our analyses demonstrate that the deconvolution algorithms DWLS and CIBERSORTx produce highly similar and accurate estimates of tissue‐ and cell‐type‐specific EV abundances in biological fluids.

Natural killer (NK) cells produce various cytokines, including interleukin (IL)-1β, IL-6, IL-10, IL-12, interferon γ, tumor necrosis factor α and transforming growth factor β, which are critical in modulating immune responses. NK cell-conditioned medium (NK-CdM), rich in cytokines, has potential applications in therapy and healing. The present study aimed to investigate the protective effect of NK-CdM against ultraviolet B (UVB)-mediated photoaging using in vitro and ex vivo models. In human keratinocyte cell line (HaCaT cells), NK-CdM mitigated UVB-induced cytotoxicity and suppressed the production of reactive oxygen species. NK-CdM enhanced the mRNA expression levels of superoxide dismutase 1 (SOD1) and catalase (CAT) and inhibited the reduction in SOD1 and CAT expression levels caused by UVB irradiation. Furthermore, NK-CdM inhibited the UVB-mediated nuclear translocation of nuclear factor erythroid 2-related factor 2. NK-CdM also prevented UVB-induced downregulation of filaggrin and involucrin and attenuated the UVB-induced reduction in hyaluronan synthase (HAS)1, HAS2, HAS3, aquaporin-3 and hyaluronan levels. Notably, NK-CdM upregulated the expression of elongation of very long chain fatty acids (ELOVL) enzymes, including ELOVL1, ELOVL5 and ELOVL6, as well as ceramide synthases (CerS), specifically CerS2 and CerS3. Furthermore, NK-CdM inhibited the UVB-induced reduction in the levels of these proteins. Overall, these findings suggested that NK-CdM has the potential to prevent UVB-mediated photoaging and promote skin health.

Background: Cyclophosphamide (CP) has some negative effects on the reproductive system. Stem cells and their metabolites are being utilized to enhance fertility after chemotherapy. Objective: This study aimed to investigate the impact of conditioned medium (CM) derived from bone marrow mesenchymal stromal stem cells (BM-MSCs) on the toxic effects of CP on testicles. Materials and Methods: BM-MSCs were isolated, a CM was collected and 25-fold concentrated. 24 male Wistar rats (8 wk, 200–250 gr) were randomly divided into following groups: control, CP, CP+DMEM, CP+CM. CP was given at a single dose of 100 mg/kg. 2 wk after the CP administration, CM was injected into the testicular efferent duct. Sperm parameters, testicular histopathology, and the level of testosterone were analyzed 2 months after treatment. The expression of B-cell lymphoma 2 (Bcl2) and Bcl2-associated X protein (Bax) genes were evaluated by real-time polymerase chain reaction. Results: CP had a negative effect on testis histology (p < 0.001) and sperm quality (p < 0.001). It changed the expression of genes associated with apoptosis (p < 0.001). Treatment with CM reduced the expression of Bax (p < 0.001), while significantly increasing the expression of Bcl2 (p = 0.01). It improved sperm count (p = 0.03), viability (p < 0.001), motility (p < 0.001), spermatogonial count (p < 0.001), and epithelial thickness of testicular tubules (p = 0.02). Conclusion: These findings suggest that CM produced from BM-MSCs may be valuable for therapeutic approaches in reproductive medicine and may lessen the side effects of CP. Key words: Bone marrow mesenchymal stem cells, Cyclophosphamide, Conditioned medium, Apoptosis, Spermatogenesis.

Objective: This study aimed to evaluate the effect of testicular cell conditioned medium (TCCM) on in vitro male germ cell differentiation and provide a proteomic profile of TCCM. Materials and Methods: TCCM was collected from 5-day-old mouse testicular tissues cultured in serum-free DMEM. Proteomic analysis was performed using liquid chromatography–tandem mass spectrometry. Germ cells were isolated from 13.5 days post-coitum (dpc) mouse fetal genital ridges and divided into three groups: (a) control (DMEM + 15% FCS), (b) 40% TCCM + 60% DMEM + 15% FCS, and (c) 60% TCCM + 40% DMEM + 15% FCS. Cells were cultured for 24 days. Gene expression of Oct4, Acr, Dazl, Vasa, Stra8, Prm1, and Gdf9 was measured using real-time PCR. Results: Proteomic analysis identified 26 proteins in TCCM. Notably, COL4A1, COL4A2, and HSPG2 are associated with the basement membrane and are essential for supporting extracellular matrix integrity, while FN1, FBN1, COL1A1, COL1A2, COL5A2, and COL3A12 are linked to the PI3K-AKT pathway, which regulates cell proliferation. In TCCM-treated groups, germ cells differentiated into spermatid-like cells by day 18 and sperm-like structures by day 24. Oct4, Dazl, Vasa, Stra8, Prm1, and Acr were expressed across all groups, but without statistically significant differences (p > 0.05), while Gdf9 was not expressed. Conclusion: The addition of TCCM, which contains extracellular matrix (ECM) proteins, enhanced in vitro differentiation of male germ cells into sperm-like structures, together with somatic cells from the genital ridge. These ECM proteins contribute to creating a microenvironment that closely mimics in vivo conditions.

Ischemic heart diseases (IHD) are one of the major causes of death worldwide. Studies have shown that mesenchymal stem cells can secrete and release conditioned medium (CM) which has biological activities and can repair tissue injury. This study aimed to investigate the effects of human amniotic membrane mesenchymal stem cells (hAMCs)-CM on myocardial ischemia/reperfusion (I/R) injury in rats by targeting oxidative stress. Male Wistar rats (40 rats, weighing 200-250 g) were randomly divided into four groups: Sham, myocardial infarction (MI), MI + culture media, and MI + conditioned medium. MI was induced by ligation of the left anterior descending coronary artery for 30 min. After 15 min of reperfusion, intramyocardial injections of hAMCs-CM or culture media (150 μl) were performed. At the end of the experiment, serum levels of cardiac troponin-I (cTn-I), myocardial levels of malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione peroxidase (GPx), as well as cardiac histological changes were evaluated. HAMCs-CM significantly decreased cTn-I and MDA levels and increased SOD and GPx activities ( <0.05). In addition, hAMCs-CM improved cardiac histological changes and decreased myocardial injury percentage ( <0.05). This study showed that hAMCs-CM has cardioprotective effects in the I/R injury condition. Reduction of oxidative stress by hAMCs-CM plays a significant role in this context. Based on the results of this study, it can be concluded that hAMCs-CM can be offered as a therapeutic candidate for I/R injury in the future, but more research is needed.

The aim of this research was to find a way to differentiate germ cells from umbilical cord Wharton's jelly mesenchymal stem cells (MSCs) to support in vitro spermatogenesis. A small piece of Wharton's jelly was cultured in high‐glucose Dulbecco's modified Eagle's medium in present of 10% foetal calf serum. After the fourth passage, the cells were isolated and cultured in Sertoli cell‐conditioned medium under induction of two different doses of retinoic acid (10−5, 10−6 m). The differentiation of MSC to germ‐like cells was evaluated by expression of Oct4, Nanog, Plzf, Stra8 and Prm1 genes during different days of culture through qPCR. The results showed that there were downregulation of Oct4 and Nanog and upregulation of pre‐meiotic germ cell marker (stra8) and haploid cell marker (Prm1) when MSCs are differentiated over time. The expression of Bax gene (an apoptotic marker) was significantly observed in high dosage of retinoic acid (RA). As a result, RA has positive effects on proliferation and differentiation of MSCs, but its effects are related to dosage. The success of this method can introduce umbilical cord MSC as a source of germ cells for treatment of infertility in future.

Background Keloid formation occurs in Caucasian, African, and Asian populations and is a severe psychosocial burden on patients. There is no permanent treatment for this problem as its pathogenesis is not properly understood. Furthermore, differences in keloid behavior between ethnic groups are not known. It has been hypothesized that keloids behave like benign tumors because of their uncontrolled growth. The present study evaluated the tumoricidal properties of human Wharton’s jelly stem cell-conditioned medium (hWJSC-CM) on fresh Asian keloid cells (AKCs). Methods Human Wharton’s jelly stem cells (hWJSCs) and AKCs were isolated based on our previous methods. hWJSCs and human skin fibroblasts (HSF) (controls) were used to collect hWJSC-CM and HSF-conditioned medium (HSF-CM). AKCs were treated with hWJSC-CM and HSF-CM in vitro and in vivo in a human keloid xenograft SCID mouse model. The inhibitory effect of hWJSC-CM on AKCs was tested in vitro using various assays and in vivo for attenuation/abrogation of AKC tumors created in a xenograft mouse model. Results qRT-PCR analysis showed that the genes FN1, MMP1, and VCAN were significantly upregulated in AKCs and ANXA1, ASPN, IGFBP7, LGALS1, and PTN downregulated. AKCs exposed to hWJSC-CM in vitro showed significant decreases in cell viability and proliferation, increases in Annexin V-FITC+ cell numbers, interruptions of the cell cycle at Sub-G1 and G2/M phases, altered CD marker expression, downregulated anti-apoptotic-related genes, and upregulated pro-apoptotic and autophagy-related genes compared to controls. When AKCs were administered together with hWJSC-CM into immunodeficient mice there were no keloid tumors formed in 7 mice ( n  = 10) compared to the untreated control mice. When hWJSC-CM was injected directly into keloid tumors created in mice there were significant reductions in keloid tumor volumes and weights in 30 days. Conclusions hWJSC-CM inhibited the growth of AKCs in vitro and in xenograft mice, and it may be a potential novel treatment for keloids in the human. The specific molecule(s) in hWJSC-CM that induce the anti-keloid effect need to be identified, characterized, and tested separately in larger preclinical and clinical studies.

Acute liver failure is a life‐threatening syndrome, for which liver transplantation is presently the most effective treatment. Unfortunately, such treatment is extremely limited by a shortage of donor organs. Stem cell therapy offers a promising treatment strategy for acute liver failure. Yet, therapeutic efficacy and potential are hampered by administration route and safety concerns. In this work, we fabricated menstrual blood‐derived stem cells‐conditioned medium/polymersome hybrid nanoparticles that were self‐assembled from amphiphilic block copolymers via the direct hydration method and encapsulated therapeutic bioactive factors within the aqueous core of vesicles. The merit of vesicular architecture enabled the loading capacity of distinct proteins and the maintenance of biological activity. These hybrid nanoparticles can be steadily taken up into cytoplasm and promote hepatocyte proliferation in vitro. Prolonged in vivo circulation time brought higher accumulation in livers. The therapeutic nanoparticles alleviated hepatic injury and promoted liver recovery in mice with carbon tetrachloride‐induced liver failure. Considering the feasibility and benefit of the hybrid nanoparticle therapy, it provided a potential strategy to treat acute liver failure. In this work, menstrual blood‐derived stem cells‐conditioned medium/polymersome nanoparticles are fabricated to treat acute liver failure. These hybrid nanoparticles are self‐assembled from amphiphilic block copolymers via the direct hydration method and encapsulated therapeutic bioactive factors within the aqueous core of vesicles. The nanoparticles alleviate hepatic injury and promote liver recovery in mice with carbon tetrachloride‐induced liver failure.