Cell-conditioned Medium Research Articles

The proteomic landscape of extracellular vesicles derived from human intervertebral disc cells.

Extracellular vesicles (EVs) function as biomarkers and are crucial in cell communication and regulation, with therapeutic potential for intervertebral disc (IVD)-related low back pain (LBP). EV cargo is often affected by tissue health, which may affect the therapeutic potential. There is currently limited knowledge of how the cargo of IVD cell-derived EVs varies with tissue health and how differences in proteomic profile affect the predicted biological functions. Our study purified EVs from human IVD cell conditioned media by size-exclusion chromatography. Nanoparticle tracking analysis was conducted to measure EV size and concentration. Transmission electron microscopy and Western blot were performed to examine EV structure and markers. Tandem mass tag-mass spectrometry was conducted to determine protein cargo. Most EVs were exosomes and intermediate microvesicles with an increasing amount linked to disease progression. Of the proteins detected, 88.6% were shared across the non-degenerate, mildly-degenerate, and degenerate samples. GO and KEGG analyses revealed that cargo from the mildly-degenerate samples was the most distinct, with the proteins in high abundance strongly associated with extracellular matrix (ECM) organization and structure. Shared proteins, highly expressed in the non-degenerate and degenerate samples, showed strong associations with cell adhesion, ECM-receptor interaction, and vesicle-mediated transport, respectively. Our findings indicate that EVs from IVD cells from tissue with different degrees of degeneration share a majority of the cargo proteins. However, the level of expression differs with degeneration grade. Cargo from the mildly-degenerate samples exhibits the most differences. A better understanding of changes in EV cargo in the degenerative process may provide novel information related to molecular mechanisms underlying IVD degeneration and suggest new potential treatment modalities for IVD-related LBP.

Therapeutic potential of hyaluronic acid hydrogel combined with bone marrow stem cells-conditioned medium on arthritic rats’ TMJs

Conditioned media (CM) is derived from mesenchymal stem cells (MSC) culture and contains biologically active components. CM is easy to handle and reduces inflammation while repairing injured joints. Combination therapy of the CM with cross-linked hyaluronic acid (HA) could ameliorate the beneficial effect of HA in treating degenerative changes of articulating surfaces associated with arthritic rats’ temporomandibular joints (TMJs). This study aimed to evaluate the therapeutic potential of HA hydrogel combined with bone marrow stem cells-conditioned medium (BMSCs-CM) on the articulating surfaces of TMJs associated with complete Freund’s adjuvant (CFA)-induced arthritis. Fifty female Sprague-Dawley rats were divided randomly into five equal groups. Rats of group I served as the negative controls and received intra-articular (IA) injections of 50 µl saline solution, whereas rats of group II were subjected to twice IA injections of 50 µg CFA in 50 µl; on day 1 of the experiment to induce persistent inflammation and on day 14 to induce arthritis. Rats of group III and IV were handled as group II and instead, they received an IA injection of 50 µl HA hydrogel and 50 µl of BMSCs-CM, respectively. Rats of group V were given combined IA injections of 50 µl HA hydrogel and BMSCs-CM. All rats were euthanized after the 4th week of inducing arthritis. The joints were processed for sectioning and histological staining using hematoxylin and eosin, Masson’s trichrome and toluidine blue special staining, and immunohistochemical staining for nuclear factor-kappa B (NF-κB). SPSS software was used to analyze the data and one-way analysis of variance followed by post-hoc Tukey statistical tests were used to test the statistical significance at 0.05 for alpha and 0.2 for beta. In the pooled BMSC-CM, 197.14 pg/ml of platelet-derived growth factor and 112.22 pg/ml of interleukin-10 were detected. Compared to TMJs of groups III and IV, TMJs of group V showed significant improvements (P = 0.001) in all parameters tested as the disc thickness was decreased (331.79 ± 0.73), the fibrocartilaginous layer was broadened (0.96 ± 0.04), and the amount of the trabecular bone was distinctive (19.35 ± 1.07). The mean values for the collagen amount were increased (12.29 ± 1.38) whereas the mean values for the NF-κB expression were decreased (0.62 ± 0.15). Combination therapy of HA hydrogel and BMSCs-CM is better than using HA hydrogel or BMSCs-CM, separately to repair degenerative changes in rats’ TMJs associated with CFA-induced arthritis.

Dental pulp mesenchymal stem cell (DPSCs)-derived soluble factors, produced under hypoxic conditions, support angiogenesis via endothelial cell activation and generation of M2-like macrophages

BackgroundCell therapy has emerged as a revolutionary tool to repair damaged tissues by restoration of an adequate vasculature. Dental Pulp stem cells (DPSC), due to their easy biological access, ex vivo properties, and ability to support angiogenesis have been largely explored in regenerative medicine.MethodsHere, we tested the capability of Dental Pulp Stem Cell-Conditioned medium (DPSC-CM), produced in normoxic (DPSC-CM Normox) or hypoxic (DPSC-CM Hypox) conditions, to support angiogenesis via their soluble factors. CMs were characterized by a secretome protein array, then used for in vivo and in vitro experiments. In in vivo experiments, DPSC-CMs were associated to an Ultimatrix sponge and injected in nude mice. After excision, Ultimatrix were assayed by immunohistochemistry, electron microscopy and flow cytometry, to evaluate the presence of endothelial, stromal, and immune cells.For in vitro procedures, DPSC-CMs were used on human umbilical-vein endothelial cells (HUVECs), to test their effects on cell adhesion, migration, tube formation, and on their capability to recruit human CD14+ monocytes.ResultsWe found that DPSC-CM Hypox exert stronger pro-angiogenic activities, compared with DPSC-CM Normox, by increasing the frequency of CD31+ endothelial cells, the number of vessels and hemoglobin content in the Ultimatrix sponges. We observed that Utimatrix sponges associated with DPSC-CM Hypox or DPSC-CM Normox shared similar capability to recruit CD45− stromal cells, CD45+ leukocytes, F4/80+ macrophages, CD80+ M1-macrophages and CD206+ M2-macropages. We also observed that DPSC-CM Hypox and DPSC-CM Normox have similar capabilities to support HUVEC adhesion, migration, induction of a pro-angiogenic gene signature and the generation of capillary-like structures, together with the ability to recruit human CD14+ monocytes.ConclusionsOur results provide evidence that DPSCs-CM, produced under hypoxic conditions, can be proposed as a tool able to support angiogenesis via macrophage polarization, suggesting its use to overcome the issues and restrictions associated with the use of staminal cells.

Controlled release of hydrogel-encapsulated mesenchymal stem cells-conditioned medium promotes functional liver regeneration after hepatectomy in metabolic dysfunction-associated steatotic liver disease

BackgroundGlobally, prevalence of metabolic dysfunction-associated steatotic liver disease (MASLD) is increasing, and there is an urgent need to develop innovative therapies that promote liver regeneration following hepatectomy for this disease. Surgical excision is a key therapeutic approach with curative potential for liver tumors. However, hepatic steatosis can lead to delayed liver regeneration and higher post-operative complication risk. Mesenchymal stem cells-conditioned medium (MSC-CM) is considered a rich source of paracrine factors that can repair tissues and restore function of damaged organs. Meanwhile, hydrogels have been widely recognized to load MSC secretome and achieve sustained release. This study aimed to evaluate the therapeutic effect of hydrogel-encapsulated MSC-CM on liver regeneration following partial hepatectomy (PHx) in a rodent model of diet-induced hepatic steatosis.MethodsMale Lewis rats were fed with a methionine and choline–deficient diet. After 3 weeks of feeding, PHx was performed and rats were randomly allocated into two groups that received hydrogel-encapsulated MSC-CM or vehicle via the intra-mesenteric space of the superior mesenteric vein (SMV).ResultsThe regeneration of the remnant liver at 30 and 168 h after PHx was significantly accelerated, and the expressions of proliferating cell nuclear antigen were significantly enhanced in the MSC-CM group. MSC-CM treatment significantly increased hepatic ATP and β-hydroxybutyrate content at 168 h after PHx, indicating that MSC-CM fosters regeneration not only in volume but also in functionality. The number of each TUNEL- and cleaved caspase-3 positive nuclei in hepatocytes at 9 h after PHx were significantly decreased in the MSC-CM group, suggesting that MSC-CM suppressed apoptosis. MSC-CM increased serum immunoregulatory cytokine interleukin-10 and interleukin-13 at 30 h after PHx. Additionally, mitotic figures and cyclin D1 expression decreased and hepatocyte size increased in the MSC-CM group, implying that this mode of regeneration was mainly through cell hypertrophy rather than cell division.ConclusionsMSC-CM represents a novel therapeutic approach for patients with MASLD requiring PHx.

Conditioned Medium from Human Amniotic Membrane-Derived Mesenchymal Stem Cells Modulates Inflammatory and Myofibrotic Factors in Vivo

Background: Heart failure (HF) is a prevalent diagnosis with a significant mortality rate. Various therapeutic approaches exist for treating HF, and human adipose-derived mesenchymal stem cells-conditioned medium (hAMSCs-CM) therapy has emerged as a promising option. Despite its potential efficacy, the precise mechanism of action underlying hAMSCs-CM treatment remains unclear. To address this knowledge gap, we conducted a novel animal study to investigate the mechanism of action of hAMSCs-CM in an HF model, with a specific focus on transforming growth factor-β (TGF-β)/galectin-3, monocyte chemoattractant protein-1 (MCP1), B-type natriuretic peptide (BNP), and aldosterone (ALD). Methods: Forty adult male Wistar rats were divided into 4 groups: control, HF, culture medium, and CM. All rats, except those in the control group, received an injection of isoproterenol to induce an animal model of HF. The CM group was administered the CM, while those in the culture medium group received standard culture media. Subsequently, serum levels of fibrotic factors, including TGF-β/galectin-3, MCP1, BNP, and ALD, were measured using ELISA. Statistical analysis was performed using one-way analysis of variance and the Tukey test. Results: Serum levels of TGF-β/galectin-3, MCP1, BNP, and ALD were significantly elevated in the HF, CM, and culture medium groups compared with the control group (P<0.001). Additionally, these fibrotic factors were significantly reduced in the CM group compared with the HF group (P<0.001). Notably, CM therapy could not restore TGF-β/galectin-3, MCP1, BNP, or ALD levels to the normal range observed in the control group. Conclusion: Our findings indicate that hAMSCs-CM modulates the expression of inflammatory and fibrotic cytokines, such as TGF-β/galectin-3, MCP1, BNP, and ALD, in isoproterenol-induced HF in male rats. These results contribute to a better understanding of the therapeutic mechanisms underlying hAMSCs-CM treatment for HF.

Mesenchymal Stem Cell-conditioned Medium Protecting Renal Tubular Epithelial Cells by Inhibiting Hypoxia-inducible Factor-1α and Nuclear Receptor Coactivator-1.

Acute kidney injury (AKI) is characterized by inflammatory infiltration and damage and death of renal tubular epithelial cells (RTECs), in which hypoxia plays an important role. Deferoxamine (DFO) is a well-accepted chemical hypoxia-mimetic agent. Mesenchymal stem cell-conditioned medium (MSC-CM) can reduce local inflammation and repair tissue. In this study, we explored the effect and molecular mechanism of MSC-CM-mediated protection of RTECs under DFO-induced hypoxia. Rat renal proximal tubule NRK-52E cells were treated with different concentrations of DFO for 24 hours, followed by evaluation of RTEC injury, using a Cell Counting Kit-8 (CCK-8) to detect cell viability and western blotting to evaluate the expression of transforming growth factor- beta 1 (TGF-β1), α-smooth muscle actin (α-SMA), and hypoxia-inducible factor-1 alpha (HIF-1α) in NRK-52E cells. Then, three groups of NRK-52E cells were used in experiments, including normal control (NC), 25 μM DFO, and 25 μM DFO + MSC-CM. MSC-CM was obtained from the human umbilical cord. MSC-CM was used to culture cells for 12 hours before DFO treatment, then fresh MSC-CM and 25 μM DFO were added, and cells were cultured for another 24 hours before analysis. Western blotting and cellular immunofluorescence staining showed culture of NRK-52E cells in 25 μM DFO for 24 hours induced HIF-1α and nuclear receptor coactivator-1 (NCoA-1), simulating hypoxia. MSC-CM could inhibit the DFO-induced up-regulation of α-SMA, TGF-β1, HIF-1α and NCoA-1. Our results suggest that MSC-CM has a protective effect on RTECs by down-regulating HIF-1α and NCoA-1, which may be the harmful factors in renal injury.

Characterization of Extracellular Vesicles by Sulfophosphovanillin Colorimetric Assay and Raman Spectroscopy.

Detailed characterization of extracellular vesicles (EVs) is crucial for their application in medical diagnostics. However, the complexity of their chemical composition and the heterogeneity of EV populations make their characterization challenging. Here we describe two analytical procedures that can help overcome this challenge. Small EVs were isolated from conditioned cell culture media using ultracentrifugation and characterized using nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). Raman spectroscopy was used to assess the overall composition of the isolated samples and lipids extracted from them. Sulfophosphovanillin (SPV) colorimetric assay was used to quantify the contents of lipid. Six samples of EVs were characterized. The lipid contents measured using SPV assay was in reasonable agreement with the quantitative estimates based on the particle size and concentration measured using NTA. The most peaks observed in the Raman spectra could be attributed to either proteins or lipids, and their origins was confirmed by lipid extraction. The protein-to-lipid ratio was estimated based on the Raman spectra. The experiential procedures described in this study will help to overcome the challenge of quick and highly informative characterization of the EVs.

Effect of Nicotinamide Riboside Against the Exhaustion of CD8+ T Cells via Alleviating Mitochondrial Dysfunction

Background: Targeting mitochondria and protecting the mitochondrial function of CD8+ T cells are crucial for enhancing the clinical efficacy of cancer immunotherapy. Objectives: In this study, our objective was to investigate the potential of nicotinamide riboside (NR) in preserving the mitochondrial function of CD8+ T cells and mitigating their exhaustion. Methods: We established two in vitro models to induce CD8+ T cell exhaustion either by tumor cell-conditioned medium (TCM) or by continuous stimulation with OVA(257–264) peptide. CD8+ T cells were treated in the absence/presence of NR. Results: Our findings demonstrated that NR supplementation effectively inhibited CD8+ T cell exhaustion and preserved mitochondrial function in both models. Moreover, apoptosis of CD8+ T cells was reduced after NR treatment. Western blot data indicated that NR treatment upregulated Silent information regulator 1 (SirT1) expression. Further inhibition of Sirt1 activity using EX527 uncovered that the inhibitory effect of NR on CD8+ T cell exhaustion and its protective effect on mitochondria were attenuated. Conclusions: In conclusion, our results indicate that NR supplementation attenuates CD8+ T cell exhaustion, and its underlying mechanism is associated with increased mitochondrial function regulated by the SirT1 pathway. Our research provides evidence that NR may assist in enhancing the clinical efficacy of immunotherapy.

Anti-tumor effects of ellagic acid and mesenchymal stem cell-conditioned medium on breast cancer cells on the 3D printed PCL/agarose scaffolds

Numerous investigations have demonstrated that natural bioactive compounds, such as flavonoids, exhibit synergistic effects with anticancer agents utilized in clinical practice. Stem cell therapies play a critical role in the domain of oncology; nevertheless, conflicting results have surfaced regarding the potential pro-cancer or anti-cancer characteristics of mesenchymal stem cells. The primary aim of the current investigation was to examine the anticancer efficacy of ellagic acid (EA) alone or in combination with adipose tissue stem cells derived conditioned medium (ADSCs-CM) on human breast cancer cells. In order to overcome the potential effects of two-dimensional (2D) culture, a three-dimensional (3D) printed polycaprolactone (PCL)/agarose scaffold is utilized to evaluate cancer cell behavior. More recently it was reported this scaffold had open and interconnected pores, as well as good water absorption and mechanical properties. Assessment of MCF-7 cancer cell viability was conducted through MTT assay, flow cytometry, cell cycle assay, and quantitative real-time PCR within a 3D cell culture framework. The outcomes revealed a reduction in cancer cell viability across all treatment cohorts in comparison to the control group. Particularly noteworthy was the significantly enhanced effect of the EA and CM combination relative to each component administered individually. Flow cytometry assessment using annexin V/PI staining indicated a decrease in cancer cells, particularly evident in the combination-treated group. Furthermore, the mRNA expression levels of BAX, BCL2, vascular endothelial growth factor (VEGF), and caspase 3 genes were consistent with pathways associated with apoptosis. Consequently, the synergistic anti-proliferative impact of EA in conjunction with ADSCs-CM on MCF-7 cells has been substantiated. These findings have two important implications for breast cancer research; highlight the utility of the PCL/agarose scaffold as a cancer model and suggest new avenues for the development of adjuvant anticancer therapeutic approaches.

P02.07.B FIBROBLAST ACTIVATION PROTEIN EXPRESSING MESENCHYMAL CELLS INFLUENCE T CELL ABUNDANCE AND FUNCTION IN GLIOBLASTOMA

Abstract BACKGROUND Glioblastomas (GBMs) are aggressive brain tumors with strong immunosuppressive properties. In epithelial cancers, mesenchymal cells expressing fibroblast activation protein (FAP) play an important role in modulating the T cell response. We have recently shown that FAP+ mesenchymal cells are present in human GBMs. The aim of this study was to determine their effect on T cell abundance and function. MATERIAL AND METHODS GBM tumors (16 samples) were mechanically and enzymatically dissociated and analyzed for immune cell subpopulations by flow cytometry. Expression of T cell related genes was analyzed by qRT-PCR in GBMs selected based on FAP protein concentration determined by ELISA (14 FAP high [upper tercile], 16 FAP low [lower tercile]). Immunohistochemistry (IHC) was used to compare the number of CD3+, CD4+, and CD8+ T cells in GBMs with high (upper tercile, n=21) and low abundance of FAP+ stroma (lower tercile, n=20). Co-localization of FAP+ mesenchymal cells and CD3, PD-L1 and PD-L2 was evaluated by immunofluorescence in frozen sections. 8 FAP+ mesenchymal and 3 glioblastoma stem-like cell cultures were derived from human GBMs and PD-L1 and PD-L2 expression was determined using ELISA. Peripheral blood mononuclear cells were isolated from healthy donors´ buffy coats. Proliferation of stimulated T cells in conditioned media from FAP+ mesenchymal cells was analyzed by flow cytometry. RESULTS The percentage of T cells in CD45+ cells from GBMs positively correlated with FAP expression. Expression of the T cell inhibitory molecule PD-L2 was higher in FAP high GBMs. Immunohistochemisty revealed that the numbers of CD3+ and CD8+ T cells were higher in GMBs with a high abundance of FAP+ stroma and T cells were frequently in close proximity to FAP+ mesenchymal cells, which were often PD-L2 positive. In vitro, FAP+ mesenchymal cells expressed more PD-L2 than glioblastoma stem-like cells. T cell proliferation was decreased after exposure to conditioned media from FAP+ mesenchymal cells. CONCLUSION FAP+ mesenchymal cells influence T cell abundance in GBMs and may affect T cell functions by limiting their proliferation via soluble factors or cell-cell contact. Acknowledgment: This work was supported by the National Institute for Cancer Research (LX22NPO5102), the Center for Tumor Ecology (CZ.02.1.01/0.0/0.0/16_019/0000785), Charles University project GAUK 365022 and Cooperatio Program Oncology and Haematology“.

Hypoxia-Induced Inflammation in In Vitro Model of Human Blood-Brain Barrier: Modulatory Effects of the Olfactory Ensheathing Cell-Conditioned Medium.

Hypoxia compromises the integrity of the blood-brain barrier (BBB) and increases its permeability, thereby inducing inflammation. Olfactory ensheathing cells (OECs) garnered considerable interest due to their neuroregenerative and anti-inflammatory properties. Here, we aimed to investigate the potential modulatory effects of OEC-conditioned medium (OEC-CM) on the response of human brain microvascular endothelial cells (HBMECs), constituting the BBB, when exposed to hypoxia. HBMECs were utilized to establish the in vitro BBB model. OECs were isolated from mouse olfactory bulbs, and OEC-CM was collected after 48h of culture. The effect of OEC-CM treatment on the HBMEC viability was evaluated under both normoxic and hypoxic conditions at 6h, 24h, and 30h. Western blot and immunostaining techniques were employed to assess NF-κB/phospho-NF-κB expression. HIF-1α, VEGF-A, and cPLA2 mRNA expression levels were quantified using digital PCR. ELISA assays were performed to measure PGE2, VEGF-A, IL-8 secretion, and cPLA2 specific activity. The in vitro formation of HBMEC capillary-like structures was examined using a three-dimensional matrix system. OEC-CM attenuated pro-inflammatory responses and mitigated the HIF-1α/VEGFA signaling pathway activation in HBMECs under hypoxic condition. Hypoxia-induced damage of the BBB can be mitigated by novel therapeutic strategies harnessing OEC potential.

YTHDF1-regulated ALOX5 in retinal pigment epithelial cells under hypoxia enhances VEGF expression and promotes viability, migration, and angiogenesis of vascular endothelial cells

Upregulation of vascular endothelial growth factor (VEGF) and enhanced angiogenesis have been implicated in the severe progression of age-related macular degeneration (AMD). Abnormal arachidonate 5-lipoxygenase (ALOX5) is associated with AMD pathogenesis. However, no reports have shown the causal role of ALOX5 in angiogenesis during AMD. In the present study, ARPE-19 cells were exposed to hypoxia, an inducer of VEGF expression. Potential proteins implicated in AMD progression were predicted using bioinformatics. RNA affinity antisense purification-mass spectrometry (RAP-MS) was applied to identify the binding proteins of ALOX5 3′UTR. Expression of ALOX5 and YTH N6-methyladenosine RNA-binding protein 1 (YTHDF1) was detected by qRT-PCR and western blotting. VEGF expression and secretion were assessed by immunofluorescence and ELISA, respectively. The chicken embryo chorioallantoic membrane (CAM) was used to analyze the effect of ALOX5 on angiogenesis. RNA stability was assayed using the Actinomycin D assay. The results show that hypoxia promoted cell growth and increased VEGF expression in ARPE-19 cells. ALOX5 was associated with AMD progression, and hypoxia upregulated ALOX5 expression in ARPE-19 cells. ALOX5 silencing reduced VEGF expression induced by hypoxia in ARPE-19 cells. Moreover, the conditioned medium of ALOX5-silenced ARPE-19 cells could suppress the viability and migration of HUVECs and diminish angiogenesis in the CAM. Furthermore, YTHDF1 was validated to bind to ALOX5 3′UTR, and YTHDF1 promoted ALOX5 expression by elevating the stability of ALOX5 mRNA. In conclusion, our findings demonstrate that YTHDF1-regulated ALOX5 increases VEGF expression in hypoxia-exposed ARPE-19 cells and enhances the viability, migration, and angiogenesis of vascular endothelial cells.

Preliminary in vivo investigation of the mesenchymal stromal cell secretome as a novel treatment for methicillin-resistant Staphylococcus aureus in equine skin wounds.

We aimed to study the antimicrobial and pro-healing potential of equine mesenchymal stromal cell secreted products (i.e. secretome), collected as conditioned media (mesenchymal stromal cell-conditioned media, MSC CM), in a novel in vivo model of methicillin-resistant Staphylococcus aureus (MRSA)-inoculated equine thorax wounds. Prospective in vivo study. Two Thoroughbred geldings. Six full-thickness cutaneous wounds were created bilaterally on the dorsal thorax of two horses (n = 12 wounds/horse). Wounds on the left thoraces were inoculated with MRSA on day 0. All wounds were then treated with either mupirocin ointment, MSC CM, or vehicle control (n = 4 wounds per group) once daily for 3 days. Photographs were taken to quantify wound scores and sizes, as well as samples to determine bacterial colony forming units (CFUs), at days 0, 1, 2, 3, 7, 14, 21, and 28. The wound edge was biopsied on days 0, 7, and 28, and scored histologically. Inoculated wounds had more bacterial CFUs at day 1 (p < .0001) and were larger in size at day 28 (p = .0009) than noninoculated wounds. Mupirocin-treated wounds were smaller than MSC CM and vehicle control-treated wounds at day 28 (p = .003). Mesenchymal stromal cell-conditioned media did not affect CFU numbers in inoculated and noninoculated wounds. Moreover, MSC CM did not affect the parameters of wound size or gross or microscopic wound scores over time. Mesenchymal stromal cell-conditioned media did not exhibit antimicrobial or pro-healing properties in the current study; however, the in vivo model of inoculated equine thorax wounds requires further optimization. This pilot study contributes to a growing understanding of the equine MSC secretome as an antimicrobial and pro-healing therapeutic for equine wounds.

Endoplasmic reticulum stress alters myelin associated protein expression and extracellular vesicle composition in human oligodendrocytes.

Myelination of the central nervous system is mediated by specialized glial cells called oligodendrocytes (OLs). Multiple sclerosis (MS) is characterized by loss of myelination and subsequent clinical symptoms that can severely impact the quality of life and mobility of those affected by the disease. The major protein components of myelin sheaths are synthesized in the endoplasmic reticulum (ER), and ER stress has been observed in patients with MS. Extracellular vesicles (EVs) have been shown to carry bioactive cargo and have the potential to be utilized as noninvasive biomarkers for various diseases. In the current study, we sought to determine how ER stress in OLs affected the production of key myelination proteins and EV release and composition. To achieve this, tunicamycin was used to induce ER stress in a human oligodendroglioma cell line and changes in myelination protein expression and markers of autophagy were assessed. EVs were also separated from the conditioned cell culture media through size exclusion chromatography and characterized. Significant reductions in the expression of myelination proteins and alterations to autophagosome formation were observed in cells undergoing ER stress. EVs released from these cells were slightly smaller relative to controls, and had strong expression of LC3B. We also observed significant upregulation of miR-29a-3p in ER stress EVs when compared to controls. Taken together, these data suggest that ER stress negatively impacts production of key myelination proteins and induces cells to release EVs that may function to preemptively activate autophagic pathways in neighboring cells.

Tumor-Activated Neutrophils Promote Lung Cancer Progression through the IL-8/PD-L1 Pathway.

Lung cancer remains a major global health threat due to its complex microenvironment, particularly the role of neutrophils, which are crucial for tumor development and immune evasion mechanisms. This study aimed to delve into the impact of lung cancer cell-conditioned media on neutrophil functions and their potential implications for lung cancer progression. Employing in vitro experimental models, this study has analyzed the effects of lung cancer cell-conditioned media on neutrophil IL-8 and IFN-γ secretion, apoptosis, PD-L1 expression, and T-cell proliferation by using techniques, such as ELISA, flow cytometry, immunofluorescence, and CFSE proliferation assay. The roles of IL-8/PD-L1 in regulating neutrophil functions were further explored using inhibitors for IL-8 and PD-L1. Lung cancer cell lines were found to secrete higher levels of IL-8 compared to normal lung epithelial cells. The conditioned media from lung cancer cells significantly reduced apoptosis in neutrophils, increased PD-L1 expression, and suppressed T-cell proliferation and IFN-γ secretion. These effects were partially reversed in the presence of IL-8 inhibitors in Tumor Tissue Culture Supernatants (TTCS), while being further enhanced by IL-8. Both apoptosis and PD-L1 expression in neutrophils demonstrated dose-dependency to TTCS. Additionally, CFSE proliferation assay results further confirmed the inhibitory effect of lung cancer cell-conditioned media on T-cell proliferation. This study has revealed lung cancer cell-conditioned media to modulate neutrophil functions through regulating factors, such as IL-8, thereby affecting immune regulation and tumor progression in the lung cancer microenvironment.

Anti-fibrogenic effect of umbilical cord-derived mesenchymal stem cell-conditioned media in human esophageal fibroblasts.

Esophageal fibrosis can develop due to caustic or radiation injuries. Umbilical cord-derived mesenchymal stem cells (UC-MSCs) are known to mitigate fibrosis in various organs. However, the potential effects of UC-MSCs on human esophageal fibrosis remain underexplored. This study investigated the anti-fibrogenic properties and mechanisms of UC-MSC-derived conditioned media (UC-MSC-CM) on human esophageal fibroblasts (HEFs). HEFs were treated with TGF-β1 and then cultured with UC-MSC-CM, and the expression levels of extracellular matrix (ECM) components, RhoA, myocardin related transcription factor A (MRTF-A), serum response factor (SRF), Yes-associated protein (YAP), and transcriptional coactivator with PDZ-binding motif (TAZ) were measured. UC-MSC-CM suppressed TGF-β1-induced fibrogenic activation in HEFs, as evidenced by the downregulation of ECM. UC-MSC-CM diminished the expression of RhoA, MRTF-A, and SRF triggered by TGF-β1. In TGF-β1-stimulated HEFs, UC-MSC-CM decreased the nuclear localization of MRTF-A and YAP. Additionally, UC-MSC-CM diminished the TGF-β1-induced nuclear expressions of YAP and TAZ, while concurrently enhancing the cytoplasmic presence of phosphorylated YAP. Furthermore, UC-MSC-CM reduced TGF-β1-induced phosphorylation of Smad2. These findings suggest that UC-MSC-CM may inhibit TGF-β1-induced fibrogenic activation in HEFs by targeting the Rho-mediated MRTF/SRF and YAP/TAZ pathways, as well as the Smad2 pathway. This indicates its potential as a stem cell therapy for esophageal fibrosis.

Proteomic Characterization of Corneal Epithelial and Stromal Cell-Derived Extracellular Vesicles.

Communication between the different layers of the cornea (epithelium and stroma) is a complex, yet crucial element in the corneal healing process. Upon corneal injury, it has been reported that the bi-directional cross talk between the epithelium and stroma via the vesicular secretome, namely, extracellular vesicles (EVs), can lead to accelerated wound closure upon injury. However, the distinct protein markers of EVs derived from human corneal epithelial (HCE) cells, keratocytes (HCKs), fibroblasts (HCFs), and myofibroblasts (HCMs) remain poorly understood. All EVs were enriched for CD81 and showed increased expression levels of ITGAV and FN1 in HCM-EVs compared to HCE- and HCF-EVs. All EVs were negative for GM130 and showed minimal differences in biophysical properties (particle concentration, median particle size, and zeta potential). At the proteomic level, we show that HCM-EVs are enriched with proteins associated with fibrosis pathways, such as COL6A1, COL6A2, MMP1, MMP2, TIMP1, and TIMP2, compared to HCE-, HCK-, and HCF-EVs. Interestingly, HCE-EVs express proteins involved with the EIF-2 signaling pathway (stress-induced signals to regulate mRNA translation), such as RPS21, RALB, EIF3H, RALA, and others, compared to HCK-, HCF-, and HCM-EVs. In this study, we isolated EVs from cell-conditioned media from HCE, HCKs, HCFs, and HCMs and characterized their biophysical and protein composition by Western blot, nanoparticle tracking analysis, and proteomics. This study supports the view that EVs from the corneal epithelium and stroma have a distinct molecular composition and may provide novel protein markers to distinguish the difference between HCE-, HCK-, HCF-, and HCM-EVs.

Geraniol intake improves age-related malnutrition in mice.

Geraniol is an acyclic monoterpenoid that is abundant in many plants, including rose, lemongrass, and lavender. As geraniol has various beneficial functions, rose oil rich in geraniol is not only used for aromatherapy but also as a supplement to promote health benefits. However, the beneficial effects of geraniol on age-related pathologies are unknown. In this study, we aimed to clarify the effects of geraniol intake on age-related pathologies. We orally administered geraniol to aged mice (age: 24-29 months) five times a week for 4 weeks and sampled their blood and various organs. We investigated age-related changes in the blood and organ samples. Furthermore, we treated HepG2 cells with geraniol and examined the expression level of the ALB gene and the amount of secreted albumin in vitro. Geraniol significantly increased blood albumin, total cholesterol, and red blood cell counts, indicating an improvement in nutritional markers in aged mice. Geraniol also transcriptionally increased the Alb gene expression in the liver of aged mice. Furthermore, treatment with geraniol significantly upregulated the ALB gene expression and the secretion of albumin in the conditioned medium of HepG2 cells. Geraniol increases serum albumin levels at the transcriptional level. Geraniol intake can be an effective strategy for age-related malnutrition. Geriatr Gerontol Int 2024; ••: ••-••.

Medicalized Aesthetic Uses of Exosomes and Cell Culture-Conditioned Media: Opening an Advanced Care Era for Biologically Inspired Cutaneous Prejuvenation and Rejuvenation

Recent advancements in aesthetic medicine offer innovative cosmetic solutions to enhance patient skin quality and appearance. Advanced treatment options enable practitioners to effectively address skin aging signs, pigmentation imbalance, and loss of elasticity in ambulatory and home-based care regimens. Exosomes (nanoscale cell-derived vesicles) transport a variety of biomolecules and are pivotal in physiological intercellular communication. Importantly, exosomes have recently emerged as key endogenous players in tissular regeneration. More broadly (from an active ingredient purity standpoint), exosomes, stem cell secretomes, and cell culture-conditioned media have been clinically proven to exert multifaceted beneficial topical effects (anti-inflammatory, antioxidant, anti-aging, skin rejuvenation). Therefore, human, animal, and plant-derived exosomes or other refined sub-cellular biological fractions are gaining substantial interest within the aesthetic and cosmetic industries. Notably, such approaches are thought to be among the most promising novel contenders for advanced, biologically inspired skin prejuvenation and rejuvenation care. The present narrative review summarizes the latest clinically oriented research on exosomes and cell culture-conditioned media, highlighting their mechanisms of action in various topical applications. Furthermore, it explores the innovation landscape and currently commercially available products on the global cosmetic market and discusses the potential future applications of advanced, biologically inspired ingredients in the medical aesthetic industry.

Biomarkers for modeling of cancer-specifc tumorassociated macrophages ex vivo

Introduction. Tumor-associated macrophages (TAMs) are essential innate immune cells in the tumor microenvironment. TAMs can stimulate cancer cell proliferation and primary tumor growth, angiogenesis, lymphangiogenesis, cancer cell invasiveness in vessels and metastatic niche formation as well as support chemotherapy resistance. TAMs are phenotypically diverse both in various cancer localizations and in intratumoral heterogeneous compartments. Tumor-specific modeling of TAMs is necessary to understand the fundamental mechanism of pro- and anti-tumor activity, to test their interaction with existing therapies, and to develop TAM- targeted immunotherapy. Aim of study: To investigate cancer-specific transcriptomic features of ex vivo human TAM models. Material and Methods. Here we compared transcriptomic profiles of TAMs for breast, colorectal, ovarian, lung, and prostate cancers ex vivo. Human monocytes were isolated from buffy coats, and then stimulated by the tumor cell conditioned medium ex vivo. Using real-time PCR, we quantified the expression of key TAM biomarkers including inflammatory cytokines, scavenger-receptors, angiogenesis-regulating genes, and matrix remodeling factors. Results. PCR analysis allowed revealing cancer-specific expression profiles of modeled TAMs. By comparing the existing knowledge about TAM phenotypes in human tumors in vivo with the collected data, we discuss the advantages and limitation of ex vivo TAM models derived from human blood monocytes. Conclusion. Monocytes-derived macrophages stimulated with cancer cell-conditioned medium can, to a certain extent, allow modeling of cancer-specific programming of TAMs. Our model system is valuable to examine agents reprogramming key TAM pro-tumoral activities, and for the reproducible analysis of mechanistic events that program tolerogenic status of TAMs towards cancer cells.