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Background: One of the obstacles to autologous chondrocyte implantation (ACI) is obtaining a large quantity of chondrocytes without depletion of their properties. The conditioned medium (CM) from different subpopulations of stem cells (mesenchymal stromal cells (MSC) or induced pluripotent stem cells (iPSC)) could be a gamechanger. MSCs' potential is related to the donor's health and age, which could be omitted when, as a source, iPSCs are used. There is a lack of data regarding their use in the chondrocyte culture expansion. Thus, we wanted to verify whether iPSC-CM could be beneficial for the cell culture of primary chondrocyte cells., Methods: We added the iPSC-CMs from GPCCi001-A and ND 41658*H cells to the culture of primary chondrocyte cell lines isolated from OA patients (n = 6) for other two passages. The composition of the CM was evaluated using Luminex technology. Then, we analysed the senescence, proliferation rate and using flow cytometry: viability, distribution of cell cycle phases, production of reactive oxygen species (ROS) and double-strand breaks. The cartilage-related markers were evaluated using Western blot and immunofluorescence. Additionally, a three-dimensional cell culture was used to determine the potential to form cartilage particles., Results: iPSC-CM increased proliferation and diminished cell ROS production and senescence. CM influenced the cartilage-related protein expression and promoted the growth of cartilage particles. The cell exposed to CM did not lose the ECM proteins, suggesting the chondroprotective effect for prolonged culture time., Conclusion: Our preliminary results suggest a beneficial effect on maintaining chondrocyte biology during in vitro expansion., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Clinical trials investigating the potential of mesenchymal stromal cells (MSCs) for the treatment of inflammatory diseases, such as acute respiratory distress syndrome (ARDS), have been disappointing, with less than 50% of patients responding to treatment. Licensed MSCs show enhanced therapeutic efficacy in response to cytokine-mediated activation signals. There are two distinct sub-phenotypes of ARDS: hypo- and hyper-inflammatory. We hypothesized that pre-licensing MSCs in a hyper-inflammatory ARDS environment would enhance their therapeutic efficacy in acute lung inflammation (ALI). Serum samples from patients with ARDS were segregated into hypo- and hyper-inflammatory categories based on interleukin (IL)-6 levels. MSCs were licensed with pooled serum from patients with hypo- or hyper-inflammatory ARDS or healthy serum controls. Our findings show that hyper-inflammatory ARDS pre-licensed MSC conditioned medium (MSC-CM Hyper ) led to a significant enrichment in tight junction expression and enhanced barrier integrity in lung epithelial cells in vitro and in vivo in a vascular endothelial growth factor (VEGF)-dependent manner. Importantly, while both MSC-CM Hypo and MSC-CM Hyper significantly reduced IL-6 and tumor necrosis factor alpha (TNF-α) levels in the bronchoalveolar lavage fluid (BALF) of lipopolysaccharide (LPS)-induced ALI mice, only MSC-CM Hyper significantly reduced lung permeability and overall clinical outcomes including weight loss and clinical score. Thus, the hypo- and hyper-inflammatory ARDS environments may differentially influence MSC cytoprotective and immunomodulatory functions., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Individuals with Alzheimer's disease (AD) often require many medications; however, these medications are dosed using regimens recommended for individuals without AD. This is despite reduced abundance and function of P-glycoprotein (P-gp) at the blood-brain barrier (BBB) in AD, which can impact brain exposure of drugs. The fundamental mechanisms leading to reduced P-gp abundance in sporadic AD remain unknown; however, it is known that the apolipoprotein E (apoE) gene has the strongest genetic link to sporadic AD development, and apoE isoforms can differentially alter BBB function. The aim of this study was to assess if apoE affects P-gp abundance and function in an isoform-dependent manner using a human cerebral microvascular endothelial cell (hCMEC/D3) model., Methods: This study assessed the impact of apoE isoforms on P-gp abundance (by western blot) and function (by rhodamine 123 (R123) uptake) in hCMEC/D3 cells. Cells were exposed to recombinant apoE3 and apoE4 at 2 - 10 µg/mL over 24 - 72 hours. hCMEC/D3 cells were also exposed for 72 hours to astrocyte-conditioned media (ACM) from astrocytes expressing humanised apoE isoforms., Results: P-gp abundance in hCMEC/D3 cells was not altered by recombinant apoE4 relative to recombinant apoE3, nor did ACM containing human apoE isoforms alter P-gp abundance. R123 accumulation in hCMEC/D3 cells was also unchanged with recombinant apoE isoform treatments, suggesting no change to P-gp function, despite both abundance and function being altered by positive controls SR12813 (5 µM) and PSC 833 (5 µM), respectively., Conclusions: Different apoE isoforms have no direct influence on P-gp abundance or function within this model, and further in vivo studies would be required to address whether P-gp abundance or function are reduced in sporadic AD in an apoE isoform-specific manner., (© 2024. The Author(s).)

Background: Innovative therapies against bacterial infections are needed. One approach is to focus on host-directed immunotherapy (HDT), with treatments that exploit natural processes of the host immune system. The goals of this type of therapy are to stimulate protective immunity while minimizing inflammation-induced tissue damage. We use non-traditional large animal models to explore the potential of the mammosphere-derived epithelial cell (MDEC) secretome, consisting of all bioactive factors released by the cells, to modulate host immune functions. MDEC cultures are enriched for mammary stem and progenitor cells and can be generated from virtually any mammal. We previously demonstrated that the bovine MDEC secretome, collected and delivered as conditioned medium (CM), inhibits the growth of bacteria in vitro and stimulates functions related to tissue repair in cultured endothelial and epithelial cells., Methods: The immunomodulatory effects of the bovine MDEC secretome on bovine neutrophils, an innate immune cell type critical for resolving bacterial infections, were determined in vitro using functional assays. The effects of MDEC CM on neutrophil molecular pathways were explored by evaluating the production of specific cytokines by neutrophils and examining global gene expression patterns in MDEC CM-treated neutrophils. Enzyme linked immunosorbent assays were used to determine the concentrations of select proteins in MDEC CM and siRNAs were used to reduce the expression of specific MDEC-secreted proteins, allowing for the identification of bioactive factors modulating neutrophil functions., Results: Neutrophils exposed to MDEC secretome exhibited increased chemotaxis and phagocytosis and decreased intracellular reactive oxygen species and extracellular trap formation, when compared to neutrophils exposed to control medium. C-X-C motif chemokine 6, superoxide dismutase, peroxiredoxin-2, and catalase, each present in the bovine MDEC secretome, were found to modulate neutrophil functions., Conclusion: The MDEC secretome administered to treat bacterial infections may increase neutrophil recruitment to the site of infection, stimulate pathogen phagocytosis by neutrophils, and reduce neutrophil-produced ROS accumulation. As a result, pathogen clearance might be improved and local inflammation and tissue damage reduced., Competing Interests: Authors LO and LH were employed by company Elanco Animal Health. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Harman, Sipka, Oxford, Oliveira, Huntimer, Nydam and Van de Walle.)

The cryopreservation procedure decreases sperm quality, causing certain changes at structural and molecular levels affecting fertilizing ability. We aimed to investigate the impacts of human adipose-derived mesenchymal stem cells (HAd-MSCs) conditioned medium (CM) on the protection of human sperm from cryoinjury. Thirty normal semen specimens were evaluated in this study. Each specimen was separated into six groups and enhanced with varying concentrations of human Ad-MSCs-CM (0, 10, 30, 50, 70, and 100%). Sperm motility, viability, morphology, apoptosis, mitochondrial potential, and lipid peroxidation, and DNA fragmentation were evaluated before freezing and after thawing. The results showed that the total motility was preserved in 10% human Ad-MSCs-CM group. Also, DNA fragmentation was significantly lower in 10% compared to 0% human Ad-MSCs-CM (63.62 ± 17.72% vs.76.46 ± 4.87%, respectively, P < 0.004). Human Ad-MSCs-CM in groups of 10, 30, 50, and 70% reduced lipid peroxidation. The normal sperm morphology rate, mitochondrial membrane potential, and apoptosis showed no significant differences across various groups. It seems that human Ad-MSCs-CM can protect the sperm parameters during the cryopreservation by decreasing cryoinjury., (© 2024. The Author(s), under exclusive licence to Society for Reproductive Investigation.)

Obesity is often accompanied by increased adipose tissue inflammation, a process that is partially driven by adipose tissue-resident macrophages. In this study, we explored the potential for plant-derived dietary compounds to exert anti-inflammatory effects in macrophages that alleviate obesity-associated adipocyte dysfunction. Capsaicin (CAP), schisandrin A (SA), enterodiol (END), and enterolactone (ENL) treatment polarized J774 macrophages to an "M2" or anti-inflammatory phenotype and inhibited responses to stimulation with lipopolysaccharide (LPS). Furthermore, these compounds blocked inflammasome activation when administered just before ATP-induced NLRP3 activation, as evidenced by the abrogation of IL-1β release in mouse macrophages and human peripheral blood monocytes. The addition of CAP, SA, or ENL during the differentiation of bone marrow-derived macrophages was also sufficient to inhibit LPS-induced IL-6 and TNFα production. Finally, CAP, END, and ENL treatment during differentiation of 3T3-L1 adipocytes induced an adiponectin-high phenotype accompanied by increases in thermogenic gene expression, and conditioned media from these adipocytes inhibited LPS-induced production of IL-1β, IL-6, and TNFα from J774 macrophages. These polarizing effects were partially mediated by the elevated adiponectin and decreased syndecan-4 in the adipocyte-conditioned media. These results implicate the contribution of plant-derived dietary components to the modulation of macrophages and adipocytes in obesity. NEW & NOTEWORTHY The utility of food-based products to prevent or alleviate chronic conditions such as obesity and its associated comorbidities is an attractive approach. Capsaicin, schisandrin A, enterodiol, and enterolactone, phytochemicals present in traditional medicinal food, decreased proinflammatory cytokine production from macrophages that, in turn, reduced obesity-associated adipocyte dysfunction. These results implicate the contribution of plant-derived dietary components to the modulation of macrophages and adipocytes in obesity.

Objective: This study aimed to check the expression profile of the C-X-C motif chemokine ligands (CXCLs)-C-X-C motif chemokine receptor 2 (CXCR2) axis in cervical cancer and to explore the cross-talk between cervical cancer cells and neutrophils via CXCLs-CXCR2 axis., Methods: Available RNA-sequencing data based on bulk tissues and single-cell/nucleus RNA-sequencing data were used for bioinformatic analysis. Cervical cancer cell lines Hela and SiHa cells were utilized for in vitro and in vivo studies., Results: Except for neutrophils, CXCR2 mRNA expression is limited in other types of cells in the cervical tumor microenvironment. CXCLs bind to CXCR2 and are mainly expressed by tumor cells. CXCL1, 2, 3, 5, 6, and 8, which are consistently associated with neutrophil infiltration, are also linked to poor prognosis. SB225002 (a CXCR2 inhibitor) treatment significantly impairs SiHa cell-induced neutrophil migration. CXCL1, CXCL2, CXCL5, or CXCL8 neutralized conditioned medium from SiHa cells have weaker recruiting effects. The conditioned medium of neutrophils from healthy donors can slow cancer cell proliferation. Conditioned medium of tumor-associated neutrophils (TANs) can drastically enhance cervical cancer cell growth in vitro and in vivo ., Conclusions: The CXCLs-CXCR2 axis is critical in neutrophil recruitment and tumor cell proliferation in the cervical cancer microenvironment.

Background & Aims: Pancreatic ductal adenocarcinoma (PDAC) is characterized by desmoplastic stroma surrounding most tumors. Activated stromal fibroblasts, namely cancer-associated fibroblasts (CAFs), play a major role in PDAC progression. We analyzed whether CAFs influence acinar cells and impact PDAC initiation, that is, acinar-to-ductal metaplasia (ADM). ADM connection with PDAC pathophysiology is indicated, but not yet established. We hypothesized that CAF secretome might play a significant role in ADM in PDAC initiation., Methods: Mouse and human acinar cell organoids, acinar cells cocultured with CAFs and exposed to CAF-conditioned media, acinar cell explants, and CAF cocultures were examined by means of quantitative reverse transcription polymerase chain reaction, RNA sequencing, immunoblotting, and confocal microscopy. Data from liquid chromatography with tandem mass spectrometry analysis of CAF-conditioned medium and RNA sequencing data of acinar cells post-conditioned medium exposure were integrated using bioinformatics tools to identify the molecular mechanism for CAF-induced ADM. Using confocal microscopy, immunoblotting, and quantitative reverse transcription polymerase chain reaction analysis, we validated the depletion of a key signaling axis in the cell line, acinar explant coculture, and mouse cancer-associated fibroblasts (mCAFs)., Results: A close association of acino-ductal markers (Ulex europaeus agglutinin 1, amylase, cytokeratin-19) and mCAFs (α-smooth muscle actin) in LSL-Kras G12D/+ ; LSL-Trp53 R172H/+ ; Pdx1 Cre (KPC) and LSL-Kras G12D/+ ; Pdx1 Cre (KC) autochthonous progression tumor tissue was observed. Caerulein treatment-induced mCAFs increased cytokeratin-19 and decreased amylase in wild-type and KC pancreas. Likewise, acinar-mCAF cocultures revealed the induction of ductal transdifferentiation in cell line, acinar-organoid, and explant coculture formats in WT and KC mice pancreas. Proteomic and transcriptomic data integration revealed a novel laminin α5/integrinα4/stat3 axis responsible for CAF-mediated acinar-to-ductal cell transdifferentiation., Conclusions: Results collectively suggest the first evidence for CAF-influenced acino-ductal phenotypic switchover, thus highlighting the tumor microenvironment role in pancreatic carcinogenesis inception., (Copyright © 2024 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Aim: The secretome of mesenchymal stem cells (MSCs) could be a potential therapeutic intervention for diabetes and associated complications like nephropathy. This study aims to evaluate the effects of conditioned mediums (CMs) collected from umbilical cord-derived MSCs incubated under 2-dimensional (2D) or 3D culture conditions on kidney functions of rats with type-I diabetes (T1D)., Main Methods: Sprague-Dawley rats were treated with 20 mg/kg streptozocin for 5 consecutive days to induce T1D, and 12 doses of CMs were applied intraperitoneally for 4 weeks. The therapeutic effects of CMs were comparatively investigated by biochemical, physical, histopathological, and immunohistochemical analysis., Key Findings: 3D-CM had significantly higher total protein concentration than the 2D-CM Albumin/creatinine ratios of both treatment groups were significantly improved in comparison to diabetes. Light microscopic evaluations showed that glomerular and cortical tubular damages were significantly ameliorated in only the 3D-CM applied group compared to the diabetes group, which were correlated with transmission electron microscopic observations. The nephrin and synaptopodin expressions increased in both treatment groups compared to diabetes. The WT1, Ki-67, and active caspase-3 expressions in glomeruli and parietal layers of the treatment groups suggest that both types of CMs suppress apoptosis and promote possible parietal epithelial cells' (PECs') transdifferentiation towards podocyte precursor cells by switching on WT1 expression in parietal layer rather than inducing new cell proliferation., Significance: 3D-CM was found to be more effective in improving kidney functions than 2D-CM by ameliorating glomerular damage through the possible mechanism of transdifferentiation of PECs into podocyte precursors and suppressing glomerular apoptosis., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Intravenous administration of conditioned medium from stem cells of human exfoliated deciduous teeth (SHED-CM) regenerates mechanically injured osteochondral tissues in mouse temporomandibular joint osteoarthritis (TMJOA). However, the underlying therapeutic mechanisms remain unclear. Here, we showed that SHED-CM alleviated injured TMJ by inducing anti-inflammatory M2 macrophages in the synovium. Depletion of M2 by Mannosylated Clodrosome abolished the osteochondral repair activities of SHED-CM. Administration of CM from M2-induced by SHED-CM (M2-CM) effectively ameliorated mouse TMJOA by inhibiting chondrocyte inflammation and matrix degradation while enhancing chondrocyte proliferation and matrix formation. Notably, in vitro, M2-CM directly suppressed the catabolic activities while enhancing the anabolic activities of interleukin-1β-stimulated mouse primary chondrocytes. M2-CM also inhibited receptor activator of nuclear factor NF-κB ligand-induced osteoclastogenesis in RAW264.7 cells. Secretome analysis of M2-CM and M0-CM revealed that 5 proteins related to anti-inflammation and/or osteochondrogenesis were enriched in M2-CM. Of these proteins, the Wnt signal antagonist, secreted frizzled-related protein 1 (sFRP1), was the most abundant and played an essential role in the shift to anabolic chondrocytes, suggesting that M2 ameliorated TMJOA partly through sFRP1. This study suggests that secretome from SHED exerted remarkable osteochondral regeneration activities in TMJOA through the induction of sFRP1-expressing tissue-repair M2 macrophages., (© The Author(s) 2024. Published by Oxford University Press.)

Objective: Spermidine (SPD) is an anti-aging natural substance, and it exerts effects through anti-apoptosis and anti-inflammation. However, the specific protective mechanism of SPD in osteoarthritis (OA) remains unclear. Here, we explored the role of SPD on the articular cartilage and the synovial tissue, and tested whether the drug would regulate the polarization of synovial macrophages by in vivo and in vitro experiments., Methods: By constructing an OA model in mice, we preliminarily explored the protective effect of SPD on the articular cartilage and the synovial tissue. Meanwhile, we isolated and cultured human primary chondrocytes and bone marrow-derived macrophages (BMDMs), and prepared a conditioned medium (CM) to explore the specific protective effect of SPD in vitro., Results: We found that SPD alleviated cartilage degeneration and synovitis, increased M2 polarization and decreased M1 polarization in synovial macrophages. In vitro experiments, SPD inhibited ERK MAPK and p65/NF-κB signaling in macrophages, and transformed macrophages from M1 to M2 subtypes. Interestingly, SPD had no direct protective effect on chondrocytes in vitro; however, the conditioned medium (CM) from M1 macrophages treated with SPD promoted the anabolism and inhibited the catabolism of chondrocytes. Moreover, this CM markedly suppressed IL-1β-induced p38/JNK MAPK signaling pathway activation in chondrocytes., Conclusions: This work provides new perspectives on the role of SPD in OA. SPD does not directly target chondrocytes, but can ameliorate the degradation of articular cartilage through regulating M1/M2 polarization of synovial macrophages. Hence, SPD is expected to be the potential therapy for OA., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: Adrenocortical carcinoma (ACC) affects approx. 2 in 1,000,000 individuals in the USA, and is more common in females than males. Adrenocortical carcinoma often presents with severe symptoms, such as abdominal pain, high blood pressure, acne, hair overgrowth, and voice deepening., Objectives: Research on ACC constitutes a large body of published data. There is an increased need for easy access to ACC-derived biological material. Moreover, there are limited numbers of human cell lines available. For this reason, we attempted to differentiate human induced pluripotent stem cells (hiPSCs) into adrenocortical-like cells to establish a new functional cell line., Material and Methods: We conducted a long-term differentiation process (35 and 70 days) in the presence of growth factors (GFs), forskolin and conditioned medium collected from the human adrenal carcinoma (HAC15) cell line. Then, we analyzed the gene expression profile of the differentiated cells., Results: The obtained cells possess features characteristic of all 3 primary germ layers. Interestingly, the differentiated cells demonstrated an extremely high level of gene expression for those involved in endocrine processes, namely glycoprotein hormones, alpha polypeptide (CGA), insulin receptor substrate 4 (IRS4), and pancreatic progenitor cell differentiation and proliferation factor-like protein (PPDPFL)., Conclusions: The results of the study indicate that we obtained progenitors derived from endoderm with some characteristics of pancreatic-like cells. The endodermal derivative differentiation is a very challenging and complicated process; thus, the results presented in this study deserve closer consideration.

Background: Identification of novel cell-based therapy sources has been of great interest in recent years to provide alternative and available therapy options in clinics. Conditioned medium (CM) can be a valuable supply for growth factors, cytokines and chemokines as a source of stem cell secretome. Exploring the role of new CM sources for tissue regeneration might be a promising approach for therapeutic purposes., Methods and Results: In the current study, neuromesodermal progenitors (NMPs) derived from induced pluripotent stem cells (iPSCs) were used to collect CM. Fibroblast derived iPSCs were successfully differentiated into NMPs and NMPs were characterized by double positive T/Bra and Sox2 staining. CM was collected from NMPs, and the content was characterized by membrane analysis. In vitro wound healing assay was used as a model system to observe potential activity of CM on cell migration. Fibroblasts, keratinocytes and endothelial cells were used to evaluate the effect of NMP-derived CM (NMP-CM) on cell migration in vitro. Several important proteins related to wound healing such as ANGPT 1, ANGPT 2, MCP-1, PDGF-AA, SDF-1α, TIMP-1 and TIMP-2 were increased in NMP-CM. NMP-CM increased cell proliferation and migration in vitro., Conclusions: In vitro data obtained from three distinct cell types suggest a promising role of NMP-CM on cell migration. NMP-CM can be used for wound management in the further future after detailed in vitro and in vivo research., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Background: The formation and accumulation of cholesterol crystals (CC) at the lesion site is a hallmark of atherosclerosis. Although studies have shown the importance of vascular smooth muscle cells (VSMCs) in the disease atherosclerosis, little is known about the molecular mechanism behind the uptake of CC in VSMCs and their role in modulating immune response., Methods: Human aortic smooth muscle cells were cultured and treated with CC. CC uptake and CC mediated signaling pathway and protein induction were studied using flow cytometry, confocal microscopy, western blot and Olink proteomics. Conditioned medium from CC treated VSMCs was used to study neutrophil adhesion, ROS production and phagocytosis. Neutrophil extracellular traps (NETs) formations were visualized using confocal microscopy., Results: VSMCs and macrophages were found around CC clefts in human carotid plaques. CC uptake in VSMCs are largely through micropinocytosis and phagocytosis via PI3K-AkT dependent pathway. The uptake of CC in VSMCs induce the release inflammatory proteins, including IL-33, an alarming cytokine. Conditioned medium from CC treated VSMCs can induce neutrophil adhesion, neutrophil reactive oxygen species (ROS) and neutrophil extracellular traps (NETs) formation. IL-33 neutralization in conditioned medium from CC treated VSMCs inhibited neutrophil ROS production and NETs formation., Conclusion: We demonstrate that VSMCs due to its vicinity to CC clefts in human atherosclerotic lesion can modulate local immune response and we further reveal that the interaction between CC and VSMCs impart an inflammatory milieu in the atherosclerotic microenvironment by promoting IL-33 dependent neutrophil influx and NETs formation., (© 2024. The Author(s).)

Background: A significant unmet need in inflammatory bowel disease is the lack of anti-fibrotic agents targeting intestinal fibrosis. This study aimed to investigate the anti-fibrogenic properties and mechanisms of the conditioned medium (CM) from human umbilical cord/placenta-derived mesenchymal stem cells (UC/PL-MSC-CM) in a murine intestinal fibrosis model and human primary intestinal myofibroblasts (HIMFs)., Methods: UC/PL-MSC-CM was concentrated 15-fold using a 3 kDa cut-off filter. C57BL/6 mice aged 7 weeks old were randomly assigned to one of four groups: (1) control, (2) dextran sulfate sodium (DSS), (3) DSS + CM (late-phase treatment), and (4) DSS + CM (early-phase treatment). Chronic DSS colitis and intestinal fibrosis was induced by three cycles of DSS administration. One DSS cycle consisted of 7 days of oral DSS administration (1.75%, 2%, and 2.5% DSS), followed by 14 days of drinking water. UC/PL-MSC-CM was intraperitoneally administered in the late phase (from day 50, 10 times) or early phase (from day 29, 10 times) of DSS cycles. HIMFs were treated with TGF-β1 and co-treated with UC/PL-MSC-CM (10% of culture media) in the cellular model., Results: In the animal study, UC/PL-MSC-CM reduced submucosa/muscularis propria thickness and collagen deposition, which improved intestinal fibrosis in chronic DSS colitis. The UC/PL-MSC-CM significantly reduced the expressions of procollagen1A1 and α-smooth muscle actin, which DSS significantly elevated. The anti-fibrogenic effect was more apparent in the UC-MSC-CM or early-phase treatment model. The UC/PL-MSC-CM reduced procollagen1A1, fibronectin, and α-smooth muscle actin expression in HIMFs in the cellular model. The UC/PL-MSC-CM downregulated fibrogenesis by suppressing RhoA, MRTF-A, and SRF expression., Conclusions: Human UC/PL-MSC-CM inhibits TGF-β1-induced fibrogenic activation in HIMFs by blocking the Rho/MRTF/SRF pathway and chronic DSS colitis-induced intestinal fibrosis. Thus, it may be regarded as a novel candidate for stem cell-based therapy of intestinal fibrosis., (© 2024. The Author(s).)

The multidrug-resistant pathogen Pseudomonas aeruginosa is a common nosocomial respiratory pathogen that continues to threaten the lives of patients with mechanical ventilation in intensive care units and those with underlying comorbidities such as cystic fibrosis or chronic obstructive pulmonary disease. For over 20 years, studies have repeatedly demonstrated that the major siderophore pyoverdine is an important virulence factor for P. aeruginosa in invertebrate and mammalian hosts in vivo . Despite its physiological significance, an in vitro , mammalian cell culture model that can be used to characterize the impact and molecular mechanisms of pyoverdine-mediated virulence has only been developed very recently. In this study, we adapt a previously-established, murine macrophage-based model to use human bronchial epithelial (16HBE) cells. We demonstrate that conditioned medium from P. aeruginosa induced rapid 16HBE cell death through the pyoverdine-dependent secretion of cytotoxic rhamnolipids. Genetic or chemical disruption of pyoverdine biosynthesis decreased rhamnolipid production and mitigated cell death. Consistent with these observations, chemical depletion of lipids or genetic disruption of rhamnolipid biosynthesis abrogated the toxicity of the conditioned medium. Furthermore, we also examine the effects of exposure to purified pyoverdine on 16HBE cells. While pyoverdine accumulated within cells, it was largely sequestered within early endosomes, resulting in minimal cytotoxicity. More membrane-permeable iron chelators, such as the siderophore pyochelin, decreased epithelial cell viability and upregulated several pro-inflammatory genes. However, pyoverdine potentiated these iron chelators in activating pro-inflammatory pathways. Altogether, these findings suggest that the siderophores pyoverdine and pyochelin play distinct roles in virulence during acute P. aeruginosa lung infection., Importance: Multidrug-resistant Pseudomonas aeruginosa is a versatile bacterium that frequently causes lung infections. This pathogen is life-threatening to mechanically-ventilated patients in intensive care units and is a debilitating burden for individuals with cystic fibrosis. However, the role of P. aeruginosa virulence factors and their regulation during infection are not fully understood. Previous murine lung infection studies have demonstrated that the production of siderophores (e.g., pyoverdine and pyochelin) is necessary for full P. aeruginosa virulence. In this report, we provide further mechanistic insight into this phenomenon. We characterize distinct and novel ways these siderophores contribute to virulence using an in vitro human lung epithelial cell culture model., Competing Interests: The authors declare no conflict of interest.

The blood-brain barrier (BBB) is mainly composed of specialized endothelial cells, which can resist harmful substances, transport nutrients, and maintain the stability of the brain environment. In this study, an endothelial cell line from tilapia (Oreochromis niloticus) named TVEC-01 was successfully established. During the earlier establishment phase of the cell line, the TVEC-01 cells were persistently exposed to an astrocyte-conditioned medium (ACM). TVEC-01 cells were identified as an endothelial cell line. TVEC-01 cells retained the multiple functions of endothelial cells and were capable of performing various experiments in vitro. Furthermore, TVEC-01 cells efficiently expressed BBB-related tight junctions and key efflux transporters. From the results of the qRT-PCR, we found that the TVEC-01 cell line did not gradually lose BBB characteristics after persistent and repetitive passages, which was different from the vast majority of immortalized endothelial cells. The results showed that ACM induced up-regulation of the expression levels of multiple BBB-related genes in TVEC-01 cells. We confirmed that Streptococcus agalactiae was capable of invading the TVEC-01 cells and initiating a series of immune responses, which provided a theoretical basis for S. agalactiae to break through the BBB of teleost through the transcellular traversal pathway. In summary, we have successfully constructed an endothelial cell line of teleost, named TVEC-01, which can be used in many experiments in vitro and even for constructing BBB in vitro. Moreover, it was confirmed that S. agalactiae broke through the BBB of teleost through the transcellular traversal pathway and caused meningitis., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Osteoimmunology has uncovered the critical role of the immune microenvironment in the bone healing process, with macrophages playing a central part in generating immune responses via chemokine production. Naringin, a flavanone glycoside extracted from various plants, has been shown to promote osteoblast differentiation, thereby enhancing bone formation and mitigating osteoporosis progression. Current research on the osteogenic mechanism primarily focuses on the direct impact of naringin on mesenchymal stem cells, while its indirect immunoregulatory effects remain elusive. In this study, we investigated the bone defect-enhancing effects of varying naringin concentrations in vivo using a cranial bone defect model in Sprague-Dawley rats. We assessed the osteoimmune modulation capacity of naringin by exposing lipopolysaccharide (LPS)-induced RAW 264.7 macrophages to different doses of naringin. To further elucidate the underlying osteogenic enhancement mechanism, Bone Marrow Stromal Cells (BMSCs) derived from mice were treated with conditioned media from naringin-treated macrophages. Our findings indicated that naringin promotes M2 phenotype polarization in macrophages, as evidenced by the downregulation of pro-inflammatory cytokines Inducible Nitric Oxide Synthase (iNOS), interleukin (IL)-1β, and Tumor Necrosis Factor (TNF)-α, and the upregulation of anti-inflammatory cytokine Transforming growth factor (TGF)-β. Transcriptome analysis revealed that differentially expressed genes were significantly enriched in osteoblast differentiation and anti-inflammatory response pathways in naringin-pretreated macrophages, with the cytokines signaling pathway being upregulated. The conditioned media from naringin-treated macrophages stimulated the expression of osteogenic-related genes Alkaline phosphatase (Alp), osteocalcin (Ocn), osteopontin (Opn), and Runt-related transcription factor (Runx) 2, as well as protein expression in BMSCs. In conclusion, naringin alleviates macrophage inflammation by promoting M2 phenotype polarization, which in turn enhances the osteogenic differentiation of BMSCs, contributing to its bone healing effects in vivo. These results suggest that naringin holds significant potential for improving bone defect healing through osteoimmune modulation., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Objective: Human gingival epithelial cells (HGECs) function as a mechanical barrier against invasion by pathogenic organisms through epithelial cell-cell junction complexes, which are complex components of integrin. Integrins play an important role in the protective functions of HGECs. Human periodontal ligament (HPL) cells regulate periodontal homeostasis. However, periodontitis results in the loss of HPL cells. Therefore, as replenishment, HPL cells or mesenchymal stem cells (MSCs) can be transplanted. Herein, HPL cells and MSCs were used to elucidate the regulatory mechanisms of HGECs, assuming periodontal tissue homeostasis., Methods: Human gingival fibroblasts (HGFs), HGECs, HPL cells, and MSCs were cultured, and the conditioned medium was collected. With or without silencing periostin mRNA, HGECs were cultured under normal conditions or in a conditioned medium. Integrin and periostin mRNA expression was determined using real-time polymerase chain reaction. Integrin protein expression was analyzed using flow cytometry, and periostin protein expression was determined via western blotting., Results: The conditioned medium affected integrin expression in HGECs. Higher expression of periostin was observed in MSCs and HPL cells than in HGFs. The conditioned medium that contained periostin protein regulated integrin expression in HGECs. After silencing periostin in MSCs and HPL cells, periostin protein was not detected in the conditioned medium, and integrin expression in HGECs remained unaffected., Conclusions: Integrins in HGECs are regulated by periostin secreted from HPL cells and MSCs. This result suggests that periostin maintains gingival cell adhesion and regulates bacterial invasion/infection. Therefore, the functional regulation of periostin-secreting cells is important in preventing periodontitis., Competing Interests: Declaration of competing interest All authors are required to disclose any COI within the period of 12 months prior to the submission of any manuscript in the subject matter of which any company, entity, or organization has an interest., (Copyright © 2023 Japanese Association for Oral Biology. Published by Elsevier B.V. All rights reserved.)

Objective: This study explores the mechanism of action of Danhongqing formula (DHQ), a compound-based Chinese medicine formula, in the treatment of cholestatic liver fibrosis., Methods: In vivo experiments were conducted using 8-week-old multidrug resistance protein 2 knockout (Mdr2 -/- ) mice as an animal model of cholestatic liver fibrosis. DHQ was administered orally for 8 weeks, and its impact on cholestatic liver fibrosis was evaluated by assessing liver function, liver histopathology, and the expression of liver fibrosis-related proteins. Real-time polymerase chain reaction, Western blot, immunohistochemistry and other methods were used to observe the effects of DHQ on long non-coding RNA H19 (H19) and signal transducer and activator of transcription 3 (STAT3) phosphorylation in the liver tissue of Mdr2 -/- mice. In addition, cholangiocytes and hepatic stellate cells (HSCs) were cultured in vitro to measure the effects of bile acids on cholangiocyte injury and H19 expression. Cholangiocytes overexpressing H19 were constructed, and a conditioned medium containing H19 was collected to measure its effects on STAT3 protein expression and cell activation. The intervention effect of DHQ on these processes was also investigated. HSCs overexpressing H19 were constructed to measure the impact of H19 on cell activation and assess the intervention effect of DHQ., Results: DHQ alleviated liver injury, ductular reaction, and fibrosis in Mdr2 -/- mice, and inhibited H19 expression, STAT3 expression and STAT3 phosphorylation. This formula also reduced hydrophobic bile acid-induced cholangiocyte injury and the upregulation of H19, inhibited the activation of HSCs induced by cholangiocyte-derived conditioned medium, and decreased the expression of activation markers in HSCs. The overexpression of H19 in a human HSC line confirmed that H19 promoted STAT3 phosphorylation and HSC activation, and DHQ was able to successfully inhibit these effects., Conclusion: DHQ effectively alleviated spontaneous cholestatic liver fibrosis in Mdr2 -/- mice by inhibiting H19 upregulation in cholangiocytes and preventing the inhibition of STAT3 phosphorylation in HSC, thereby suppressing cell activation. Please cite this article as: Li M, Zhou Y, Zhu H, Xu LM, Ping J. Danhongqing formula alleviates cholestatic liver fibrosis by downregulating long non-coding RNA H19 derived from cholangiocytes and inhibiting hepatic stellate cell activation. J Integr Med. 2024; 22(2): 188-198., Competing Interests: Declaration of competing interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Shanghai Yueyang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine. Published by Elsevier B.V. All rights reserved.)

Mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) have shown great promise for regeneration and immunomodulation. However, efficient and scalable methods for their preparation are still lacking. In this study, we present the adoption of a label-free technique known as "EXODUS" to isolate and purify MSC-EVs from the conditioned medium. Our findings indicate that EXODUS can rapidly isolate EVs from 10 mL of conditioned medium with a 5-fold higher yield compared to conventional approaches, including ultracentrifugation (UC) and polyethylene glycol precipitation (PEG) methods. Additionally, pre-storing the conditioned medium at 4°C for 1 week resulted in a ~2-fold higher yield of MSC-EVs compared to the freshly prepared medium. However, storing the purified EV particles at 4°C for 1 month led to a 2-fold reduction in particle concentration. Furthermore, we found that MSC-EVs isolated using EXODUS exhibit higher expression levels of EV markers such as Alix, Flotillin1, CD81, and TSG101 in comparison to PEG and UC methods. We also discovered that MSC-EVs isolated using EXODUS are enriched in response to cytokine, collagen-containing extracellular matrix, and calcium ion binding compared to PEG method and enriched in extracellular structure organization, extracellular matrix, and extracellular matrix structure constituents compared to UC. Finally, we demonstrated that MSC-EVs isolated using EXODUS exhibit greater potential in animal organ development, tissue development, and anatomical structure morphogenesis compared to the UC. These findings suggest that EXODUS is a suitable method for the large-scale preparation of high-quality MSC-EVs for various clinical applications., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH, DE part of Springer Nature.)

Background: Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by repetitive behaviors, a limited range of activities, and deficiencies in social communications. Bone marrow mesenchymal stem cells (BM-MSCs), which secrete factors that stimulate surrounding microenvironment, and BM-MSCs conditioned medium (BM-MSCs-CM), which contains cell-secreted products, have been speculated to hold potential as a therapy for ASD. This study aimed to compare the therapeutic effects of BM-MSCs and BM-MSCs-CM on behavioral and microglial changes in an animal model of autism induced by valproic acid (VPA)., Methods and Results: Pregnant Wistar rats were administered by VPA at a dose of 600 mg/kg at 12.5 days post-conception. After birth, male pups were included in the study. At 6 weeks of age, one group of rats received intranasal administration of BM-MSCs, while another group received BM-MSCs-CM. The rats were allowed to recover for 2 weeks. Behavioral tests, quantitative real-time polymerase chain reaction (qRT-PCR), and immunohistochemistry were performed. Both BM-MSCs and BM-MSCs-CM administration significantly improved some behavioral deficits. Furthermore, these treatments notably reduced Iba-1 marker associated with microgliosis. Additionally, there was a significant reduction in the expression of pro-inflammatory cytokines IL-1β and IL-6, and an increase in the levels of the anti-inflammatory cytokine IL-10 in rats administered by BM-MSCs and BM-MSCs-CM., Conclusions: Post-developmental administration of BM-MSCs and BM-MSCs-CM can ameliorate prenatal neurodevelopmental deficits, restore cognitive and social behaviors, and modulate microglial and inflammatory markers. Results indicated that the improvement rate was higher in the BM-MSCs group than BM-MSCs-CM group., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Embryonic transfer of bovine blastocysts produced using in vitro fertilization (IVF) is widely used, although the challenge of compromised conception rates remains. Using bovine oviduct epithelial cells (BOEC) to improve embryo culture conditions has attracted attention, particularly since the recent discovery of extracellular vesicles from BOEC. The selection of embryos for transfer has also been the subject of various studies, and a set of evaluation criteria to predict pregnancy success has been suggested, in which the embryos are judged by their kinetics and morphology at the early stages. In the present study, we established a spontaneously immortalized BOEC line (SI-BOEC) and examined the effects of conditioned medium on IVF embryos, focusing on the results of the recommended criteria. A modified KSOM (mKSOM) was used to prepare conditioned media. Presumptive zygotes were cultured in mKSOM (control), SI-BOEC-conditioned medium, mKSOM supplemented with sediment (pellet) collected after the ultracentrifugation of the conditioned medium (mKSOM/sediment), and the supernatant. A significantly higher percentage of embryos satisfied the recommended criteria when grown in the conditioned medium than in the mKSOM. A higher proportion of embryos developed into blastocysts after achieving the four criteria. A similar tendency was observed when grown in mKSOM/sediment compared to mKSOM; however, this was not observed in the supernatant. Vesicles with a size similar to that of exosomes were observed in the sediment. In conclusion, the culture medium conditioned by SI-BOEC promoted the production of bovine blastocysts that satisfied the four evaluation criteria recommended for embryo selection.

Lipofilling is an option for breast reconstruction after tumor resection to avoid the complications of an implant-based reconstruction. Although some concerns exist regarding the oncological safety of tissue rich in mesenchymal stem cells with their proangiogenic and proliferation-supportive properties, there are also reports that adipose-tissue-derived stem cells can exhibit antitumoral properties. We isolated primary adipose-tissue-derived stem cells. Both conditioned medium and exosomes were harvested from the cell culture and used to treat the breast cancer cell line MCF-7. Cell viability, cytotoxicity, and gene expression of MCF-7 cells in response to the indirect co-culture were evaluated. MCF-7 cells incubated with exosomes from adipose-tissue-derived stem cells show reduced cell viability in comparison to MCF-7 cells incubated with adipose-tissue-derived stem-cell-conditioned medium. Expression of proapoptotic genes was upregulated, and expression of antiapoptotic genes was downregulated. The debate about the oncological safety of autologous fat grafting after tumor resection continues. Here, we show that exosomes from adipose-tissue-derived stem cells exhibit some antitumoral properties on breast cancer cell line MCF-7.

Introduction: Loss of NADPH oxidase activity results in proinflammatory macrophages that contribute to hyperinflammation in Chronic Granulomatous Disease (CGD). Previously, it was shown in a zymosan-induced peritonitis model that gp91 phox-/- (CGD) monocyte-derived macrophages (MoMacs) fail to phenotypically mature into pro-resolving MoMacs characteristic of wild type (WT) but retain the ability to do so when placed in the WT milieu. Accordingly, it was hypothesized that soluble factor(s) in the CGD milieu thwart appropriate programming., Methods: We sought to identify key constituents using ex vivo culture of peritoneal inflammatory leukocytes and their conditioned media. MoMac phenotyping was performed via flow cytometry, measurement of efferocytic capacity and multiplex analysis of secreted cytokines. Addition of exogenous TNFα, TNFα neutralizing antibody and TNFR1-/- MoMacs were used to study the role of TNFα: TNFR1 signaling in MoMac maturation., Results: More extensive phenotyping defined normal MoMac maturation and demonstrated failure of maturation of CGD MoMacs both ex vivo and in vivo . Protein components, and specifically TNFα, produced and released by CGD neutrophils and MoMacs into conditioned media was identified as critical to preventing maturation. Exogenous addition of TNFα inhibited WT MoMac maturation, and its neutralization allowed maturation of cultured CGD MoMacs. TNFα neutralization also reduced production of IL-1β, IL-6 and CXCL1 by CGD cells though these cytokines played no role in MoMac programming. MoMacs lacking TNFR1 matured more normally in the CGD milieu both ex vivo and following adoptive transfer in vivo ., Discussion: These data lend mechanistic insights into the utility of TNFα blockade in CGD and to other diseases where such therapy has been shown to be beneficial., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Gibbings, Haist, Redente, Henson and Bratton.)

Osteonecrosis of the femoral head (ONFH) is a type of ischemic osteonecrosis that causes pain, loss of function, and femoral head collapse. Here, we analyzed samples of femoral heads excised from patients with ONFH to clarify the relationship between ischemic osteonecrosis and cellular senescence. X-gal staining was strong and p16INK4a-positive cells were abundant in the transitional region of ONFH. The β-galactosidase-positive cells in the transitional region were also positive for nestin, periostin, or DMP-1. In contrast, no β-galactosidase-positive cells were detected in the healthy region. The senescence-associated p16INK4a, p21, and p53 were upregulated in ONFH tissue. We also examined and analyzed a mouse ischemic femoral osteonecrosis model in vivo to verify the association between ONFH and cellular senescence. Human mesenchymal stem cell-conditioned medium (MSC-CM) was administered to determine its therapeutic efficacy against cellular senescence and bone collapse. MSC-CM reduced the number of senescent cells and downregulated the aforementioned senescence-related genes. It also decreased the number of empty lacunae 4 weeks after ischemia induction and promoted bone formation. At 6 weeks post-surgery, MSC-CM increased the trabecular bone volume, thereby suppressing bone collapse. We conclude that cellular senescence is associated with ONFH and that MSC-CM suppresses bone collapse in this disorder., (© 2024. The Author(s).)

Background: Pericytes are a vital component of the blood-brain barrier, and their involvement in acute inflammation was recently suggested. However, it remains unclear whether pericytes contribute to hypothalamic chronic inflammation and energy metabolism in obesity. The present study investigated the impact of pericytes on the pathophysiology of obesity by focusing on platelet-derived growth factor (PDGF) signaling, which regulates pericyte functions., Methods: Tamoxifen-inducible systemic conditional PDGF receptor β knockout mice (Pdgfrb ∆SYS -KO) and Calcium/calmodulin-dependent protein kinase type IIa (CaMKIIa)-positive neuron-specific PDGF receptor β knockout mice (Pdgfrb ∆CaMKII -KO) were fed a high-fat diet, and metabolic phenotypes before and 3 to 4 weeks after dietary loading were examined. Intracellular energy metabolism and relevant signal transduction in lipopolysaccharide- and/or platelet-derived growth factor-BB (PDGF-BB)-stimulated human brain pericytes (HBPCs) were assessed by the Seahorse XFe24 Analyzer and Western blotting. The pericyte secretome in conditioned medium from HBPCs was studied using cytokine array kit, and its impact on polarization was examined in bone marrow-derived macrophages (BMDMs), which are microglia-like cells., Results: Energy consumption increased and body weight gain decreased after high-fat diet loading in Pdgfrb ∆SYS -KO mice. Cellular oncogene fos (cFos) expression increased in proopiomelanocortin (POMC) neurons, whereas microglial numbers and inflammatory gene expression decreased in the hypothalamus of Pdgfrb ∆SYS -KO mice. No significant changes were observed in Pdgfrb ∆CaMKII -KO mice. In HBPCs, a co-stimulation with lipopolysaccharide and PDGF-BB shifted intracellular metabolism towards glycolysis, activated mitogen-activated protein kinase (MAPK), and modulated the secretome to the inflammatory phenotype. Consequently, the secretome showed an increase in various proinflammatory chemokines and growth factors including Epithelial-derived neutrophil-activating peptide 78 (C-X-C motif chemokine ligand (CXCL)5), Thymus and activation-regulated chemokine (C-C motif chemokine (CCL)17), Monocyte chemoattractant protein 1 (CCL2), and Growth-regulated oncogene α (CXCL1). Furthermore, conditioned medium from HBPCs stimulated the inflammatory priming of BMDMs, and this change was abolished by the C-X-C motif chemokine receptor (CXCR) inhibitor. Consistently, mRNA expression of CXCL5 was elevated by lipopolysaccharide and PDGF-BB treatment in HBPCs, and the expression was significantly lower in the hypothalamus of Pdgfrb ∆SYS -KO mice than in control Pdgfrb flox/flox mice (FL) following 4 weeks of HFD feeding., Conclusions: PDGF receptor β signaling in hypothalamic pericytes promotes polarization of macrophages by changing their secretome and contributes to the progression of obesity., (© 2024. The Author(s).)

Aim: To investigate the effect of IWP-2, Wnt inhibitor, on human dental pulp stem cells (hDPSCs) responses., Methodology: hDPSCs were isolated from human dental pulp tissues. Cells were treated with 25 μM IWP-2 for 24 h, and subsequently, the gene expression profile was examined using high-throughput RNA sequencing. The mRNA expression was analysed using qPCR. The effect of IWP-2 was investigated in both normal and LPS-induced hDPSCs (inflamed hDPSCs). CD4 + T cells and CD14 + monocyte-derived macrophages were cultured with conditioned media of IWP-2 treated hDPSCs to observe the immunosuppressive property., Results: RNA sequencing indicated that IWP-2 significantly downregulated several KEGG pathways, including cytokine-cytokine receptor interaction, IL-17 signalling pathway, and TNF signalling pathway. In both normal and inflamed conditions, IWP-2 markedly upregulated TGFB1 mRNA expression while the mRNA expression of pro-inflammatory cytokines, TNFA, IL1B, IFNG, and IL6, was inhibited. In the inhibition experiment, the pretreatment with p38, MAPK, or PI3K inhibitors abolished the effects of IWP-2 in LPS-induced inflammation. In terms of immune cells, IWP-2-treated-inflamed hDPSCs conditioned media attenuated T cell proliferation and regulated regulatory T cell differentiation. In addition, the migratory property of macrophage was decreased after being exposed to IWP-2-treated inflamed hDPSCs conditioned media., Conclusion: IWP-2 suppressed inflammatory cytokine expression in both normal and inflamed hDPSCs. Moreover, hDPSCs exerted the immunosuppressive property after IWP-2 treatment. These results suggest the role of Wnt in inflammatory responses and immunomodulation in dental pulp tissues., (© 2023 British Endodontic Society. Published by John Wiley & Sons Ltd.)

Insufficient healing of aneurysms following treatment with vascular occlusion devices put patients at severe risk of fatal rupture. Therefore, promoting healing and not just occlusion is vital to enhance aneurysm healing. Following occlusion device implantation, healing is primarily orchestrated by macrophage immune cells, ending with fibroblasts depositing collagen to stabilize the aneurysm neck and dome, preventing rupture. Several modified occlusion devices are available currently on-market. Previous in vivo work demonstrated that modifications of occlusion devices with a shape memory polymer foam had enhanced aneurysm healing outcomes. To better understand cellular response to occlusion devices and improve aneurysm occlusion device design variables, we developed an in vitro assay to isolate prominent interactions between devices and key healing players: macrophages and fibroblasts. We used THP-1 monocyte derived macrophages and human dermal fibroblasts in our cell culture models. Macrophages were allowed device contact with on-market competitor aneurysm occlusion devices for up to 96 h, to allow for any spontaneous device-driven macrophage activation. Macrophage secreted factors were captured in the culture media, in response to device-specific activation. Fibroblasts were then exposed to device-conditioned macrophage media (with secreted factors alone), to determine if there were any device-induced changes in collagen secretion. Our in vitro studies were designed to test the direct effect of devices on macrophage activation, and the indirect effect of devices on collagen secretion by fibroblasts to promote aneurysm healing and stabilization. Over 96 h, macrophages displayed significant migration toward and interaction with all tested devices. As compared to other devices, shape memory polymer foams (SMM, Shape Memory Medical) induced significant changes in gene expression indicating a shift toward an anti-inflammatory pro-healing M2-like phenotype. Similarly, macrophages in contact with SMM devices secreted more vascular endothelial growth factor (VEGF) compared with other devices. Macrophage conditioned media from SMM-contacted macrophages actively promoted fibroblast secretion of collagen, comparable to amounts observed with exogenous stimulation via VEGF supplementation. Our data indicate that SMM devices may promote good aneurysm healing outcomes, because collagen production is an essential step to ultimately stabilize an aneurysm., (© 2024 Wiley Periodicals LLC.)

Objectives: Secretomes of mesenchymal stromal cells (MSC) represent a novel strategy for growth-factor delivery for tissue regeneration. The objective of this study was to compare the efficacy of adjunctive use of conditioned media of bone-marrow MSC (MSC-CM) with collagen barrier membranes vs. adjunctive use of conditioned media of leukocyte- and platelet-rich fibrin (PRF-CM), a current growth-factor therapy, for guided bone regeneration (GBR)., Methods: MSC-CM and PRF-CM prepared from healthy human donors were subjected to proteomic analysis using mass spectrometry and multiplex immunoassay. Collagen membranes functionalized with MSC-CM or PRF-CM were applied on critical-size rat calvaria defects and new bone formation was assessed via three-dimensional (3D) micro-CT analysis of total defect volume (2 and 4 weeks) and 2D histomorphometric analysis of central defect regions (4 weeks)., Results: While both MSC-CM and PRF-CM revealed several bone-related proteins, differentially expressed proteins, especially extracellular matrix components, were increased in MSC-CM. In rat calvaria defects, micro-CT revealed greater total bone coverage in the MSC-CM group after 2 and 4 weeks. Histologically, both groups showed a combination of regular new bone and 'hybrid' new bone, which was formed within the membrane compartment and characterized by incorporation of mineralized collagen fibers. Histomorphometry in central defect sections revealed greater hybrid bone area in the MSC-CM group, while the total new bone area was similar between groups., Conclusion: Based on the in vitro and in vivo investigations herein, functionalization of membranes with MSC-CM represents a promising strategy to enhance GBR., (© 2023 The Authors. Clinical Oral Implants Research published by John Wiley & Sons Ltd.)

Introduction: Liposarcoma constitutes a prevalent subtype of soft tissue sarcoma, represents approximately 20% of all sarcomas. However, conventional chemotherapeutic agents have shown restricted effectiveness in treating liposarcoma patients. Accumulating evidence indicates that mesenchymal stem cells (MSCs) have the characteristic of migration to tumor site, promote or suppress tumors. How human bone marrow mesenchymal stem cells (BMSCs) contribute to liposarcoma phenotype remains poorly understood. This study aims to investigate the effects of human bone marrow mesenchymal stem cell-conditioned medium (BMSC-CM) on the proliferation and migration of liposarcoma cell lines 93T449 and SW872, as well as explore potential underlying mechanisms of BMSC-CM action on these cells., Materials and Methods: We transfected BMSCs with lentiviral constructs to knock down the transcriptional co-activator Yes-associated protein 1 (YAP1), conditioned medium (CM) obtained from BMSCs and shYAP1-BMSC, respectively. Liposarcoma cell lines 93T449 and SW872 were co-cultured with BMSC-CM or shYAP1-BMSC-CM. Cell proliferation ability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cell apoptosis was evaluated using flow cytometric assay. A wound healing assay was used to analyze cell migration. The expression levels of YAP1, Bcl-2, and matrix metalloproteinase-2 (MMP-2) were determined by western blot assay., Results: Co-culturing liposarcoma cell lines 93T449 and SW872 with BMSC-CM promoted tumor cell proliferation, while shYAP1-BMSC-CM significantly inhibited cell viability and migration, induced apoptosis, and downregulated Bcl-2 and MMP-2 expression., Conclusions: These findings provide new insights into the impact of BMSC-CM on liposarcoma and suggest its possible involvement in liposarcoma cell growth.

Background: Sesamol (SEM), a natural lignan compound isolated from sesame, has strong anti-oxidant property, regulating lipid metabolism, decreasing cholesterol and hepatoprotection. However, its anti-hepatic fibrosis effect and mechanisms have not been comprehensively elucidated., Hypothesis/purpose: This study aims to investigate the anti-hepatic fibrosis of SEM and its underlying mechanisms., Method: C57BL/6 mice with hepatic fibrosis were induced by TAA, then administrated with SEM or curcumin, respectively. HSCs were stimulated by TGF-β or conditioned medium, and then cultured with SEM, GW4064, GW3965, Rapamycin (RA) or 3-methyladenine (3-MA), respectively. Mice with hepatic fibrosis also were administrated with SEM, RA or 3-MA to estimate the effect of SEM on autophagy., Results: In vitro, SEM significantly inhibited extracellular matrix deposition, P2 × 7r-NLRP3, and inflammatory cytokines. SEM increased FXR and LXRα/β expressions and decreased MAPLC3α/β and P62 expressions, functioning as 3-MA (autophagy inhibitor). In vivo, SEM reduced serum transaminase, histopathology changes, fibrogenesis, autophagy markers and inflammatory cytokines caused by TAA. LX-2 were activated with conditioned medium from LPS-primed THP-1, which resulted in significant enhance of autophagy markers and inflammatory cytokines and decrease of FXR and LXRα/β expressions. SEM could reverse above these changes and function as 3-MA, GW4064, or GW3965. Deficiency of FXR or LXR attenuated the regulation of SEM on α-SMA, MAPLC3α/β, P62 and IL-1β in activated LX-2. In activated THP-1, deficiency of FXR could decrease the expression of LXR, and vice versa. Deficiency of FXR or LXR in activated MΦ decreased the expressions of FXR and LXR in activated LX-2. Deficiency FXR or LXR in activated MΦ also attenuated the regulation of SEM on α-SMA, MAPLC3α/β, P62, caspase-1 and IL-1β. In vivo, SEM significantly reversed hepatic fibrosis via FXR/LXR and autophagy., Conclusion: SEM could regulate hepatic fibrosis by inhibiting fibrogenesis, autophagy and inflammation. FXR/LXR axis-mediated inhibition of autophagy contributed to the regulation of SEM against hepatic fibrosis, especially based on involving in the crosstalk of HSCs-macrophage. SEM might be a prospective therapeutic candidate, and its mechanism would be a new direction or strategy for hepatic fibrosis treatment., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier GmbH.)

Background: Recently, the importance of the interactions between liver cancer cells and fibroblasts has been increasingly recognized; however, many details remain to be explored., Methods: In this work, we first studied their intercellular interactions using conditioned medium from mouse embryonic fibroblasts (MEFs), then through a previously established coculture model., Results: Culturing in a conditioned medium from MEFs could significantly increase the growth, migration, and invasion of liver cancer cells. The coculture model further demonstrated that a positive feedback loop was formed between transforming growth factor-β (TGF-β) from HepG2 cells and mHGF (mouse hepatocyte growth factor) from MEFs during coculture. In this feedback loop, c-Met expression in HepG2 cells was significantly increased, and its downstream signaling pathways, such as Src/FAK, PI3K/AKT, and RAF/MEK/ERK, were activated. Moreover, the proportion of activated MEFs was also increased. More importantly, the growth-promoting effects caused by the interaction of these two cell types were validated in vitro by a 3D spheroid growth assay and in vivo by a xenograft mouse model., Conclusion: Collectively, these findings provide valuable insights into the interactions between fibroblasts and liver cancer cells, which may have therapeutic implications for the treatment of liver cancer., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Several reports assume that myocardial necroptotic cell death is induced during the development of chronic heart failure. Although it is well accepted that angiotensin II induces apoptotic cell death of cardiac myocytes, the involvement of angiotensin II in the induction of myocardial necroptosis during the development of heart failure is still unknown. Therefore, we examined the role of angiotensin II in myocardial necroptosis using rat failing hearts following myocardial infarction and cultured cardiomyocytes. We found that administration of azilsartan, an angiotensin II AT 1 receptor blocker, or trandolapril, an angiotensin-converting enzyme inhibitor, to rats from the 2nd to the 8th week after myocardial infarction resulted in preservation of cardiac function and attenuation of mixed lineage kinase domain-like (MLKL) activation. Furthermore, the ratio of necroptotic cell death was increased in neonatal rat ventricular cardiomyocytes cultured with conditioned medium from rat cardiac fibroblasts in the presence of angiotensin II. This increase in necroptotic cells was attenuated by pretreatment with azilsartan. Furthermore, activated MLKL was increased in cardiomyocytes cultured in conditioned medium. Pretreatment with azilsartan also prevented the conditioned medium-induced increase in activated MLKL. These results suggest that angiotensin II contributes to the induction of myocardial necroptosis during the development of heart failure.

Mesenchymal stem/stromal cells (MSCs) hold significant therapeutic value due to their regeneration abilities, migration capacity, and immunosuppressive and immunomodulatory properties. These cells secrete soluble and insoluble factors, and this complex secretome contributes to their therapeutic effect. Furthermore, stimulation of cells by various external stimuli lead to secretome modifications that can increase the therapeutic efficacy. So, this study examined the effect of dimethyloxalylglycine (DMOG), a hypoxia-mimetic agent, on secretome profiles and exosome secretions of MSCs by evaluating conditioned medium (CM) and ultrastructural morphologies of the cells in comparison with unpreconditioned MSCs. The appropriate dose and duration of the use of DMOG were determined as 1000 μM and 24 h by evaluating the HIF-1α expression. DMOG-CM and N-CM were collected from MSCs incubated in serum-free medium with/without DMOG for 24 h, respectively. The content analysis of conditioned mediums (CMs) revealed that VEGF, NGF, and IL-4 levels were increased in DMOG-CM. Subsequently, exosomes were isolated from the CMs and were shown by transmission electron microscopy and Western blot analysis in both groups. The effects of CMs on proliferation and migration were determined by in vitro wound healing tests; both CMs increased the fibroblast's migratory and proliferative capacities. According to the ultrastructural evaluation, autophagosome, autolysosome, myelin figure, and microvesicular body structures were abundant in DMOG-preconditioned MSCs. Consistent with the high number of autophagic vacuoles, Beclin-1 expression was increased in those cells. These findings suggested that DMOG could alter MSCs' secretion profile, modify their ultrastructural morphology accordingly, and make the CM a more potent therapeutic tool. RESEARCH HIGHLIGHTS: Preconditioning mesenchymal stem/stromal cells with dimethyloxalylglycine, a hypoxia-mimetic agent, could modify cellular metabolism. Hypoxic mechanisms lead to alterations in the ultrastructural characteristics of mesenchymal stromal/stem cells. Preconditioning with dimethyloxalylglycine leads to ultrastructural and metabolic changes of mesenchymal stromal/stem cells along with modifications in their secretome profiles. Preconditioning of mesenchymal stromal/stem cells could render them a more potent therapeutic tool., (© 2023 The Authors. Microscopy Research and Technique published by Wiley Periodicals LLC.)

Introduction: Hematopoietic stem cells are cells that differentiate into blood cell types. Although the placenta secretes hormones, proteins and other factors important for maternal/fetal health, cross-talk between placental and hematopoietic stem cells is poorly understood. Moreover, toxicant impacts on placental-hematopoietic stem cell communication is understudied. The goals of this study were to determine if factors secreted from placental cells alter transcriptomic responses in hematopoietic stem cells and if monoethylhexyl phthalate (MEHP), the bioactive metabolite of the pollutant diethylhexyl phthalate, modifies these effects., Methods: We used K-562 and BeWo cells as in vitro models of hematopoietic stem cells and placental syncytiotrophoblasts, respectively. We treated K-562 cells with medium conditioned by incubation with BeWo cells, medium conditioned with BeWo cells treated with 10 μM MEHP for 24 h, or controls treated with unconditioned medium. We extracted K-562 cell RNA, performed RNA sequencing, then conducted differential gene expression and pathway analysis., Results: Relative to controls, K-562 cells treated with BeWo cell conditioned medium differentially expressed 173 genes (FDR<0.05 and fold-change>2.0), including 2.4-fold upregulatation of tropomyosin 4 (TPM4, a cytoskeletal regulator involved in processes such as cell morphology and migration) and 3.3-fold upregulatation of sphingosine-1-phosphate receptor 3 (S1PR3, a mediator of myeloid cell differentiation and inflammatory responses). Upregulated genes were enriched for pathways including stem cell maintenance, cell proliferation and immune processes. Downregulated genes were enriched for terms involved in protein translation and transcriptional regulation. MEHP treatment differentially expressed eight genes (FDR<0.05), including genes involved in lipid metabolism (e.g., Perilipin 2, fold-change: 1.4; Carnitine Palmitoyltransferase 1A, fold-change: 1.4)., Discussion: K-562 cells, a model of hematopoietic stem cells, are responsive to media conditioned by placental cells, potentially impacting pathways like stem cell maintenance., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2023. Published by Elsevier Ltd.)

Gastric cancer (GC) is one of the most aggressive tumors and has a poor prognosis. It has been demonstrated that gastric cancer mesenchymal stem cells (GC-MSCs) can promote the progression, metastasis, and chemoresistance of GC through various mechanisms, but the effect of GC-MSCs on GC during chemotherapy is still unknown. In this study, flow cytometry, CCK8 assay, migration assay, colony formation assay, and western blot were conducted. We also analyzed GC patients from the cancer genome atlas (TCGA). Our results showed that GC-MSCs were resistant to 5-FU and Taxol at the IC50 concentration for GC cells, and 5-FU could promote the migration of GC-MSCs at low doses. Furthermore, the conditioned medium of GC-MSCs pretreated with chemotherapeutic drugs was more effective in promoting the proliferation, migration, and stemness of GC cell lines than the conditioned medium of GC-MSCs without chemotherapeutic drugs treatment. These effects were dependent on the activation of phosphorylated AKT (p-AKT) in GC cell lines. Correspondingly, the inhibition of p-AKT reversed the tumor-promoting effect of the conditioned medium of GC-MSCs pretreated with chemotherapeutic drugs. Additionally, the expression of AKT1 was higher in GC tissues than in both paracancerous tissues and normal tissues, and patients resistant to chemotherapy expressed more AKT1 compared to those who were sensitive. Taken together, our data demonstrated that GC-MSCs gained more tumor-promoting abilities during chemotherapy.

Rationale: Platinum-based chemotherapy is commonly used for treating solid tumors, but drug resistance often limits its effectiveness. Cancer-associated fibroblast (CAF)-derived extracellular vesicle (EV), which carry various miRNAs, have been implicated in chemotherapy resistance. However, the molecular mechanism through which CAFs modulate cisplatin resistance in oral squamous cell carcinoma (OSCC) is not well understood. We employed two distinct primary CAF types with differential impacts on cancer progression: CAF-P, representing a more aggressive cancer-promoting category, and CAF-D, characterized by properties that moderately delay cancer progression. Consequently, we sought to investigate whether the two CAF types differentially affect cisplatin sensitivity and the underlying molecular mechanism. Methods: The secretion profile was examined by utilizing an antibody microarray with conditioned medium obtained from the co-culture of OSCC cells and two types of primary CAFs. The effect of CAF-dependent factors on cisplatin resistance was investigated by utilizing conditioned media (CM) and extracellular vesicle (EVs) derived from CAFs. The impacts of candidate genes were confirmed using gain- and loss-of-function analyses in spheroids and organoids, and a mouse xenograft. Lastly, we compared the expression pattern of the candidate genes in tissues from OSCC patients exhibiting different responses to cisplatin. Results: When OSCC cells were cultured with conditioned media (CM) from the two different CAF groups, cisplatin resistance increased only under CAF-P CM. OSCC cells specifically expressed insulin-like growth factor binding protein 3 ( IGFBP3 ) after co-culture with CAF-D. Meanwhile, IGFBP3 -knockdown OSCC cells acquired cisplatin resistance in CAF-D CM. IGFBP3 expression was promoted by GATA-binding protein 1 ( GATA1 ), a transcription factor targeted by miR-876-3p, which was enriched only in CAF-P-derived EV. Treatment with CAF-P EV carrying miR-876-3p antagomir decreased cisplatin resistance compared to control miRNA-carrying CAF-P EV. On comparing the staining intensity between cisplatin-sensitive and -insensitive tissues from OSCC patients, there was a positive correlation between IGFBP3 and GATA1 expression and cisplatin sensitivity in OSCC tissues from patients. Conclusion: These results provide insights for overcoming cisplatin resistance, especially concerning EVs within the tumor microenvironment. Furthermore, it is anticipated that the expression levels of GATA1 and miR-876-3p, along with IGFBP3 , could aid in the prediction of cisplatin resistance., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Acute lung injury (ALI) is a progressive inflammatory injury, and mesenchymal stem cells (MSCs) can be used to treat ALI. MSC-conditioned medium (MSC-CM) contains many cytokines, in which keratinocyte growth factor (KGF) is a soluble factor that plays a role in lung development. We aim to explore the protective effects of MSCs secreted KGF on ALI, and investigate the involvement of epithelial sodium channel (ENaC), which are important in alveolar fluid reabsorption. Both lipopolysaccharides (LPS)-induced mouse and alveolar organoid ALI models were established to confirm the potential therapeutic effect of MSCs secreted KGF. Meanwhile, the expression and regulation of ENaC were determined in alveolar type II epithelial (ATII) cells. The results demonstrated that MSC-CM and KGF could alleviate the extent of inflammation-related pulmonary edema in ALI mice, which was abrogated by a KGF neutralizing antibody. In an alveolar organoid ALI model, KGF in MSC-CM could improve the proliferation and decrease the differentiation of ATII cells. At the cellular level, the LPS-inhibited protein expression of ENaC could be reversed by KGF in MSC-CM. In addition, bioinformatics analysis and our experimental data provided the evidence that the NF-κB signaling pathway may be involved in the regulation of ENaC. Our research confirmed that the therapeutic effect of MSC-CM on edematous ALI was closely related to KGF, which may be involved in the proliferation and differentiation of ATII cells, as well as the upregulation of ENaC expression by the inhibition of NF-κB signaling pathway., Competing Interests: Declaration of Competing Interest The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Objectives: This study aimed to investigate the functions of 19 types of Wnt ligands during the process of osteogenic differentiation in human periodontal ligament stem cells (hPDLSCs), with particular attention to WNT3A and WNT4., Materials and Methods: The expression levels of 19 types of Wnt ligands were examined using real-time quantitative polymerase chain reaction (real-time qPCR) during hPDLSCs osteogenic differentiation at 7, 10, and 14 days. Knockdown of WNT3A and WNT4 expression was achieved using adenovirus vectors, and conditioned medium derived from WNT3A and WNT4 overexpression plasmids was employed to investigate their roles in hPDLSCs osteogenesis. Osteogenic-specific genes were analyzed using real-time qPCR. Alkaline phosphatase (ALP) and alizarin red S activities and staining were employed to assess hPDLSCs' osteogenic differentiation ability., Results: During hPDLSCs osteogenic differentiation, the expression of 19 types of Wnt ligands varied, with WNT3A and WNT4 showing significant upregulation. Inhibiting WNT3A and WNT4 expression hindered hPDLSCs' osteogenic capacity. Conditioned medium of WNT3A promoted early osteogenic differentiation, while WNT4 facilitated late osteogenesis slightly., Conclusion: Wnt ligands, particularly WNT3A and WNT4, play an important role in hPDLSCs' osteogenic differentiation, highlighting their potential as promoters of osteogenesis., Clinical Relevance: Given the challenging nature of alveolar bone regeneration, therapeutic strategies that target WNT3A and WNT4 signaling pathways offer promising opportunities. Additionally, innovative gene therapy approaches aimed at regulating of WNT3A and WNT4 expression hold potential for improving alveolar bone regeneration outcomes., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Purpose: To investigate human corneal epithelial cell and fibroblast migration and growth factor secretion after rose bengal photodynamic therapy (RB-PDT) and the effect of conditioned medium (CM)., Methods: A human corneal epithelial cell line (HCE-T), human corneal fibroblasts (HCF) and keratoconus fibroblasts (KC-HCF) have been used. Twenty-four hours after RB-PDT (0.001% RB concentration, 565 nm wavelength illumination, 0.17 J/cm2 fluence) cell migration rate using scratch assay and growth factor concentrations in the cell culture supernatant using ELISA have been determined. In addition, the effect of CM has been observed., Results: RB-PDT significantly reduced migration rate in all cell types, compared to controls (p≤0.02). Migration rate of HCE-T cultures without RB-PDT (untreated) was significantly higher using HCF CM after RB-PDT, than using HCF CM without RB-PDT (p<0.01). Similarly, untreated HCF displayed a significantly increased migration rate with HCE-T CM after RB-PDT, compared to HCE-T CM without treatment (p<0.01). Furthermore, illumination alone and RB-PDT significantly decreased keratinocyte growth factor (KGF) concentration in HCF and KC-HCF supernatant, and RB-PDT significantly decreased soluble N-Cadherin (SN-Cad) concentration in HCF supernatant, compared to controls (p<0.01 for all). In HCE-T CM, RB-PDT increased hepatocyte growth factor (HGF) and basic fibroblast growth factor (FGFb) concentration (p≤0.02), while decreasing transforming growth factor β (TGF-β) concentration (p<0.01). FGFb concentration increased (p<0.0001) and TGF-β concentration decreased (p<0.0001) in HCF CM, by RB-PDT. Epidermal growth factor (EGF), HGF, and TGF-β concentration decreased (p≤0.03) and FGFb concentration increased (p<0.01) in KC-HCF CM, using RB-PDT., Conclusions: HCE-T, HCF and KC-HCF migration rate is reduced 24 hours after RB-PDT. In contrast, HCE-T migration is enhanced using HCF CM after RB-PDT, and HCF migration rate is increased through HCE-T CM following RB-PDT. Modulation of EGF, KGF, HGF, FGFb, TGF-β and N-Cadherin secretion through RB-PDT may play an important role in corneal wound healing., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Chai et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Radiotherapy (RT) is one of three major treatments for malignant tumors, and one of its most common side effects is skin and soft tissue injury. However, the treatment of these remains challenging. Several studies have shown that mesenchymal stem cell (MSC) treatment enhances skin wound healing. In this study, we extracted human dermal fibroblasts (HDFs) and adipose-derived stem cells (ADSCs) from patients and generated an in vitro radiation-induced skin injury model with HDFs to verify the effect of conditioned medium derived from adipose-derived stem cells (ADSC-CM) and extracellular vesicles derived from adipose-derived stem cells (ADSC-EVs) on the healing of radiation-induced skin injury. The results showed that collagen synthesis was significantly increased in wounds treated with ADSC-CM or ADSC-EVs compared with the control group, which promoted the expression of collagen-related genes and suppressed the expression of inflammation-related genes. These findings indicated that treatment with ADSC-CM or ADSC-EVs suppressed inflammation and promoted extracellular matrix deposition; treatment with ADSC-EVs also promoted fibroblast proliferation. In conclusion, these results demonstrate the effectiveness of ADSC-CM and ADSC-EVs in the healing of radiation-induced skin injury.

Mesenchymal stromal cells (MSCs) have recently been shown to play an important role in the growth and progression of many solid tumors, including cholangiocarcinoma (CCA). The human placental amniotic membrane (hPAM) is one of the most favorable sources of MSCs due to its availability and non-invasive harvesting procedure. However, the role of human placental amniotic membrane mesenchymal stromal cells (hPAMSCs) in the growth and progression of human CCA has not yet been determined. This study investigates the effects of conditioned medium derived from hPAMSCs (PA-CM) on the properties of three human CCA cell lines and explores possible mechanisms of action. Varying concentrations of PA-CM were used to treat CCA cells to determine their effects on the proliferation and apoptosis of CCA cells. The results showed that PA-CM inhibited the proliferation and colony-forming capacity of KKU100, KKU213A, and KKU213B cells. PA-CM also promoted the apoptosis of these CCA cells by causing the loss of mitochondrial membrane potential. Western Blotting confirmed that PA-CM induced CCA cell apoptosis by increasing the levels of the Bax/Bcl-2 ratio, cleaved caspase 3, and cleaved PARP, possibly by inhibiting the IL-6/JAK2/STAT3 signaling pathway. Moreover, our in vivo study also confirmed the suppressive effect of hPAMSCs on CCA cells by showing that PA-CM reduced tumor volume in nude mice transplanted with human CCA cells. Taken together, our results demonstrate that PA-CM has potent tumor-suppressive effects on human CCA cells and could potentially be used in combination with chemotherapy to develop a more effective treatment for CCA patients.

In treating brain diseases, such as canine cognitive dysfunction (CCD), most currently available potent drugs have weak therapeutic efficacy. One of the causes is the inability of the substance to reach the brain in therapeutic quantities. These pharmaceuticals lacked targeted mechanisms for drug delivery, coming about in an elevated drug concentration in imperative organs, which drove to drug harmfulness. In recent years, cell-free treatment (conditioned medium) determined from animal and human stem cells has provided new promise for treating brain diseases, as CM can stimulate the regeneration of neurons and prevent the inflammation and apoptotic of neurons caused by pathology or aging. On the other hand, it is well known that chitosan-hydrogel (CH) is a polymer derived from natural sources. It has been authorized for use in biomedical use because of its uncommon biodegradability, biocompatibility, and mucoadhesive properties. CH modification has been utilized to generate nanoparticles (NPs) for intranasal and intravenous brain targeting. NPs shown upgraded drug take-up to the brain with decreased side impacts due to their drawn out contact time with the nasal mucosa, surface charge, nanosize, and capacity to extend the tight intersections inside the mucosa. Due to the aforementioned distinctive characteristics, developing Chitosan Hydrogel Nanoparticles load with bovine umbilical mesenchymal stem cell conditioned medium is crucial as a new therapeutic strategy for CCD.

In vitro fabrication of 3D cell culture systems that could provide in vivo tissue-like, structural, and biochemical environments to neural cells is essential not only for fundamental studies on brain function and behavior, but also for tissue engineering and regenerative medicine applicable to neural injury and neurodegenerative diseases. In particular, for astrocytes-which actively respond to the surroundings and exhibit varied morphologies based on stimuli (e.g., stiffness and chemicals) in vitro, as well as physiological or pathological conditions in vivo-it is crucial to establish an appropriate milieu in in vitro culture platforms. Herein, we report the induction of in vivo-relevant, stellate-shaped astrocytes derived from cortices of Rattus norvegicus by constructing the 3D cell culture systems of brain-derived, decellularized extracellular matrices (bdECMs). The bdECM hydrogels were mechanically stable and soft, and the bdECM-based 3D scaffolds supplied biochemically active environments that astrocytes could interact with, leading to the development of in vivo-like stellate structures. In addition to the distinct morphology with actively elongated endfeet, the astrocytes, cultured in 3D bdECM scaffolds, would have neurosupportive characteristics, indicated by the accelerated neurite outgrowth in the astrocyte-conditioned media. Furthermore, next-generation sequencing showed that the gene expression profiles of astrocytes cultured in bdECMs were significantly different from those cultured on 2D surfaces. The stellate-shaped astrocytes in the bdECMs were analyzed to have reached a more mature state, for instance, with decreased expression of genes for scaffold ECMs, actin filaments, and cell division. The results suggest that the bdECM-based 3D culture system offers an advanced platform for culturing primary cortical astrocytes and their mixtures with other neural cells, providing a brain-like, structural and biochemical milieu that promotes the maturity and in vivo-like characteristics of astrocytes in both form and gene expression. STATEMENT OF SIGNIFICANCE: Decellularized extracellular matrices (dECMs) have emerged as strong candidates for the construction of three-dimensional (3D) cell cultures in vitro, owing to the potential to provide native biochemical and physical environments. In this study, we fabricated hydrogels of brain-derived dECMs (bdECMs) and cultured primary astrocytes within the bdECM hydrogels in a 3D context. The cultured astrocytes exhibited a stellate morphology distinct from conventional 2D cultures, featuring tridimensionally elongated endfeet. qRT-PCR and NGS-based transcriptomic analyses revealed gene expression patterns indicative of a more mature state, compared with the 2D culture. Moreover, astrocytes cultured in bdECMs showed neurosupportive characteristics, as demonstrated by the accelerated neurite outgrowth in astrocyte-conditioned media. We believe that the bdECM hydrogel-based culture system can serve as an in vitro model system for astrocytes and their coculture with other neural cells, holding significant potential for neural engineering and therapeutic applications., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Background: High-functional cosmetic products combined with the concept of "treatment" cosmetics are being introduced to the market. Cosmetic products containing a skin-derived microbiome, a three-dimensional (3D) stem cell culture medium, and low-molecular-weight collagen are being introduced, and these products are leading the cosmeceutical market. We aimed to confirm the potential of a 3D stem cell culture medium-containing cream as a skin-whitening and moisturizing product., Aim: To determine the enhancing effects of a cream containing 3D adipose tissue-derived mesenchymal stem cell-conditioned media (3D ADMSC-CM) on whitening and moisturization., Methods: The inhibitory activities of tyrosinase (TYR) and melanin were confirmed using 3D ADMSC-CM. Furthermore, hyaluronic acid expression in 3D ADMSC-CM was verified. The clinical efficacy of the cream containing 3D ADMSC-CM was established by evaluating its antioxidant properties and effects on skin tone, radiance, freckles, and moisturization., Results: The use of 3D ADMSC-CM suppressed the inhibitory effects of TYR and melanin by approximately 24% and 33%, respectively, and increased the expression of hyaluronic acid synthase. A significant difference was observed after 4 weeks of using 3D ADMSC-CM in the skin antioxidant evaluation. After 2 and 4 weeks of use, skin tone and radiance increased and skin freckles decreased significantly. Under extremely cold and dry weather conditions, the use of the cream increased skin moisturization., Conclusions: The 3D ADMSC-CM cream evaluated in an environment similar to the human body was found to enhance skin whitening and moisturization and can therefore be used in the skin care and cosmetic industries as a biocosmetic product., (© 2023 The Authors. Journal of Cosmetic Dermatology published by Wiley Periodicals LLC.)

Multipotent Mesenchymal stem cells (MSCs) have vigorous immunomodulatory activity, apoptotic effects, and the capacity to migrate to inflammatory and tumor sites. This study focuses on the apoptotic effects of MSCs conditioned medium (CM) on colorectal cancer cell lines. MSCs were preconditioned with lipopolysaccharide (LPS) to induce apoptosis in colorectal cancer cells. The conditioned medium (LPS-CM) from the preconditioned cells was isolated and used to treat colorectal cancer cells (HT29 and SW48). The survival and proliferation of cancer cells were assessed using Trypan blue staining and MTT assay. The apoptosis rate was evaluated through flow cytometry analysis and caspase-3 activity. Additionally, Real-Time PCR was used to measure the mRNA level of apoptotic and anti-apoptotic factors, including bcl2, bax, and p53 genes. The results showed that LPS-CM significantly increased (p < 0.001) the percentage of apoptosis in the SW48 and HT29 cell lines. Caspase-3 activity significantly increased (p < 0.001) in these cell lines after treatment with LPS-CM. The mRNA level of bcl2 was significantly decreased (p < 0.001), while bax and p53 genes were significantly overexpressed (p < 0.001) in the LPS-CM treated cell lines. Notably, the mRNA level of bcl2 and bax genes was significantly altered at a higher concentration of LPS-CM. In conclusion, the conditioned medium from LPS-preconditioned MSCs can effectively induce apoptosis in colorectal cancer cells. This finding suggests that LPS-CM could be a potential strategy for inhibiting the proliferation and progression of colorectal cancer cells., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing interests. There are no other persons who satisfied the criteria for authorship., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Polycystic ovary syndrome (PCOS) is a disorder with endocrinal and metabolic problems in reproductive aged women. Evidence shows that PCOS is in a high prone trend to develop kidney diseases. In this study, we investigated the mediators responsible for PCOS-related kidney injury. We found that tumor necrosis factor (TNF-α) levels were significantly increased in serum and primary cultured granulosa cells (GCs) from PCOS patients. Serum TNF-α levels were positively correlated with serum testosterone and luteinizing hormone (LH)/follicle-stimulating hormone (FSH) ratio, suggesting its positive role in the severity of PCOS. Serum TNF-α levels were also positively correlated with the levels of urinary KapU, LamU, α1-MU and β2-MU, the markers for renal tubular cell-derived proteinuria. We established a PCOS mouse model by resection of the right kidney, followed by daily administration of dihydrotestosterone (DHT, 27.5 μg, i.p.) from D7 for 90 days. We found that TNF-α levels were significantly increased in the ovary and serum of the mice, accompanied by increased renal tubular cell apoptosis, inflammation and fibrosis in kidneys. Furthermore, the receptor of TNF-α, tumor necrosis factor receptor 1 (TNFR1), was significantly upregulated in renal tubular cells. We treated human ovarian granulosa-like tumor cells (KGN) with DHT (1 μg/ml) in vitro, the conditioned medium derived from the granulosa cell culture greatly accelerated apoptotic injury in human proximal tubular epithelial cells (HKC-8), which was blocked after knockdown of TNF-α in KGN cells. Furthermore, knockdown of TNFR1 in renal tubular epithelial cells greatly ameliorated cell injury induced by granulosa cell-derived conditioned medium. These results suggest that serum TNF-α plays a key role in mediating inflammation and apoptosis in renal tubular cells associated with PCOS-related kidney injury., (© 2023. The Author(s), under exclusive licence to Shanghai Institute of Materia Medica, Chinese Academy of Sciences and Chinese Pharmacological Society.)

Objectives: This study aimed to investigate the site-specific characteristics of rat mandible periosteal cells (MPCs) and tibia periosteal cells (TPCs) to assess the potential application of periosteal cells (PCs) in bone tissue engineering (BTE)., Materials and Methods: MPCs and TPCs were isolated and characterized. The potential of proliferation, migration, osteogenesis and adipogenesis of MPCs and TPCs were evaluated by CCK-8, scratch assay, Transwell assay, alkaline phosphatase staining and activity, Alizarin Red S staining, RT‒qPCR, and Western blot (WB) assays, respectively. Then, these cells were cocultured with human umbilical vein endothelial cells (HUVECs) to investigate their angiogenic capacity, which was assessed by scratch assay, Transwell assay, Matrigel tube formation assay, RT‒qPCR, and WB assays., Results: MPCs exhibited higher osteogenic potential, higher alkaline phosphatase activity, and more mineralized nodule formation, while TPCs showed a greater capability for proliferation, migration, and adipogenesis. MPCs showed higher expression of angiogenic factors, and the conditioned medium of MPCs accelerated the migration of HUVECs, while MPC- conditioned medium induced the formation of more tubular structure in HUVECs in vitro. These data suggest that compared to TPCs, MPCs exert more consequential proangiogenic effects on HUVECs., Conclusions: PCs possess skeletal site-specific differences in biological characteristics. MPCs exhibit more eminent osteogenic and angiogenic potentials, which highlights the potential application of MPCs for BTE., Clinical Relevance: Autologous bone grafting as the main modality for maxillofacial bone defect repair has many limitations. Constituting an important cell type in bone repair and regeneration, MPCs show greater potential for application in BTE, which provides a promising treatment option for maxillofacial bone defect repair., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The conditioned medium of mesenchymal stem cells (MSCs) has controversial roles in cancer, either promoting or suppressing tumor growth. Our research on the results of adipose tissue-derived MSC (AD-MSC)-conditioned media on U87 glioma cells was motivated by the disputed role of mesenchymal stem cells (MSCs) in cancer, which may either promote or inhibit tumor growth. Using flow cytometry, AD-MSCs were identified, verified, and their conditioned media was used to treat U87 cells. Through RT-qPCR, scratch assay, and apoptosis analysis, we evaluated gene expression (SOX4, H19, and CCAT1), cell migration, and apoptosis in U87 cells.The conditioned media greatly increased the expression of SOX4 and H19, but not CCAT1. Although there were few differences in migration and apoptosis, both were slightly increased in the treated group.These outcomes have drawn attention to the complexity of the interactions between MSCs and glioma cells. This complexity requires further research to identify the specific mechanisms governing MSC-mediated impacts on the development of glioblastoma multiforme (GBM)., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interests., (Copyright © 2023 Elsevier Ltd. All rights reserved.)