Cell-conditioned Medium Research Articles

Novel therapeutic targets, including IGFBP3, of umbilical cord mesenchymal stem-cell-conditioned medium in intrauterine adhesion.

Mesenchymal stem cells play important roles in repairing injured endometrium. However, the molecular targets and potential mechanism of the endometrial recipient cells for stem cell therapy in intrauterine adhesion (IUA) are poorly understood. In this study, umbilical cord mesenchymal stem-cell-conditioned medium (UCMSCs-CM) produced positive effects on a Transforming growth factor beta (TGF-β) induced IUA cell model. RNA-sequencing was performed on clinical IUA tissues, and the top 40 upregulated and top 20 downregulated mRNAs were selected and verified using high-throughput (HT) qPCR in both tissues and cell models. Based on a bioinformatic analysis of RNA-sequencing and HT-qPCR results, 11 mRNAs were uncovered to be the intervention targets of UCMSCs-CM on IUA endometrium cell models. Among them, IGFBP3 was striking as a key pathogenic gene and a potential diagnostic marker of IUA, which exhibited the area under the curve (AUC), sensitivity, specificity were 0.924, 93.1% and 80.6%, respectively in 60 endometrial tissues. The silencing of IGFBP3 exerted positive effects on the IUA cell model through partially upregulating MMP1 and KLF2. In conclusion, RNA-sequencing combined with HT qPCR based on clinical tissues and IUA cell models were used in IUA research and our results may provide some scientific ideas for the diagnosis and treatment of IUA.

Cellular senescence is associated with osteonecrosis of the femoral head while mesenchymal stem cell conditioned medium inhibits bone collapse

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.

Tumor-specific activation of folate receptor beta enables reprogramming of immune cells in the tumor microenvironment.

Folate receptors can perform folate transport, cell adhesion, and/or transcription factor functions. The beta isoform of the folate receptor (FRβ) has attracted considerable attention as a biomarker for immunosuppressive macrophages and myeloid-derived suppressor cells, however, its role in immunosuppression remains uncharacterized. We demonstrate here that FRβ cannot bind folate on healthy tissue macrophages, but does bind folate after macrophage incubation in anti-inflammatory cytokines or cancer cell-conditioned media. We further show that FRβ becomes functionally active following macrophage infiltration into solid tumors, and we exploit this tumor-induced activation to target a toll-like receptor 7 agonist specifically to immunosuppressive myeloid cells in solid tumors without altering myeloid cells in healthy tissues. We then use single-cell RNA-seq to characterize the changes in gene expression induced by the targeted repolarization of tumor-associated macrophages and finally show that their repolarization not only changes their own phenotype, but also induces a proinflammatory shift in all other immune cells of the same tumor mass, leading to potent suppression of tumor growth. Because this selective reprogramming of tumor myeloid cells is accompanied by no systemic toxicity, we propose that it should constitute a safe method to reprogram the tumor microenvironment.

Conditioned Medium of Intervertebral Disc Cells Inhibits Osteo-Genesis on Autologous Bone-Marrow-Derived Mesenchymal Stromal Cells and Osteoblasts.

Low back pain (LBP) is associated with the degeneration of human intervertebral discs (IVDs). Despite progress in the treatment of LBP through spinal fusion, some cases still end in non-fusion after the removal of the affected IVD tissue. In this study, we investigated the hypothesis that the remaining IVD cells secrete BMP inhibitors that are sufficient to inhibit osteogenesis in autologous osteoblasts (OBs) and bone marrow mesenchymal stem cells (MSCs). A conditioned medium (CM) from primary human IVD cells in 3D alginate culture was co-cultured with seven donor-matched OB and MSCs. After ten days, osteogenesis was quantified at the transcript level using qPCR to measure the expression of bone-related genes and BMP antagonists, and at the protein level by alkaline phosphatase (ALP) activity. Additionally, cells were evaluated histologically using alizarin red (ALZR) staining on Day 21. For judging ALP activity and osteogenesis, the Noggin expression in samples was investigated to uncover the potential causes. The results after culture with the CM showed significantly decreased ALP activity and the inhibition of the calcium deposit formation in alizarin red staining. Interestingly, no significant changes were found among most bone-related genes and BMP antagonists in OBs and MSCs. Noteworthy, Noggin was relatively expressed higher in human IVD cells than in autologous OBs or MSCs (relative to autologous OB, the average fold change was in 6.9, 10.0, and 6.3 in AFC, CEPC, and NPC, respectively; and relative to autologous MSC, the average fold change was 2.3, 3.4, and 3.2, in AFC, CEPC, and NPC, respectively). The upregulation of Noggin in residual human IVDs could potentially inhibit the osteogenesis of autologous OB and MSC, thus inhibiting the postoperative spinal fusion after discectomy surgery.

Effects of conditioned medium of alveolar epithelial cells on vascular endothelial cell damage

To observe the effect of lipopolysaccharide (LPS) induced conditioned medium of alveolar epithelial cells on the inflammatory response and cell damage of vascular endothelial cells, and explore its mechanism. The LPS induced type II alveolar epithelial cells (A549) conditioned medium was used as a stimulus to induce human umbilical vein endothelial cells (HUVEC) damage. The cell counting kit-8 (CCK-8) was used to detect the effect of 0% (blank group), 12.5%, 25%, 50%, 75% and 100% A549 cell conditioned medium cultured for 6, 12, 24 and 48 hours on the cell viability of HUVEC. Enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of inflammatory factors [interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α)] and vasoactive substances [vascular endothelial growth factor (VEGF), endothelin-1 (ET-1)] in the supernatant. Phalloidin staining was used to observe the effects of A549 cells conditioned medium on cell morphology. The expressions of protein kinase B/nuclear factor-κB (AKT/NF-κB) pathway in HUVEC induced by conditioned medium was detected by Western blotting. Compared with the blank group, A549 cells conditioned medium with concentrations of 12.5%, 25%, and 50% had no significant effects on cell viability of HUVEC after 6, 12, and 24 hours, but the activity of HUVEC decreased significantly after 48 hours. Therefore, 12.5%, 25%, 50% A549 cell conditioned medium stimulated for 24 hours was selected as the induction condition for follow-up experiments. Compared with the blank group, the level of IL-6 was significantly increased in 12.5% and 50% conditioned medium groups (ng/L: 2 438.95±64.89, 3 036.41±96.69 vs. 1 736.75±20.99, both P < 0.05), the level of TNF-α was significantly increased in 12.5% and 25% conditioned medium groups (ng/L: 174.08±11.09, 81.37±8.17 vs. 50.03±0.26, both P < 0.01), the levels of VEGF and ET-1 were significantly increased in 12.5%, 25% and 50% conditioned medium groups [VEGF (ng/L): 173.60±41.44, 192.49±12.38, 318.89±27.90 vs. 66.68±19.65; ET-1 (ng/L): 54.88±1.37, 36.69±0.29, 24.07±0.73 vs. 10.67±0.25, all P < 0.01]. Phalloidin staining showed that HUVEC induced by 25% A549 cells conditioned medium were irregular in shape, uneven in size, disordered in arrangement, widened in gap, dense and unclear in microfilament structure and serrated in cell membrane. Furthermore, the average fluorescence intensity of 25% conditioned medium group significantly increased compared to the blank group (67 205.60±3 430.40 vs. 56 272.67±7 650.95, P < 0.05). Western blotting showed that compared with the blank group, the expression of HUVEC cells phosphonated inhibitor α of NF-κB (p-IκBα) was significantly decreased in the 12.5%, 25%, and 50% conditioned medium groups (p-IκBα/IκBα: 0.38±0.08, 0.67±0.12, 0.31±0.07 vs. 1.00±0.00, all P < 0.01), the expressions of phosphonated-AKT (p-AKT) and VEGF were significantly increased (p-AKT/AKT: 1.50±0.18, 1.42±0.27, 1.61±0.14 vs. 1.00±0.00, VEGF/GAPDH: 1.37±0.10, 1.53±0.22, 1.40±0.12 vs. 1.00±0.00, all P < 0.05), the expression of phosphonated NF-κB p65 (p-P65) was significantly increased in the 25% conditioned medium group (p-P65/P65: 1.45±0.14 vs. 1.00±0.00, P < 0.05). LPS induced conditional culture medium of alveolar epithelial cells induced endothelial cell damage via activating AKT/NF-κB pathway.

Photobiomodulation and conditioned medium of adipose-derived stem cells for enhancing wound healing in rats with diabetes: an investigation on the proliferation phase.

This investigation tried to evaluate the combined and solo effects of photobiomodulation (PBM) and conditioned medium derived from human adipose tissue-derived stem cells (h-ASC-CM) on the inflammatory and proliferative phases of an ischemic infected delayed healing wound model (IIDHWM) in rats with type I diabetes mellitus (TIDM). The present investigation consisted of four groups: group 1 served as the control, group 2 treated with h-ASC-CM, group 3 underwent PBM treatment, and group 4 received a combination of h-ASC-CM and PBM. Clinical and laboratory assessments were conducted on days 4 and 8. All treatment groups exhibited significantly higher wound strength than the group 1 (p = 0.000). Groups 4 and 3 demonstrated significantly greater wound strength than group 2 (p = 0.000). Additionally, all therapeutic groups showed reduced methicillin -resistant Staphylococcus aureus (MRSA) in comparison with group 1 (p = 0.000). While inflammatory reactions, including neutrophil and macrophage counts, were significantly lower in all therapeutic groups rather than group 1 on days 4 and 8 (p < 0.01), groups 4 and 3 exhibited superior results compared to group 2 (p < 0.01). Furthermore, proliferative activities, including fibroblast and new vessel counts, as well as the measurement of new epidermal and dermal layers, were significantly increased in all treatment groups on 4 and 8 days after the surgery (p < 0.001). At the same times, groups 4 and 3 displayed significantly higher proliferative activities compared to group 2 (p < 0.001). The treatment groups exhibited significantly higher mast cell counts and degranulation phenotypes in comparison with the group 1 on day 4 (p < 0.05). The treatment groups showed significantly lower mast cell counts and degranulation phenotypes than group 1 on day 8 (p < 0.05).The combined and individual application of h-ASC-CM and PBM remarkably could accelerate the proliferation phase of wound healing in the IIDHWM for TIDM in rats, as indicated by improved MRSA control, wound strength, and stereological evaluation. Furthermore, the combination of h-ASC-CM and PBM demonstrated better outcomes compared to the individual application of either h-ASC-CM or PBM alone.

Interconversion of Cancer Cells and Induced Pluripotent Stem Cells.

Cancer cells, especially cancer stem cells (CSCs), share many molecular features with induced pluripotent stem cells (iPSCs) that enable the derivation of induced pluripotent cancer cells by reprogramming malignant cells. Conversely, normal iPSCs can be converted into cancer stem-like cells with the help of tumor microenvironment components and genetic manipulation. These CSC models can be utilized in oncogenic initiation and progression studies, understanding drug resistance, and developing novel therapeutic strategies. This review summarizes the role of pluripotency factors in the stemness, tumorigenicity, and therapeutic resistance of cancer cells. Different methods to obtain iPSC-derived CSC models are described with an emphasis on exposure-based approaches. Culture in cancer cell-conditioned media or cocultures with cancer cells can convert normal iPSCs into cancer stem-like cells, aiding the examination of processes of oncogenesis. We further explored the potential of reprogramming cancer cells into cancer-iPSCs for mechanistic studies and cancer dependencies. The contributions of genetic, epigenetic, and tumor microenvironment factors can be evaluated using these models. Overall, integrating iPSC technology into cancer stem cell research holds significant promise for advancing our knowledge of cancer biology and accelerating the development of innovative and tailored therapeutic interventions.

Retracted: Antioxidative Capacity of Liver- and Adipose-Derived Mesenchymal Stem Cell-Conditioned Media and Their Applicability in Treatment of Type 2 Diabetic Rats.

[This retracts the article DOI: 10.1155/2021/8833467.].

Bakuchiol targets mitochondrial proteins, prohibitins and voltage-dependent anion channels: New insights into developing antiviral agents

We previously reported that bakuchiol, a phenolic isoprenoid anti-cancer compound, and its analogs exert anti-influenza activity. However, the proteins targeted by bakuchiol remain unclear. Here, we investigated the chemical structures responsible for the anti-influenza activity of bakuchiol and found that all functional groups and C6 chirality of bakuchiol were required for its anti-influenza activity. Based on these results, we synthesized a molecular probe containing a biotin tag bound to the C1 position of bakuchiol. With this probe, we performed a pulldown assay for Madin-Darby canine kidney (MDCK) cell lysates and purified the specific bakuchiol-binding proteins with sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Using nano-liquid chromatography with tandem mass spectrometry analysis, we identified prohibitin (PHB) 2, voltage dependent anion channel (VDAC) 1, and VDAC2 as binding proteins of bakuchiol. We confirmed the binding of bakuchiol to PHB1, PHB2, and VDAC2 in vitro using western blot analysis. Immunofluorescence analysis showed that bakuchiol was bound to PHBs and VDAC2 in cells and co-localized in the mitochondria. The knockdown of PHBs or VDAC2 by transfection with specific siRNAs, along with bakuchiol cotreatment, led to significantly reduced influenza nucleoprotein (NP) expression levels and viral titers in the conditioned medium of virus-infected MDCK cells, compared to the levels observed with transfection or treatment alone. These findings indicate that reducing PHBs or VDAC2 protein, combined with bakuchiol treatment, additively suppressed the growth of influenza virus. Our findings indicate that bakuchiol exerts anti-influenza activity via a novel mechanism involving these mitochondrial proteins, providing new insight for developing anti-influenza agents.

Carnosol ameliorated cancer cachexia-associated myotube atrophy by targeting P5CS and its downstream pathways.

Introduction: Carnosol exhibited ameliorating effects on muscle atrophy of mice developed cancer cachexia in our previous research. Method: Here, the ameliorating effects of carnosol on the C2C12 myotube atrophy result from simulated cancer cachexia injury, the conditioned medium of the C26 tumor cells or the LLC tumor cells, were observed. To clarify the mechanisms of carnosol, the possible direct target proteins of carnosol were searched using DARTS (drug affinity responsive target stability) assay and then confirmed using CETSA (cellular thermal shift assay). Furthermore, proteomic analysis was used to search its possible indirect target proteins by comparing the protein expression profiles of C2C12 myotubes under treatment of C26 medium, with or without the presence of carnosol. The signal network between the direct and indirect target proteins of carnosol was then constructed. Results: Our results showed that, Delta-1-pyrroline-5-carboxylate synthase (P5CS) might be the direct target protein of carnosol in myotubes. The influence of carnosol on amino acid metabolism downstream of P5CS was confirmed. Carnosol could upregulate the expression of proteins related to glutathione metabolism, anti-oxidant system, and heat shock response. Knockdown of P5CS could also ameliorate myotube atrophy and further enhance the ameliorating effects of carnosol. Discussion: These results suggested that carnosol might ameliorate cancer cachexia-associated myotube atrophy by targeting P5CS and its downstream pathways.

Improvements in Diabetic Neuropathy with Topical Human Mesenchymal Stem Cell Conditioned Media

Improvements in Diabetic Neuropathy with Topical Human Mesenchymal Stem Cell Conditioned Media

Differential response of placental cells to high D‐glucose and its impact on extracellular vesicle biogenesis and trafficking via small GTPase Ras‐related protein RAB‐7A

AbstractPlacental extracellular vesicles (EVs) can be found in the maternal circulation throughout gestation, and their concentration, content and bioactivity are associated with pregnancy outcomes, including gestational diabetes mellitus (GDM). However, the effect of changes in the maternal microenvironment on the mechanisms associated with the secretion of EVs from placental cells remains to be fully established. Here, we evaluated the effect of high glucose on proteins associated with the trafficking and release of different populations of EVs from placental cells. BeWo and HTR8/SVneo cells were used as placental models and cultured under 5‐mM D‐glucose (i.e. control) or 25‐mM D‐glucose (high glucose). Cell‐conditioned media (CCM) and cell lysate were collected after 48 h. Different populations of EVs were isolated from CCM by ultracentrifugation (i.e. pellet 2K‐g, pellet 10K‐g, and pellet 100K‐g) and characterised by Nanoparticle Tracking Analysis. Quantitative proteomic analysis (IDA/SWATH) and multiple reaction monitoring protocols at high resolution (MRMHR) were developed to quantify 37 proteins related to biogenesis, trafficking/release and recognition/uptake of EVs. High glucose increased the secretion of total EVs across the pellets from BeWo cells, an effect driven mainly by changes in the small EVs concentration in the CCM. Interestingly, no effect of high glucose on HTR8/SVneo cells EVs secretion was observed. High glucose induces changes in proteins associated with vesicle trafficking in BeWo cells, including Heat Shock Protein Family A (Hsp70) Member 9 (HSPA9) and Member 8 (HSPA8). For HTR8/SVneo, altered proteins including prostaglandin F2α receptor regulatory protein (FPRP), RAB5A, RAB35, RAB5B, and RB11B, STAM1 and TSG101. These proteins are associated with the secretion and trafficking of EVs, which could explain in part, changes in the levels of circulating EVs in diabetic pregnancies. Further, we identified that proteins RAB11B, PDCD6IP, STAM, HSPA9, HSPA8, SDCBP, RAB5B, RAB5A, RAB7A and ERAP1 regulate EV release in response to high and low glucose when overexpressed in cells. Interestingly, immunohistochemistry analysis of RAB7A revealed distinct changes in placental tissues obtained from women with normal glucose tolerance (NGT, n = 6) and those with GDM (n = 6), influenced by diet or insulin treatment. High glucose regulation of proteins involved in intercellular dynamics and the trafficking of multivesicular bodies to the plasma membrane in placental cells is relevant in the context of GDM pregnancies.

Conditioned culture medium of bone marrow mesenchymal stem cells promotes phenotypic transformation of microglia by regulating mitochondrial autophagy.

To study the mechanism by which conditioned medium of bone marrow mesenchymal stem cells (BMSCs-CM) facilitates the transition of pro-inflammatory polarized microglia to an anti-inflammatory phenotype. BV2 cells, a mouse microglia cell line, were transformed into a pro-inflammatory phenotype using lipopolysaccharide. The expression of phenotypic genes in BV2 cells was detected using real-time quantitative PCR (RT-qPCR). Enzyme-linked immunosorbent assay was used to measure inflammatory cytokine levels in BV2 cells co-cultured with BMSCs-CM. The expressions of mitophagy-associated proteins were determined using western blot. The mitochondrial membrane potential and ATP levels in BV2 cells were measured using JC-1 staining and an ATP assay kit, respectively. Additionally, we examined the proliferation, apoptosis, and migration of C8-D1A cells, a mouse astrocyte cell line, co-cultured with BV2 cells. After co- culture with BMSCs -CM, the mRNA expression of tumor necrosis factor-α (TNF-α) and inducible nitric oxide synthase significantly decreased in pro-inflammatory BV2 cells, whereas the expression of CD206 and arginase-1 significantly increased. Moreover, TNF-α and interleukin-6 levels significantly decreased, whereas transforming growth factor-β and interleukin-10 levels significantly increased. Furthermore, co-culture with BMSCs-CM increased mitophagy-associated protein expression, ATP levels, mitochondrial and lysosomal co-localization in these cells and decreased reactive oxygen species levels. Importantly, BMSCs-CM reversed the decrease in the proliferation and migration of C8-D1A cells co-cultured with pro-inflammatory BV2 cells and inhibited the apoptosis of C8-D1A cells. BMSCs-CM may promote the transition of polarized microglia from a pro-inflammatory to an anti-inflammatory phenotype by regulating mitophagy and influences the functional state of astrocytes.

Isolation and Characterization of Extracellular Vesicles from Cell Culture Media.

Extracellular vesicles (EVs) were once believed to serve as a means of disposing of cellular waste. However, recent discoveries have identified their crucial roles in intercellular communication between neighboring and distant cells. Almost all cell types have now been identified to produce EVs, which play a vital role in transporting cellular cargo. The functional roles of EVs, along with their implications in (patho)physiology of various diseases, are still being explored. In the last decade, the identification of EV roles in pathophysiology, pharmacology, and diagnostics has gained significant interest, albeit the development of universal methods for the isolation and characterization of EVs has been the limiting factor. A further challenge is ensuring that EVs of various size categories, which are thought to be produced via independent cellular mechanisms and often differ in their cargo and physiological purpose, can be separated and studied in isolation.This protocol provides an efficient and accessible method for isolating and characterizing EV samples from conditioned cell culture media. The combination of differential centrifugation and the use of a commercial EV-precipitation kit allows for the rapid isolation of a highly pure sample of EVs separated by size. A microfluidic resistive pulse sensing (MRPS)-based method is then used to quantify the particles, as well as to assess the size distribution of the EV sample. As a result, this protocol provides a reproducible means to isolate and characterize EVs of a variety of sizes from nearly any cultured cells.

Platycodin D ameliorates ammonia-induced pulmonary fibrosis by repressing TGF-β1-mediated extracellular matrix remodeling.

Ammonia can induce pulmonary fibrosis in humans and animals. Platycodin D (PLD) possesses various bioactive activities including anti-fibrotic properties. In this study, we aimed to explore the activity and mechanism of PLD in pulmonary fibrosis induced by ammonia. The mouse model of ammonia-induced lung fibrosis was established, and the role of PLD was assessed by H&E and Masson's trichrome staining. The differentially expressed genes (DEGs) were identified by RNA-seq and subjected to GO and KEGG pathway analyses. BEAS-2B cells were treated with NH4 Cl alone or along with PLD. Results showed that PLD attenuated ammonia-induced pulmonary inflammation and fibrosis invivo. The extracellular matrix (ECM)-receptor interaction pathway was predicted as a prominent pathway underlying the anti-fibrotic function of PLD. In ammonia-induced mouse models and NH4 Cl-treated BEAS-2B cells, PLD could repress the activation of the TGF-β1 pathway. By incubating lung fibroblast HFL1 cells with the conditioned medium of BEAS-2B cells treated with NH4Cl alone or along with PLD, PLD was confirmed to attenuate NH4 Cl-induced ECM deposition in HFL1 cells. Our findings demonstrate that PLD exerts a protective function in ammonia-induced pulmonary fibrosis by repressing TGF-β1-mediated ECM remodeling, suggesting the potential therapeutic value of PLD in this disease.

Experimental study of the nephroprotective properties of cryoextracts of the placenta and spleen, and the conditioned medium of mesenchymal stem cells on the background of autoimmune membranous nephropathy

Experimental study of the nephroprotective properties of cryoextracts of the placenta and spleen, and the conditioned medium of mesenchymal stem cells on the background of autoimmune membranous nephropathy

Therapeutic Effects of Mesenchymal Stem Cell Conditioned Medium in Rat Varicocele Model.

This study aimed to examine the therapeutic effects of injection of conditioned medium of adipose-derived mesenchymal stem cells (ADMSC-CM) in a surgically created varicocele model in comparison with varicocelectomy. Twenty-eight male Wistar Albino rats were randomly divided into four groups: sham group, varicocele group, varicocelectomy group, and ADMSC-CM injection group. Sperm parameters were analyzed in samples taken from the epididymis after treatment. Malondialdehyde and superoxide dismutase (SOD) levels in blood samples were examined by biochemical analysis. The testicular tissues were stained with hematoxylin-eosin for histological examination (Johnsen's Score). Additionally, Western Blot analyzes were performed to detect Claudin-11 levels, the functional protein of the blood-testis barrier, in testicular tissues. Varicocelectomy and ADMSC-CM treatments significantly improved mean sperm parameters (concentration, progressive motility, motility, normal sperm morphology) (p≤0.05 for all). Both treatment groups had increased SOD levels along with a decrease in malondialdehyde levels (p≤0.05 for all). No significant difference was observed between the ADMSC-CM group and the varicocelectomy group in preserving normal testicular histology according to Johnsen's Score (p=0.114). Levels of Claudin-11 were significantly higher in the varicocelectomy and ADMSC-CM groups compared to the varicocele group (p≤0.05 for all). The therapeutic effects of ADMSC-CM in varicocele model may involve secretion of anti-inflammatory and regenerative factors from ADMSC. ADMSC-CM injection appears to be a promising new strategy in the treatment of varicocele.

Cancer-associated fibroblasts reprogram cysteine metabolism to increase tumor resistance to ferroptosis in pancreatic cancer.

Background: Pancreatic ductal adenocarcinoma (PDAC) is an insidious, rapidly progressing malignancy of the gastrointestinal tract. Due to its dense fibrous stroma and complex tumor microenvironment, neither of which is sensitive to radiotherapy, pancreatic adenocarcinoma is one of the malignancies with the poorest prognosis. Therefore, detailed elucidation of the inhibitory microenvironment of PDAC is essential for the development of novel therapeutic strategies. Methods: We analyzed the association between cancer-associated fibroblasts (CAFs) and resistance to ferroptosis in PDAC using conditioned CAF medium and co-culture of pancreatic cancer cells. Abnormal cysteine metabolism was observed in CAFs using non-targeted metabolomics analysis with liquid chromatography-tandem mass spectrometry (LC-MS/MS). The regulatory effects of cysteine were investigated in PDAC cells through measurement of cell cloning, cell death, cell function, and EdU assays. The effects of exogenous cysteine intake were examined in a mouse xenograft model and the effects of the cysteine pathway on ferroptosis in PDAC were investigated by western blotting, measurement of glutathione and reactive oxygen species levels, among others. Results: It was found that CAFs played a critical role in PDAC metabolism by secreting cysteine, which could increase tumor resistance to ferroptosis. A previously unrecognized function of the sulfur transfer pathway in CAFs was identified, which increased the extracellular supply of cysteine to support glutathione synthesis and thus inducing ferroptosis resistance. Cysteine secretion by CAFs was found to be mediated by the TGF-β/SMAD3/ATF4 signaling axis. Conclusion: Taken together, the findings demonstrate a novel metabolic relationship between CAFs and cancer cells, in which cysteine generated by CAFs acts as a substrate in the prevention of oxidative damage in PDAC and thus suggests new therapeutic targets for PDAC.

Identification of circulating miRNAs as fracture-related biomarkers.

Fracture non-unions affect many patients worldwide, however, known risk factors alone do not predict individual risk. The identification of novel biomarkers is crucial for early diagnosis and timely patient treatment. This study focused on the identification of microRNA (miRNA) related to the process of fracture healing. Serum of fracture patients and healthy volunteers was screened by RNA sequencing to identify differentially expressed miRNA at various times after injury. The results were correlated to miRNA in the conditioned medium of human bone marrow mesenchymal stromal cells (BMSCs) during in vitro osteogenic differentiation. hsa-miR-1246, hsa-miR-335-5p, and miR-193a-5p were identified both in vitro and in fracture patients and their functional role in direct BMSC osteogenic differentiation was assessed. The results showed no influence of the downregulation of the three miRNAs during in vitro osteogenesis. However, miR-1246 may be involved in cell proliferation and recruitment of progenitor cells. Further studies should be performed to assess the role of these miRNA in other processes relevant to fracture healing.

Dynamic regulation of EXO1 promotes the progression from liver fibrosis to HCC through TGF-β1/Smad signaling feedback loop.

HSCs are the main stromal cells in the process of liver fibrosis and accelerate HCC progression. Previous studies determined that highly expressed exonuclease 1 (EXO1) increases the malignant behavior of HCC cells and is closely related to liver cirrhosis. This study aimed to explore the roles and mechanisms of EXO1 in the development of liver cirrhosis and HCC. We fully demonstrated that EXO1 expression was positively correlated with liver fibrosis and cirrhotic HCC by combining bioinformatics, hepatic fibrosis mouse models, and human HCC tissues. The role of EXO1 in a murine HCC model induced by activated forms of AKT and Ras oncogenes (AKT/Ras) was investigated by employing an adeno-associated virus-mediated EXO1 knockdown technique. The knockdown of EXO1 promoted a regression of HCC in AKT/Ras mice and reduced the degree of liver fibrosis. Downregulated EXO1 inhibited LX-2 cell activation and inhibited the proliferation and migration of HCC cells. Moreover, conditioned medium of LX-2 cells with EXO1 overexpression increased the proliferation and migration of HCC cells, which was attenuated after EXO1 knockout in LX-2 cells. EXO1 knockdown attenuated the role of LX-2 in promoting HepG2 xenograft growth in vivo. Mechanistically, EXO1 promotes the activation of the downstream TGF-β-smad2/3 signaling in LX-2 and HCC cells. Interestingly, increased TGF-β-smad2/3 signaling had a feedback effect on EXO1, which sustains EXO1 expression and continuously stimulates the activation of HSCs. EXO1 forms a positive feedback circuit with TGF-β-Smad2/3 signaling and promotes the activation of HSCs, which accelerates HCC progression. Those findings indicate EXO1 may be a promising target for the diagnosis and treatment of cirrhotic HCC.