Human Mesenchymal Stem Cells Research Articles

Inflammatory priming of human mesenchymal stem cells induces osteogenic differentiation via the early response gene IER3.

Mesenchymal stem cells (MSCs) have gained tremendous interest due to their overall potent pro-regenerative and immunomodulatory properties. In recent years, various invitro and preclinical studies have investigated different priming ("licensing") approaches to enhance MSC functions for specific therapeutic purposes. In this study, we primed bone marrow-derived human MSCs (hMSCs) with an inflammation cocktail designed to mimic the elevated levels of inflammatory mediators found in serum of patients with severe injuries, such as bone fractures. We observed a significantly enhanced osteogenic differentiation potential of primed hMSCs compared to untreated controls. By RNA-sequencing analysis, we identified the immediate early response 3 (IER3) gene as one of the top-regulated genes upon inflammatory priming. Small interfering RNA knockdown experiments established IER3 as a novel positive regulator of osteogenic differentiation. Mechanistic analysis further revealed that IER3 deletion significantly downregulated bone marrow stromal cell antigen 2 (BST2) expression and extracellular signal-related kinase 1/2 (ERK1/2) phosphorylation in hMSCs, suggesting that IER3 regulates osteogenic differentiation through BST2 and ERK1/2 signaling pathway activation. On the basis of these findings, we propose IER3 as a novel therapeutic target to promote hMSC osteoblastogenesis, which might be of high clinical relevance, for example, in patients with osteoporosis or compromised fracture healing.

EPO modified MSCs protects SH-SY5Y cells against ischemia/hypoxia-induced apoptosis via REST-dependent epigenetic remodeling

Hypoxic-ischemic encephalopathy (HIE) is a diffuse brain tissue injury caused by acute ischemia and hypoxia, and it is most commonly found in newborn infants but can also occur in adults. Mesenchymal stem cell (MSC) therapies have showed improved outcomes for treating HIE-induced neuronal defects. However, many key issues associated with poor cell viability and tolerance of grafted MSCs after HIE remain to be resolved. Genetic engineering could endow MSCs with more robust regenerative capacities. Our research, along with that of other scientists, has found that the expression of intracellular erythropoietin (EPO) in human umbilical cord MSCs (hUC-MSCs) increases proportionally with the duration of hypoxia exposure. Furthermore, we observed that EPO, when introduced into the EPO gene-modified hUC-MSCs, can be secreted into the extracellular space. However, the underlying mechanisms that support the neuroprotective effects of EPO-MSCs remain unclear. EPO-MSCs, hUC-MSCs, and NC-MSCs were identified by flow cytometry, osteogenic, and adipogenic differentiation assays. The oxygen-glucose deprivation (OGD)-induced SH-SY5Y cell-line was established, and five groups were set up: control, 24-h ischemia-hypoxia, co-cultured with hUC-MSCs, NC-MSCs, and EPO-MSCs after hypoxia. LEGENDplex™ multi-factor flow cytometry was used to detect the secretion of inflammatory factors in cell supernatants and cerebrospinal fluid. Chromosome-targeted excision and tagging (CUT&Tag) sequencing was applied to detect genomic H3K4me2 modifications, and conjoint analysis with transcriptome sequencing (RNA-seq) was performed. Lentiviral vector infection was used to construct SH-SY5Y cells with stable knockdown of RE1-silencing transcription factor (REST), and flow cytometry was used to detect alterations in apoptosis. Finally, the molecular mechanism underlying the neuroprotective and anti-apoptotic effects of EPO-MSCs was investigated using RNA sequencing, qRT-PCR, and western blot assays. Our results suggest that EPO-MSCs are genetically engineered to secrete significantly more EPO. EPO-MSCs treatment has anti-apoptotic properties and offers neuronal protection during ischemic-hypoxic injury. Furthermore, RNA-seq results suggest that multiple inflammation-related genes were down-regulated after EPO-MSCs treatment. Application of RNA-seq and CUT&Tag combined analysis found that the expressions of REST were significantly up-regulated. Lentiviral vector infection to construct REST knockdown SH-SY5Y failed to rescue apoptosis after hypoxia and co-culture with EPO-MSCs, and SETD2-mediated H3K36me3 protein level expression was reduced. EPO-MSCs may promote neuronal survival by affecting H3K4me2 and thus activating the expression of REST and TET3. EPO-MSCs also upregulated the modification level of SETD2-mediated H3K36me3 and regulated the expression of inflammation-related genes such as PLCG2, as well as apoptosis genes BCL2A1. To investigate the neuroprotective effects of EPO-modified hUC-MSCs and the underlying epigenetic regulatory mechanisms, this study aims to provide a theoretical foundation for the potential application of EPO gene-modified hUC-MSCs in the treatment of HIE.

Magnesium oxide nanoparticle reinforced pumpkin-derived nanostructured cellulose scaffold for enhanced bone regeneration

Considering global surge in bone fracture prevalence, limitation in use of traditional healing approaches like bone grafts highlights the need for innovative regenerative strategies. Here, a novel green fabrication approach has reported for reinforcement of physicochemical performances of sustainable bioinspired extracellular matrix (ECM) based on decellularized pumpkin tissue coated with Magnesium oxide nanoparticles (hereafter called DM-Pumpkin) for enhanced bone regeneration. Compared to uncoated scaffold, DM-Pumpkin exhibited significantly improved surface roughness, mechanical stiffness, porosity, hydrophilicity, swelling, and biodegradation rate. Obtained nanoporous structure provides an ideal three-dimensional microenvironment for the attachment, migration and osteo-induction in human adipose-derived mesenchymal stem cells (h- AdMSCs). Calcium deposition and mineralization, alkaline phosphatase activity, and SEM imaging of the cells as well as increased expression of bone-related genes after 21 days incubation confirmed capability of DM-Pumpkin in mimicking the biological properties of bone tissue. The presence of MgONPs had a silencing effect on inflammatory factors and improved wound closure, verified by in vivo studies. Increased expression of collagen type I and osteocalcin in the h- AdMSCs cultured on DM-Pumpkin compared to control further corroborated gained results. Altogether, boosting physicochemical and biological properties of DM-Pumpkin due to surface modification is a promising approach for guided bone regeneration.

Exosomes from human bone marrow MSCs alleviate PD-1/PD-L1 inhibitor-induced myocardial injury in melanoma mice by regulating macrophage polarization and pyroptosis

Myocarditis, which can be triggered by immune checkpoint inhibitor (ICI) treatment, represents a critical and severe adverse effect observed in cancer therapy. Thus, elucidating the underlying mechanism and developing effective strategies to mitigate its harmful impact is of utmost importance. The objective of this study is to investigate the potential role and regulatory mechanism of exosomes derived from human bone marrow mesenchymal stem cells (hBMSC-Exos) in providing protection against myocardial injury induced by ICIs. We observed that the administration of programmed death 1/programmed death-ligand 1 (PD-1/PD-L1) inhibitor BMS-1 in tumor-bearing mice led to evident cardiac dysfunction and myocardial injury, which were closely associated with M1 macrophage polarization and cardiac pyroptosis. Remarkably, these adverse effects were significantly alleviated through tail-vein injection of hBMSC-Exos. Moreover, either BMS-1 or hBMSC-Exos alone demonstrated the ability to reduce tumor size, while the combination of hBMSC-Exos with BMS-1 treatment not only effectively improved the probability of tumor inhibition but also alleviated cardiac anomalies induced by BMS-1.

Revolutionizing pressure ulcer regeneration: Unleashing the potential of extracellular matrix-derived temperature-sensitive injectable antioxidant hydrogel for superior stem cell therapy

Pressure ulcers are a common issue in elderly and medically compromised individuals, posing significant challenges in healthcare. Human umbilical cord mesenchymal stem cells (HUMSCs) offer therapeutic benefits like inflammation modulation and tissue regeneration, yet challenges in cell survival, retention, and implantation rates limit their clinical application. Hydrogels in three-dimensional (3D) stem cell culture mimic the microenvironment, improving cell survival and therapeutic efficacy. A thermosensitive injectable hydrogel (adEHG) combining gallic acid-modified hydroxybutyl chitosan (HBC-GA) with soluble extracellular matrix (adECM) has been developed to address these challenges. The hybrid hydrogel, with favorable physical and chemical properties, shields stem cells from oxidative stress and boosts their therapeutic potential by clearing ROS. The adEHG hydrogel promotes angiogenesis, cell proliferation, and collagen deposition, further enhancing inflammation modulation and wound healing through the sustained release of therapeutic factors and cells. Additionally, the adEHG@HUMSC composite induces macrophage polarization towards an M2 phenotype, which is crucial for wound inflammation inhibition and successful healing. Our research significantly propels the field of stem cell-based therapies for pressure ulcer treatment and underscores the potential of the adEHG hydrogel as a valuable tool in advancing regenerative medicine.

The extracellular matrix with a continuous gradient of SDF1α guides the oriented migration of human umbilical cord mesenchymal stem cells

The extracellular matrix (ECM) plays a crucial role in maintaining cell morphology and facilitating intercellular signal transmission within the human body. ECM has been extensively utilized for tissue injury repair. However, the consideration of factor gradients during ECM preparation has been limited. In this study, we developed a novel approach to generate sheet-like ECM with a continuous gradient of stromal cell-derived factor-1 (SDF1α). Briefly, we constructed fibroblasts to overexpress SDF1αfused with the collagen-binding domain (CBD-SDF1α), and cultured these cells on a slanted plate to establish a gradual density cell layer at the bottom surface. Subsequently, excess parental fibroblasts were evenly distributed on the plate laid flat to fill the room between cells. Following two weeks of culture, the monolayer cells were lyophilized to form a uniform ECM sheet possessing a continuous gradient of SDF1α. This engineered ECM material demonstrated its ability to guide oriented migration of human umbilical cord mesenchymal stem cells on the ECM sheet. Our simple yet effective method holds great potential for advancing research in regenerative medicine.

Effect of the polyphenol flavonoids fisetin and quercetin on the adipogenic differentiation of human mesenchymal stromal cells.

Fisetin and quercetin, polyphenol flavonoids, have been shown to have a wide range of beneficial pharmacological effects including anti-inflammatory, anti-oxidative, and anti-cancer. Our previous work shows that fisetin also affects the specification of the adipogenic-osteogenic lineage of human mesenchymal stem cells (hMSCs) by modulating the Hippo-YAP signaling pathway. Although quercetin has a structure similar to that of fisetin, its effects on the functional properties of hMSCs have not yet been investigated. The objective of this study is to determine the effects of quercetin on the various properties of hMSCs, including proliferation, migration, and differentiation capacity toward adipogenic and osteogenic lineages. The results show that while fisetin increases hMSC adipogenic differentiation, quercetin inhibited adipogenic differentiation of hMSCs. The inhibition is mediated, at least in part, by the activation of hippo signaling and up-regulation of miR-27b, which inhibits the expression of genes involved in all critical steps of lipid droplet biogenesis, resulting in a decrease in the number of lipid droplets in hMSCs. It is possible that the lack of hydroxylation of the 5 position on the A ring of quercetin could be responsible for its different effect on the adipogenic-osteogenic lineage specification of hMSCs compared to fisetin. Molecular docking and molecular dynamics simulation suggested that fisetin and quercetin possibly bind to serine / threonine protein kinases 4 (STK4/MST1), which is an upstream kinase responsible for LATS phosphorylation. Taken together, our results demonstrate more insight into the mechanism underlying the role of flavonoid fisetin and quercetin in the regulation of adipogenesis.

Mechanistic understanding of enhancing bioactivity via bio-ionic liquid functionalization of biomaterials

The selection of biomaterials is a pivotal criterion for designing tissue engineering scaffolds, crucial for diverse biomedical applications. While natural biomaterials offer inherent bioactivity and biocompatibility, fine-tuning their properties for specific applications poses challenges. Conversely, synthetic biomaterials, while highly customizable, are often bio-inert. Functionalizing materials with bioactive molecules can improve cell-material interactions. Prevailing approaches utilizing RGD peptides or natural/synthetic composites have drawbacks including cost and immunogenicity. Bio-ionic liquids (BILs), a class of biocompatible ionic liquids derived from natural or synthetic biomolecules, offer a potential alternative. Herein, we present the biofunctionalization of a bio-inert polyethylene glycol diacrylate (PEGDA) with an acrylated choline-based BIL (“BioPEG”) as a method for enhancing the bioactivity of biomaterials. Cell studies using mouse myoblast C2C12 and human mesenchymal stem cells (hMSCs) revealed the excellence of BioPEG hydrogels in promoting cell attachment, growth, proliferation, and differentiation. Computational molecular dynamics (MD) simulations elucidated the mechanism by which BIL facilitates cell interaction with PEGDA. 3D printability of BioPEG hydrogel was showcased using a digital light processing (DLP) bioprinter, with printed scaffolds demonstrating excellent cytocompatibility. Collectively, these findings present BIL as a versatile bioactive agent for augmenting cell adhesion to materials, thus enriching the repertoire of biomaterials for advanced tissue engineering.

Human amniotic membrane hydrogel loaded with exosomes derived from human placental mesenchymal stem cells accelerate diabetic wound healing

Diabetic wound is one of the most common and costly complication in diabetic patients. Hence, numerous studies have been carried out to discover a suitable approach to enhance the process of wound healing. Biological hydrogels are commonly utilized for wound healing due to their suitable properties among different materials available. Herein we investigated whether human amniotic membrane hydrogel (hAMH) loaded with human placental mesenchymal stem cells (PlaMSCs)-derived exosomes could promote healing in diabetic rats. Sixty diabetic rats were randomly assigned into the control group, hAMH group, exosome group, and hAMH+Exosome group. According to the phases of wound healing, sampling was done on days 7, 14, and 21 for further assessments. Our findings showed a significant increase in wound contraction rate, new epidermal length, fibroblast and blood vessel count, collagen density, and the levels of antioxidative factors (GSH, SOD, and CAT) in the treatment groups compared to the control group, with more pronounced effects observed in the hAMH+Exosome group. Furthermore, the levels of bFGF and VEGF gene expression significantly increased in each treatment group when compared to the control group, with the highest levels observed in the hAMH+Exosome group. This occurred as the hAMH+Exosome group showed a greater decrease in neutrophil count, the expression of TNF-α and IL-1β genes, and the levels of an oxidative factor (MDA) compared to the other groups. In summary, the combination of hAMH and PlaMSCs-derived exosomes was determined to have a more significant effect on healing diabetic wounds.

Determination of adipogenesis stages of human umbilical cord-derived mesenchymal stem cells using three-dimensional label-free holotomography

Adipogenesis involves complex changes in gene expression, morphology, and cytoskeletal organization. However, the quantitative analysis of live cell images to identify their stages through morphological markers is limited. Distinct adipogenesis markers on human umbilical cord-derived mesenchymal stem cells (UC-MSCs) were identified through holotomography, a label-free live cell imaging technique. In the MSC-to-preadipocyte transition, the nucleus-to-cytoplasm ratio (0.080 vs. 0.052) and lipid droplet (LD) refractive index variation decreased (0.149 % vs. 0.061 %), whereas the LD number (20 vs. 65) increased. This event was also accompanied by the downregulation and upregulation of THY1 and Preadipocyte Factor-1 (PREF-1), respectively. In the preadipocyte to immature adipocyte shift, cell sphericity (0.20 vs. 0.43) and LD number (65 vs. 200) surged, large LDs (>10 μm3) appeared, and the major axis of the cell was reduced (143.7 μm vs. 83.12 μm). These findings indicate features of preadipocyte and immature adipocyte stages, alongside the downregulation of PREF-1 and upregulation of Peroxisome Proliferator-Activated Receptor gamma (PPARγ). In adipocyte maturation, along with PPARγ and Fatty Acid-Binding Protein 4 upregulation, cell compactness (0.15 vs. 0.29) and sphericity (0.43 vs. 0.59) increased, and larger LDs (>30 μm3) formed, marking immature and mature adipocyte stages. The study highlights the distinct adipogenic morphological biomarkers of adipogenesis stages in UC-MSCs, providing potential applications in biomedical and clinical settings, such as fostering innovative medical strategies for treating metabolic disease.

Direct conversion of human umbilical cord mesenchymal stem cells into dopaminergic neurons for Parkinson's disease treatment

Parkinson's disease (PD) is a neurodegenerative disorder characterized by motor deficits due to the depletion of nigrostriatal dopamine. Stem cell differentiation therapy emerges as a promising treatment option for sustained symptom relief. In this study, we successfully developed a one-step differentiation system using the YFBP cocktail (Y27632, Forskolin, SB431542, and SP600125) to effectively convert human umbilical cord mesenchymal stem cells (hUCMSCs) into dopaminergic neurons without genetic modification. This approach addresses the challenge of rapidly and safely generating functional neurons on a large scale. After a 7-day induction period, over 80 % of the cells were double-positive for TUBB3 and NEUN. Transcriptome analysis revealed the dual roles of the cocktail in inducing fate erasure in mesenchymal stem cells and activating the neuronal program. Notably, these chemically induced cells (CiNs) did not express HLA class II genes, preserving their immune-privileged status. Further study indicated that YFBP significantly downregulated p53 signaling and accelerated the differentiation process when Pifithrin-α, a p53 signaling inhibitor, was applied. Additionally, Wnt/β-catenin signaling was transiently activated within one day, but the prolonged activation hindered the neuronal differentiation of hUCMSCs. Upon transplantation into the striatum of mice, CiNs survived well and tested positive for dopaminergic neuron markers. They exhibited typical action potentials and sodium and potassium ion channel activity, demonstrating neuronal electrophysiological activity. Furthermore, CiNs treatment significantly increased the number of tyrosine hydroxylase-positive cells and the concentration of dopamine in the striatum, effectively ameliorating movement disorders in PD mice. Overall, our study provides a secure and reliable framework for cell replacement therapy for Parkinson's disease.

Influence of budesonide and fluticasone propionate in the anti-osteoporotic potential in human bone marrow-derived mesenchymal stem cells via stimulation of osteogenic differentiation

Osteoporosis is a prevalent bone condition with adverse effects observed in patients undergoing long-term glucocorticoid therapy, resulting in bone demineralization and tissue loss. There has been limited studies on the global response to dexamethasone in terms of comparing its expression profile to other common glucocorticoids during osteogenic differentiation. This study focused on the downregulated gene expression profile of glucocorticoid compounds; dexamethasone, budesonide, and fluticasone propionate, during osteogenic differentiation to elucidate the related target genes and pathways associated with the anti-osteoporotic potential of telomerase-immortalized human bone marrow-derived mesenchymal stem cells using a bioinformatics approach. Based on gene expression microarrays experiments and bioinformatics analysis, several key genes involved in the regulation of osteogenic differentiation and osteoporosis development in mesenchymal stem cells that were targeted by these specific glucocorticoids were determined. Network analysis using GeneCards, OMIM, and CTD databases were performed and osteoporosis-related genes were identified. LIMMA and moderated Welch test R packages were performed to determine significant downregulated differentially expressed genes for each glucocorticoid treatment. A total of 479 (dexamethasone), 84 (budesonide), and 889 (fluticasone propionate) differentially expressed genes were identified for each glucocorticoid, of which 35 common genes overlapped. Enrichment pathway analysis was conducted using Metascape, and protein-protein interaction networks were constructed using the STRING database and Cytoscape software to determine potential target genes involved with osteoporosis. Enrichment pathway analysis revealed genes involved in 3 Reactome pathways namely cytokine signaling in immune system, immune system and the interferon alpha/beta signaling pathways and identified 10 hub genes based on the PPI network to determine potential target pathways associated with osteoporosis. These findings provide preliminary insights into the relationship between the key target genes of dexamethasone, budesonide, and fluticasone propionate, and the pathways associated with regulated osteoporosis metabolism during osteogenic differentiation.

Human Umbilical Cord Mesenchymal Stem Cell Exosome-derived miR-335-5p Alleviated Lipopolysaccharide-induced Acute Lung Injury by Regulating the m6A Level of ITGβ4 Gene.

Acute lung injury (ALI) is a serious complication that may accompany severe pneumonia in children. Derived from human umbilical cord mesenchymal stem cell exosome (HucMSC-Exo) can contribute to the regeneration of damaged lung tissue. This study aims to investigate the impact of HucMSC-Exo on ALI and its potential mechanisms. Firstly, RT-qPCR was performed to assess the expression of miR-335-5p. Subsequently, Pearson correlation analysis was performed to examine the correlation between METTL14 and miR-335-5p, as well as the correlation between METTL14 and ITGβ4, while RNA immunoprecipitation (RIP) was used to determine the m6A modification level of ITGβ4. Additionally, molecular biology techniques were employed to evaluate the expression of glycolysis-related factors. Definitively, an LPS-induced ALI model was established to investigate the effect of miR-335-5p on mice lung tissue. miR-335-5p was found to be highly expressed in HucMSC-Exo. Transfection with miR-335-5p mimics resulted in increased glucose uptake. Pearson correlation analysis revealed a negative correlation between METTL14 and miR-335-5p, as well as between METTL14 and ITGβ4. The m6A level of ITGβ4 was elevated in ALI. Overexpression of METTL14 was found to reduce the expression of ITGβ4 and glucose levels, while overexpression of ITGβ4 reversed the effects of METTL14 overexpression. In vivo, results demonstrated that miR-335-5p could improve the extent of lung tissue lesions and reduce glycolytic levels. This study revealed the mechanism by which miR-335-5p derived from HucMSC-Exo could alleviate LPS-induced ALI by regulating the m6A modification of ITGβ4, providing a new direction for the treatment of ALI.

NaV1.1 contributes to the cell cycle of human mesenchymal stem cells by regulating AKT and CDK2.

Non-excitable cells express sodium voltage-gated channel alpha subunit 1 gene and protein (known as SCN1A and NaV1.1, respectively); however, the functions of NaV1.1 are unclear. In this study, we investigated the role of SCN1A and NaV1.1 in human mesenchymal stem cells (MSCs). We found that SCN1A was expressed in MSCs, and abundant expression of NaV1.1 was observed in the endoplasmic reticulum; however, this expression was not found to be related to Na+ currents. SCN1A-silencing reduced MSC proliferation and delayed the cell cycle in the S phase. SCN1A silencing also suppressed the protein levels of CDK2 and AKT (herein referring to total AKT), despite similar mRNA expression, and inhibited AKT phosphorylation in MSCs. A cycloheximide-chase assay showed that SCN1A-silencing induced CDK2 but not AKT protein degradation in MSCs. A proteolysis inhibition assay using epoxomicin, bafilomycin A1 and NH4Cl revealed that both the ubiquitin-proteasome system and the autophagy and endo-lysosome system were irrelevant to CDK2 and AKT protein reduction in SCN1A-silenced MSCs. The AKT inhibitor LY294002 did not affect the degradation and nuclear localization of CDK2 in MSCs. Likewise, the AKT activator SC79 did not attenuate the SCN1A-silencing effects on CDK2 in MSCs. These results suggest that NaV1.1 contributes to the cell cycle of MSCs by regulating the post-translational control of AKT and CDK2.

Targeting PAR2-mediated inflammation in osteoarthritis: a comprehensive in vitro evaluation of oleocanthal’s potential as a functional food intervention for chondrocyte protection and anti-inflammatory effects

BackgroundOsteoarthritis (OA) is a prevalent degenerative joint disease characterized by chronic inflammation and progressive cartilage degradation, ultimately leading to joint dysfunction and disability. Oleocanthal (OC), a bioactive phenolic compound derived from extra virgin olive oil, has garnered significant attention due to its potent anti-inflammatory properties, which are comparable to those of non-steroidal anti-inflammatory drugs (NSAIDs). This study pioneers the investigation into the effects of OC on the Protease-Activated Receptor-2 (PAR-2) mediated inflammatory pathway in OA, aiming to validate its efficacy as a functional food-based therapeutic intervention.MethodsTo simulate cartilage tissue in vitro, human bone marrow-derived mesenchymal stem cells (BMSCs) were differentiated into chondrocytes. An inflammatory OA-like environment was induced in these chondrocytes using lipopolysaccharide (LPS) to mimic the pathological conditions of OA. The therapeutic effects of OC were evaluated by treating these inflamed chondrocytes with various concentrations of OC. The study focused on assessing key inflammatory markers, catabolic enzymes, and mitochondrial function to elucidate the protective mechanisms of OC. Mitochondrial function, specifically mitochondrial membrane potential (ΔΨm), was assessed using Rhodamine 123 staining, a fluorescent dye that selectively accumulates in active mitochondria. The integrity of ΔΨm serves as an indicator of mitochondrial and bioenergetic function. Additionally, Western blotting was employed to analyze protein expression levels, while real-time polymerase chain reaction (RT-PCR) was used to quantify gene expression of inflammatory cytokines and catabolic enzymes. Flow cytometry was utilized to measure cell viability and apoptosis, providing a comprehensive evaluation of OC’s therapeutic effects on chondrocytes.ResultsThe results demonstrated that OC significantly downregulated PAR-2 expression in a dose-dependent manner, leading to a substantial reduction in pro-inflammatory cytokines, including TNF-α, IL-1β, and MCP-1. Furthermore, OC attenuated the expression of catabolic markers such as SOX4 and ADAMTS5, which are critically involved in cartilage matrix degradation. Importantly, OC was found to preserve mitochondrial membrane potential (ΔΨm) in chondrocytes subjected to inflammatory stress, as evidenced by Rhodamine 123 staining, indicating a protective effect on cellular bioenergetics. Additionally, OC modulated the Receptor Activator of Nuclear Factor Kappa-Β Ligand (RANKL)/Receptor Activator of Nuclear Factor Kappa-Β (RANK) pathway, suggesting a broader therapeutic action against the multifactorial pathogenesis of OA.ConclusionsThis study is the first to elucidate the modulatory effects of OC on the PAR-2 mediated inflammatory pathway in OA, revealing its potential as a multifaceted therapeutic agent that not only mitigates inflammation but also protects cartilage integrity. The preservation of mitochondrial function and modulation of the RANKL/RANK pathway further underscores OC’s comprehensive therapeutic potential in counteracting the complex pathogenesis of OA. These findings position OC as a promising candidate for integration into nutritional interventions aimed at managing OA. However, further research is warranted to fully explore OC’s therapeutic potential across different stages of OA and its long-term effects in musculoskeletal disorders.

Key Magnetized Exosomes for Effective Targeted Delivery of Doxorubicin Against Breast Cancer Cell Types in Mice Model.

Exosomes (Exos) are promising drug delivery systems due to their low immunogenicity, minimal toxicity, high biocompatibility, and effective delivery capabilities. However, addressing the cardiotoxicity and other toxic side effects associated with anthracyclines has proven challenging. In this study, we loaded doxorubicin (Dox) into Exos derived from human placental mesenchymal stem cells (MSCs) and modified them with carboxylated Fe3O4 nanoparticles (NPs) to create an Exo-Dox-NP delivery system. Using an external magnetic force (MF), we regulated the distribution of Exos for targeted Dox delivery in breast cancer treatment. We characterized and determined the drug-loading efficiency of Exo-Dox-NPs, their uptake by tumor cells, and the modulation of drug release. The therapeutic efficacy of Exo-Dox-NPs was evaluated through both in vitro and in vivo anti-tumor experiments. Our results indicated that Exo-Dox-NPs remain stable in the bloodstream while releasing the drug in the acidic environment of tumor cells and their lysosomes. As a drug delivery system, Exo-Dox-NPs enhanced Dox absorption by tumor cells, demonstrating high targeting specificity. Moreover, Exo-Dox-NPs inhibited the migration of breast cancer cells, as confirmed by scratch migration and Transwell Matrigel invasion assays. In vivo experiments confirmed the effective targeting and delivery of Dox to malignant tumors using Exo-Dox-NPs/MFs, with the Exo-Dox-NP/MF formulation exhibiting the most potent anti-tumor activity. The utilization of Exos as carriers for Dox showed promising efficacy in breast cancer management. Carboxylated Fe3O4 NPs demonstrated to be suitable targeting agents, potentially advancing the development of natural nanocarriers for combination cancer therapy.

HUC-MSC-derived exosomes repaired the damage induced by hydroquinone to 16HBE cells via miR-221/PTEN pathway

Mesenchymal stem cell - originated exosomes (MSC-exo) are promising non-cellular treatment agents for various diseases. The present study aimed to explore whether human umbilical cord MSC - originated exosomes (HUC-MSC-exo) have the function of protecting human cells (16HBE) against the damage caused by HQ and the related mechanism. HUC-MSC-exo was isolated with differential gradient ultracentrifugation method and characterized by using transmission electron microscope (TEM). 16HBE cells were used as the tool cells and co-cultured with HUC-MSC-exo. Confocal laser scanning microscope was employed to confirm the ingestion of HUC-MSC-exo by 16HBE. Cell proliferation, migration, oxidative stress, DNA and chromosome damages of 16HBE were analyzed under HQ stress, and the role of miR-221/PTEN axis was investigated. Our data showed that under HQ stress, different groups of cells exhibited significantly decreased proliferation and migration abilities, and significant oxidative stress, DNA and chromosome damage effects. HUC-MSC-exo could alleviate the cytotoxic, oxidative stress and genotoxic damage effects of HQ on 16HBE cells. Mechanistically, HQ exposure up-regulated the level of miR-221 and down-regulated PTEN, while HUC-MSC-exo could significantly reduce the level of miR-221 and promote PTEN expression, which was involved in alleviating the toxic effects of HQ on 16HBE cells. Our data indicates that HUC-MSC-exo can alleviate the oxidative stress, cytotoxic and genotoxic effects of HQ on 16HBE cells via miR-221/PTEN pathway, and it may be a promising agent for protecting against the toxicity of HQ.

Piezoelectric barium titanate/hydroxyapatite composite coatings on Ti-6Al-4V alloy via electrophoretic deposition

Electrophoretic deposition (EPD) is a widely accepted, cost-effective and simple method to obtain conformal coatings of dense nanoparticles via application of a DC voltage. This paper reports the physical and mechanical properties of nanostructured barium titanate/hydroxyapatite (BT/HA) ceramic composites coated onto Ti-6Al-4V alloy via cathodic EPD in various ratios of 40:60, 50:50 and 60:40 (HB4, HB5 and HB6) respectively. Homogenous BT/HA coatings were obtained at a notably low voltage of 10 V. The crystallinity and phase analysis confirmed the formation of tetragonal phase of BT indicating the piezoelectric property. HB6 exhibits maximum piezoelectric coefficient (d33) of 159 pC/N and Vicker's hardness of 242.31 HV. The cross-sectional electron micrographs show well connected and homogeneous coatings with increasing amounts of BT. Human mesenchymal stem cell lines were utilized in biocompatibility experiments, which revealed that HB composites had greater viability of HW-MSCs cells than pure BT, with HB6 exhibiting a maximum cell viability of more than 90 %.

Using qPCR and ddPCR to study biodistribution of cell therapy products: a multi-site evaluation

Backgroud aimsRegenerative therapies employing cell therapy products (CTPs) have attracted considerable attention. Biodistribution (BD) evaluation of CTPs is mainly performed to clarify the cell survival time, engraftment, and distribution site. This evaluation is crucial for predicting the efficacy and safety profiles of clinical studies based on non-clinical BD study outcomes. However, no internationally unified method has been established for assessing cell BD after administration. Here, we aimed to standardize the BD assay method used for CTPs, conducting the following evaluations using the same protocol across multiple study facilities: (1) in vitro validation of quantitative polymerase chain reaction (qPCR) and droplet digital PCR (ddPCR) analyses using the primate-specific Alu gene, and (2) in vivo BD studies after the intravenous administration of human mesenchymal stem cells (hMSCs) to immunodeficient mice, commonly used in non-clinical tumorigenicity studies. MethodsQuality control samples were prepared and analyzed by adding a fixed number of human-derived cells to several mouse tissues. The respective quantitative performances of the qPCR and ddPCR methods were compared for accuracy and precision. hMSCs were intravenously administered to immunodeficient mice, and tissues were collected at 1, 4, and 24 h after administration. ResultsBoth methods demonstrated an accuracy (relative error) generally within ±50% and a precision (coefficient of variation) generally less than 50%. While differences in calibration curve ranges were observed between qPCR and ddPCR, no significant differences in quantification were found among the assay facilities. The BD of hMSCs in mice was evaluated at seven facilities (qPCR at three facilities; ddPCR at four facilities), revealing similar tissue distribution profiles in all facilities, with the lungs showing the highest cell distribution among the tissues tested. ConclusionsQuantitative evaluation of qPCR and ddPCR using Alu sequences was conducted, demonstrating that the test method can be adapted for BD evaluation.

Nervonic acid improves fat transplantation by promoting adipogenesis and angiogenesis.

Adipose tissue engraftment has become a promising strategy in the field of regenerative surgery; however, there are notable challenges associated with it, such as resorption of 50‑90% of the transplanted fat or cyst formation due to fat necrosis after fat transplantation. Therefore, identifying novel materials or methods to improve the engraftment efficiency is crucial. The present study investigated the effects of nervonic acid (NA), a monounsaturated very long‑chain fatty acid, on adipogenesis and fat transplantation, as well as its underlying mechanisms. To assess this, NA was used to treat cells during adipogenesis in vitro, and the expression levels of markers, including PPARγ and CEBPα, and signaling molecules were detected through reverse transcription‑quantitative PCR and western blotting. In addition, NA was mixed with fat grafts in in vivo fat transplantation, followed by analysis through Oil Red O staining, hematoxylin & eosin staining and immunohistochemistry. It was demonstrated that NA treatment accelerated adipogenesis through activation of the Akt/mTOR pathway and inhibition of Wnt signaling. NA treatment enriched the expression of Akt/mTOR signaling‑related genes, and increased the expression of genes involved in angiogenesis and fat differentiation in human mesenchymal stem cells (MSCs). Additionally, NA effectively improved the outcome of adipose tissue engraftment in mice. Treatment of grafts with NA at transplantation reduced the resorption of transplanted fat and increased the proportion of perilipin‑1+ adipocytes with a lower portion of vacuoles in mice. Moreover, the NA‑treated group exhibited a reduced pro‑inflammatory response and had more CD31+ vessel structures, which were relatively evenly distributed among viable adipocytes, facilitating successful engraftment. In conclusion, the present study demonstrated that NA may not only stimulate adipogenesis by regulating signaling pathways in human MSCs, but could improve the outcome of fat transplantation by reducing inflammation and stimulating angiogenesis. It was thus hypothesized that NA could serve as an adjuvant strategy to enhance fat engraftment in regenerative surgery.