Search Results (117)

Background/Objectives: The effects of silver-coated dressing on wound healing, including cytotoxicity, are controversial due to the limited and incongruous results of in vitro versus in vivo research. Multiple factors intervene in wound healing processes and scarring, including pro/anti-inflammatory and pro/anti-fibrosis markers. Herein, to elucidate reported differences between in vitro and in vivo results, the effects of silver-coated dressing on 2D and 3D mono- and cocultures of fibroblasts and adipose-derived stem cells (ADSC) were investigated. Methods: Migration profiles in 2D and 3D assays, α-smooth muscle actin and proliferation marker Ki-67 expression, TGF-β1, TGF-β3, IL-6 and IL-10 levels and/or gene expression were assessed on four culture constructs. Results: In 2D systems at 24 h, silver-treated ADSC monocultures displayed better migration abilities compared to cocultures with high fibroblast ratio. In contrast, changes in the sprouting pattern between treated and untreated samples were non-significant in 3D constructs. TGFβ-1 levels decreased post-treatment, while TGFβ-3 increased, especially in 3D models. IL-6 gene expression was up-regulated following silver exposure in 3D models, mainly for stem cells in mono- and cocultures. Conclusions: Experiment data on 3D constructs suggest that silver-coated dressings do not significant impede wound healing, whereas cytotoxic effects were more pronounced in the 2D cultures. These inconsistencies, also noted in the literature, invite a methodological discussion of the 2D setup implications, recommending 3D constructs as a more appropriate evaluation standard where observable effects are closer to in vivo conditions and more relevant for transfer to clinical applications. Full article

Purpose: Castration-resistant prostate cancer (CRPC) responds poorly to conventional chemotherapy. We evaluated a cell-based enzyme–prodrug therapy using adipose-derived stem cells (ADSCs) engineered to express cytosine deaminase (CD) or carboxylesterase 2 (CE2), paired with their respective prodrugs 5-fluorocytosine (5-FC) or irinotecan (CPT-11), to compare their antitumor efficacy. Materials and Methods: Human telomerase reverse transcriptase (hTERT)-immortalized ADSCs were transduced with CD or CE2, and transgene expression and stem cell phenotype were confirmed. CD expression was verified at the transcript level and by functional 5-FC-to-5-fluorouracil (5-FU) conversion, whereas CE2 expression was verified by transcript analysis and immunoblotting. Tumor tropism toward PC3 prostate cancer cells was tested using migration assays and analysis of chemoattractant ligand/receptor expression. Prodrug-induced self-killing and bystander tumor cell killing were assessed through viability assays and co-culture with PC3 cells. For the CE2/CPT-11 system, SN-38 was not directly quantified; functional activity was inferred from prodrug-dependent cytotoxicity and in vivo efficacy. In vivo efficacy was evaluated in nude mice with PC3 tumors treated systemically with engineered ADSCs plus prodrug. Results: CD- and CE2-expressing ADSCs were successfully established and retained mesenchymal stem cell (MSC) characteristics. Both cell types exhibited significant migration toward PC3 cells. The CE2/CPT-11 system produced stronger prodrug-mediated cytotoxicity than CD/5-FC, with CE2-modified ADSCs showing higher sensitivity to CPT-11 and inducing greater apoptosis in co-cultured PC3 cells. In vivo, both treatments suppressed tumor growth, but CE2/CPT-11 achieved greater inhibition (tumor volume ~26% of control vs. ~32% for CD/5-FC at day 14). No overt clinical toxicity was observed based on body weight and daily clinical monitoring; however, hematology/serum chemistry were not assessed. Conclusions: Engineered ADSCs home to CRPC tumors and enable local prodrug activation, producing significant antitumor effects. Within the constraints of our in vitro assays and subcutaneous xenograft model, CE2/CPT-11 demonstrated stronger efficacy outcomes than CD/5-FC. Mechanistic attribution to intratumoral SN-38 exposure should be confirmed by direct metabolite measurements in future studies. Full article

The number of pet dogs is increasing, and the number of working dogs (e.g., guide dogs, police dogs) is also gradually increasing. Skin wounds are a common clinical problem in dogs and tend to be more common in the clinic as mechanical wounds. The healing process of skin wounds is often influenced by a variety of factors, including infection, nutritional status, and immune response, while wound healing is more difficult in dogs with diabetes or aging dogs. Mesenchymal stem cells (MSCs) play an important role in skin healing and regeneration with their multidirectional differentiation potential and immunomodulatory function. However, the application of MSCs alone for the treatment of skin wounds may have certain limitations, such as low cell survival and a lack of localization. Therefore, it is important to find methods that can enhance the therapeutic effect of MSCs. Secreted protein acidic and rich in cysteine (SPARC), an extracellular matrix protein widely involved in regulating biological processes such as cell proliferation, migration, and matrix production, may enhance the efficacy of MSCs in skin wound healing. This study aims to systematically evaluate the therapeutic efficacy of SPARC-overexpressing adipose-derived mesenchymal stem cells (ADSCs) in promoting skin wound healing by establishing wound models in normal, diabetic, and aged mice and dogs, thereby validating their potential under diverse physiological and pathological conditions. For in vitro validation, we used hydrogen peroxide (H₂O₂) to induce Human Umbilical Vein Endothelial Cell (HUVEC) and Human Keratinocyte Cell (HaCaT) injury. All animals were randomly assigned to six experimental groups as follows: (1) Model group: Untreated wound (negative control); (2) HY group: Hydrogel alone (vehicle control); (3) Con group: Control-ADSCs (cell control); (4) Con-Exo&HY group: Control-ADSC exosomes in hydrogel; (5) SPARC group: oe-SPARC-ADSCs (treatment); (6) SPARC-Exo&HY group: oe-SPARC-ADSC exosomes in hydrogel (treatment). Separately, HUVEC and HaCaT cells were assigned to four experimental conditions: a blank control group, a model group, a control-ADSC-treated group, and an oe-SPARC-ADSC-treated group. ADSCs modified by SPARC significantly promoted re-epithelialization integrity, collagen deposition, inflammation reduction, angiogenesis, and hair follicle regeneration during wound healing in dog skin. HUVEC and HaCaT cells proliferated after adding oe-SPARC-ADSCs cell supernatant. Meanwhile, quantitative proteomic sequencing data analysis showed that SPARC could promote skin wound healing by enhancing cell adhesion, hyaluronic acid binding, and vascular smooth muscle contraction of ADSCs. Both in vitro cellular assays and in vivo wound-healing models suggest that the combination of SPARC and ADSCs for the treatment of skin wounds has broad application prospects. Full article

Background: Peripheral nerve regeneration relies on Schwann cell activation and neurotrophic support. Although adipose-derived stem cells (ADSCs) show therapeutic potential through paracrine mechanisms, their clinical application is often limited by donor-dependent heterogeneity in therapeutic efficacy. Accordingly, strategies to standardize and potentiate their secretory function are essential. This study investigated a safety-optimized strategy to achieve this by combining three-dimensional (3D) spheroid culture with ibudilast, a clinically approved phosphodiesterase inhibitor. Methods: Human ADSCs were cultured in 2D or 3D conditions with varying ibudilast concentrations. Safety was confirmed via CCK-8 assays, and trophic factor secretion was quantified by RT-qPCR and ELISA. To rigorously validate functional outcomes, conditioned media were applied to a dual-model system comprising immortalized rat (RSC96) and primary human Schwann cells (HSwCs), assessing migration and the expression of regeneration-associated genes. Results: Ibudilast demonstrated no cytotoxicity. While 3D culture alone enhanced secretion compared to 2D controls, the addition of ibudilast provided a synergistic boost, resulting in a 6- to 14-fold increase in NGF, VEGF, and IGF-1 levels compared to 3D spheroids alone. Notably, conditioned media from these primed spheroids significantly accelerated HSwCs migration and induced robust upregulation of myelination-related genes (specifically PMP22 and EGR2), with trophic effects sustained for up to 72 h. Conclusions: Ibudilast-primed 3D spheroids synergistically amplify the neuroregenerative secretome of ADSCs. By utilizing a repurposed, safe small molecule to overcome functional variability and maximize potency without genetic manipulation, this strategy represents a highly translatable candidate for peripheral nerve repair. Full article

Stem cells can selectively migrate toward cancer cells, and therapeutic genes can be introduced into stem cells. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in cancer cells without harming normal cells. In this study, we evaluated the inhibition of tumor growth in castration-resistant prostate cancer (CRPC) using human adipose-derived stem cells (ADSCs) engineered to express cytosine deaminase (CD) and soluble TRAIL (sTRAIL), combined with the prodrug 5-fluorocytosine (5-FC). An immortalized human ADSC line (hTERT-ADSC) was transduced with a lentiviral vector encoding CD and sTRAIL, generating ADSC.CD.sTRAIL cells. Expression of chemoattractant ligands and receptors was assessed by RT-PCR. The suicide gene effect was evaluated by 5-FC treatment, measuring cell viability and apoptosis markers in vitro. A subcutaneous CRPC mouse model was used for in vivo studies. ADSC.CD.sTRAIL cells showed enhanced migration toward prostate cancer cells. Treatment with 5-FC significantly reduced cell viability, and co-culture with PC3 cells plus 5-FC increased apoptosis marker expression. In vivo, mice treated with ADSC.CD.sTRAIL and 5-FC had significantly smaller tumor volumes than control groups, with no treatment-related toxicity observed. These findings suggest that ADSCs overexpressing CD and sTRAIL, combined with 5-FC, effectively inhibit CRPC tumor growth and represent a promising targeted therapeutic strategy. Full article

Diabetic nephropathy (DN) is the most common cause of kidney failure worldwide. Mesenchymal stromal cells (MSCs) have demonstrated promise for treating DN by promoting kidney repair and regulating inflammation. Allogeneic (Allo)-MSCs may have similar or superior anti-inflammatory effects to autologous (Auto)-MSCs but also have potential to elicit adverse immune responses due to major histocompatibility complex (MHC) mismatches. To better understand how MSC-delivered allo-antigens influence therapeutic effects of Allo-MSCs compared to Auto-MSCs in DN, lentiviral transduction was used to generate adipose-derived MSCs (ADSCs) from DBA/2J (H-2^d) mice which expressed an allogeneic class I MHC protein (H-2K^b). H-2K^b-ADSCs were injected intravenously into male DBA/2J mice at 11 and 13 weeks after initiation of diabetes, and their effects on renal functional and structural indices were compared at week 15 with those of diabetic DBA/2J recipients of vehicle alone or of empty vector-transduced DBA/2J ADSCs (EV-ADSCs). Both EV-ADSCs and H-2K^b-ADSCs resulted in reduced kidney/total body weight ratio, blood urea nitrogen (BUN), urine albumin creatinine ratio (uACR), mesangial matrix expansion (MME) and renal fibrosis compared to vehicle alone, without influencing glycemia or survival. However, H-2K^b-ADSCs recipients had greater reductions in BUN and uACR, reduced intra-renal myeloid cell infiltration, increased splenic regulatory T cell (Treg) proportions and increased intra-renal Treg infiltration and FOXP3 and IL-10 mRNA. Nonetheless, recipients of H-2K^b-ADSCs also had decreased splenic CD4/CD8 T cell ratios, increased circulating anti-H-2K^b IgG antibodies and histological and biochemical evidence of inflammatory liver injury. These novel findings demonstrated that ADSCs expressing an MHC-I allo-antigen had superior beneficial effects on DN than fully autologous ADSCs. Improved DN severity was associated with immune modulation, including Treg enhancement, but also had potentially detrimental immunological effects in mice with established diabetes. The results highlight the need for further investigation of the immune modulatory effects of Allo-MSCs in diabetes and its organ-specific complications. Full article

Breast cancer remains the most common malignancy worldwide and the leading cause of cancer-related mortality. Recently, extracellular vesicles (EVs) derived from adipose tissue-derived stem cells (ADSCs) have attracted increasing attention for their potential to modulate inflammatory signaling and influence tumor cell behavior. This in vitro study was designed to investigate the effects of ADSC-EVs on MCF-7 breast cancer cells. EVs were isolated from ADSC culture supernatants and applied to MCF-7 cells at concentrations ranging from 0 to 80% (v/v). Cell viability, migration, and expression of IL-6/STAT3 pathway-related genes were evaluated using MTT, scratch assays, and qRT-PCR. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test, with significance set at p < 0.05. The results showed that 20% EV treatment markedly inhibited MCF-7 cell activity, significantly reducing viability and almost completely blocking migration, with wound closure rates of 35.4% ± 3.80 at 24 h and 47.6% ± 4.2 at 48 h, compared with 48% ± 4.6 and 67% ± 4.2 in the control group, respectively. Notably, expression levels of IL-6, IL-6RST, and STAT3 were significantly downregulated (fold changes 0.155 ± 0.02 and 0.258 ± 0.012, p < 0.01), accompanied by severe disruption of the microtubule network. Immunofluorescence imaging revealed a disorganized microtubule architecture and irregular filament distribution in EV-treated cells, corresponding with decreased expression of TubA1 and CALR genes. These findings indicate that ADSC-EVs not only suppress IL-6/STAT3 inflammatory signaling but also destabilize the intracellular microtubule system, collectively contributing to the inhibition of MCF-7 breast cancer cell migration and survival. This provides an important molecular basis for developing novel EV-based therapeutic strategies in breast cancer treatment. Full article

Mesenchymal stem cells (MSCs) are multipotent cells capable of differentiating into various connective tissue cell types. Adipose tissue provides a rich source of MSCs (ADSCs), which can differentiate into osteoblasts, adipocytes, and chondroblasts. Pluripotency factors such as SOX2, NANOG, and OCT4 maintain MSC stemness, whereas human endogenous retroviruses (HERVs) and their epigenetic regulators TRIM28 and SETDB1 have been implicated in transcriptional regulation and cell fate decisions. This study investigated the transcriptional expression of HERV-H, -K, and -W, TRIM28, SETDB1, and pluripotency markers (NANOG, OCT4, SOX2) during chondrogenic differentiation of ADSCs using Real-Time PCR. Chondrogenesis was confirmed by aggrecan (ACAN) upregulation and aggrecan immunostaining. Although no statistically significant differences were observed for HERV-H, HERV-K, or HERV-W, HERV-K and HERV-W showed a trend toward decreased expression in differentiated cells, consistent with the overall shift in transcriptional profile during lineage commitment. TRIM28 expression was significantly reduced, while SETDB1 showed a decreasing trend. Among pluripotency markers, OCT4 was significantly downregulated, whereas NANOG and SOX2 remained stable. Correlation analyses revealed that in differentiated ADSCs, HERV-W expression correlated negatively with TRIM28 and positively with SETDB1, while no correlations were found for HERV-H or HERV-K. These findings suggest that specific HERV families and their epigenetic regulators may undergo coordinated modulation during chondrogenic differentiation, supporting a complex and family-specific interplay between retroelement regulation, pluripotency factors, and MSC lineage commitment. Full article

Dry Eye Disease (DED) is a multifactorial condition of the ocular surface, with one potential cause being damage from eye drops containing preservatives such as benzalkonium chloride (BAC). Current treatments for DED are unsatisfactory; therefore, it is worth exploring new therapies based on the secretome derived from stem cells. Human stem cells are important sources of growth factors and cytokines that promote tissue regeneration. The secretome of these cells can be obtained in vitro in conditioned medium (CM). The aim of the study was to evaluate the effect of CM derived from adipose-derived stem cells (hADSCs) and amniotic membrane-derived cells expressing mesenchymal and/or epithelial markers on limbal stem cells (LSCs) damaged by BAC, focusing on their regenerative potential. The study used two experimental models: the first focused on neutralizing the toxic effects of BAC when each CM was administered concurrently, and the second on the therapeutic effects of CM after prior cell damage by BAC. The effects of CM on LSCs were assessed, including apoptosis, cell cycle progression, proliferation, migration, and inflammation. CM from ADSCs and amniotic cells were shown to significantly reduce BAC-induced damage to LSCs. All tested CM promoted LSC regeneration, although their efficacy varied among treatments. The application of CM during BAC exposure yielded stronger and more consistent benefits than post-injury treatment. Full article

Castration-resistant prostate cancer (CRPC) remains difficult to treat with conventional chemotherapy. We evaluated a stem cell-based enzyme-prodrug strategy using hTERT-immortalized adipose-derived stem cells engineered to express rabbit carboxylesterase 2 (hTERT-ADSC.CE2) in combination with irinotecan (CPT-11). hTERT-ADSC.CE2 cells were generated via lentiviral transduction and confirmed to overexpress CE2. Their tumor-homing capacity toward PC3 prostate cancer cells was assessed, along with prodrug activation, apoptosis induction, and in vivo tumor suppression in a CRPC mouse model. hTERT-ADSC.CE2 cells demonstrated enhanced migration toward PC3 cells and higher expression of tumor-homing factors than the controls. Under CPT-11, they exhibited a strong “suicide” effect and induced selective killing of PC3 cells, with upregulation of BAX and cleaved caspase-3 and downregulation of BCL-2. By day 14, the combination arm showed significantly lower tumor burden than both the control and irinotecan-alone arms (p < 0.05). The pattern is consistent with intratumoral activation and localized SN-38 exposure. hTERT-ADSC.CE2 combined with irinotecan provides potent, tumor-targeted cytotoxicity and markedly suppresses CRPC progression. This cell-mediated prodrug activation system may represent a promising therapeutic approach for advanced prostate cancer. Full article

This study reports the construction and validation of a CAFLUX (Chemically Activated Fluorescent Expression) HepG2 reporter cell line engineered to express a histone H2B–green fluorescent protein (H2B–GFP) fusion protein under the control of a dioxin-responsive cytochrome P450 1A1 (CYP1A1) promoter. A lentiviral construct containing a synthetic promoter with multiple dioxin-responsive elements (DREs) upstream of the H2B–EGFP coding sequence was cloned into the pFUGW vector, packaged in human embryonic kidney (HEK) 293FT cells, and used to transduce HepG2 hepatocellular carcinoma cells. Stable clones obtained by limiting dilution were screened for GFP expression in response to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The resulting CAFLUX HepG2 cells exhibited dose-dependent nuclear GFP fluorescence when exposed to aryl hydrocarbon receptor (AhR) agonists, with limits of detection of approximately 0.01 pM for TCDD and 0.1 pM for benzo[a]pyrene (B[a]P), a polycyclic aromatic hydrocarbon (PAH). This reporter activity correlated with endogenous CYP1A1 mRNA expression as determined by quantitative polymerase chain reaction (qPCR), confirming that GFP signals reflected native transcriptional responses. In functional assays, curcumin suppressed GFP expression in a concentration-dependent manner and induced apoptotic morphology at higher doses, while extracellular vesicles (EVs) derived from adipose-derived stem cells (ADSCs) significantly reduced both GFP fluorescence and CYP1A1 mRNA levels, suggesting an inhibitory effect on AhR-driven transcription. The CAFLUX HepG2 reporter system therefore provides a sensitive and reproducible platform for real-time, nuclear-localized monitoring of AhR-mediated gene expression. Its responsiveness to both agonists and antagonists underscores its potential utility in toxicological evaluation, drug discovery, and the investigation of EV-mediated signaling in liver cancer models. Full article

Bone-related defects present a key challenge in orthopaedics. The current gold standard, autografts, poses significant limitations, such as donor site morbidity, limited supply, and poor morphological adaptability. This study investigates the potential of scaffold geometry to induce osteogenic differentiation of human adipose-derived stem cells (hADSCs) through mechanotransduction, without the use of chemical inducers. Four distinct poly-(L)-lactic acid (PLA) scaffold architectures—Traditional Cross (Tc), Triangle (T), Diamond (D), and Gyroid (G)—were fabricated using fused filament fabrication (FFF) 3D printing. hADSCs were cultured on these scaffolds, and their response was evaluated utilising an alkaline phosphatase (ALP) assay, immunofluorescence, and extensive proteomic analyses. The results showed the D scaffold to have the highest ALP activity, followed by Tc. Proteomics results showed that more than 1200 proteins were identified in each scaffold with unique proteins expressed in each scaffold, respectively Tc—204, T—194, D—244, and G—216. Bioinformatics analysis revealed structures with complex curvature to have an increased expression of proteins involved in mid- to late-stage osteogenesis signalling and differentiation pathways, while the Tc scaffold induced an increased expression of signalling and differentiation pathways pertaining to angiogenesis and early osteogenesis. Full article

The global obese population accounts for approximately 30% of the total population and continues to increase. White adipocytes, which accumulate in the body for energy storage, are associated with obesity. Mechanisms that activate browning of white adipocytes are an attractive therapeutic target for obesity and metabolic disorders. Exosomes are nano-sized biovesicles that play a role in cell-to-cell communication though the transfer of cargos such as microRNAs. Although milk exosomes contain many endogenous microRNA molecules, the role of microRNAs in milk exosomes is limited. Therefore, the aim of this study was to investigate the effects of milk exosomes on the browning of white adipocyte. Mouse pre-adipocytes (3T3-L1) and human adipose-derived stem cells (hADSCs) were differentiated and exposed to milk exosomes. Compared to control, milk exosomes promoted the expression of thermogenic genes and cellular mitochondrial energy metabolism in both 3T3-L1 cells and hADSCs. Additionally, milk exosomes were orally administered to mice fed a high-fat diet. As the intake of milk exosomes increased, the mice’s body weight decreased. Milk exosomes also increased the protein levels of thermogenic genes and mitochondrial-related genes in mouse adipose tissue. The overexpression of miR-11987, which is abundant in milk exosomes, in both 3T3-L1 cells and hADSCs led to the increased expression of thermogenic genes and mitochondrial activity. Our results support that bovine-specific miR-11987 in milk exosomes promotes the browning of white adipocytes. Therefore, milk exosome and milk exosomal miR-11987 could have significant clinical implications for obesity and metabolic syndrome. Full article

Adipocyte differentiation is a complex process in which pluripotent mesenchymal stem cells (MSCs) differentiate and develop into mature fat cells, also known as adipocytes. This process is controlled by various transcription factors, hormones, and signaling molecules that regulate the development of these cells. Recently, an increasing number of non-coding RNAs (ncRNAs), especially microRNAs (miRNAs), have been established to be involved in the regulation of many biological processes, including adipocyte differentiation, development, metabolism, and energy homeostasis of white and brown adipose tissue. Several in vitro and in vivo studies reported the significant role of ncRNAs in either promoting or inhibiting adipocyte differentiation into white or brown fat cells by targeting specific transcription factors and regulating the expression of key adipogenic genes. Identifying the function of ncRNAs and their subsequent targets contributes to our understanding of how these molecules can be used as potential biomarkers and tools for therapies against obesity, diabetes, and other diseases related to obesity. This could also contribute to advancements in tissue-engineering based treatments. In this review, we intended to present an up-to-date comprehensive literature overview of the role of ncRNAs, including miRNAs, long non-coding RNAs (lncRNAs), and circular RNAs (circRNAs), focusing particularly on miRNAs, in regulating the differentiation and development of cells into white and brown adipose tissue. In addition, we further discuss the potential use of these molecules as biomarkers for the development of novel therapeutic strategies for future personalized treatment options for patients. Full article