Search Results (115)

Human adipose-derived stem cells (hASCs) are widely used in regenerative medicine due to their accessibility and high proliferative capacity. Platelet lysate (PL) has recently emerged as a promising alternative to fetal bovine serum (FBS), offering superior cell expansion potential; however, the molecular basis for its efficacy remains insufficiently elucidated. In this study, we performed RNA sequencing to compare hASCs cultured with PL or FBS, revealing a significant upregulation of genes related to stress response and cell proliferation under PL conditions. These findings were validated by RT–qPCR and supported by functional assays demonstrating enhanced cellular resilience to oxidative and genotoxic stress, reduced doxorubicin-induced senescence, and improved antiapoptotic properties. In a murine wound model, PL-treated wounds showed accelerated healing, characterized by thicker dermis-like tissue formation and increased angiogenesis. Immunohistochemical analysis further revealed elevated expression of chk1, a DNA damage response kinase encoded by CHEK1, which plays a central role in maintaining genomic integrity during stress-induced repair. Collectively, these results highlight PL not only as a viable substitute for FBS in hASC expansion but also as a bioactive supplement that enhances regenerative efficacy by promoting proliferation, stress resistance, and antiaging functions. Full article

Mesenchymal stem cells have been widely investigated in the field of regenerative medicine and also used as a model to study the differentiation-induction properties of a variety of biomaterials. This study evaluates the osteoinductive potential of novel hydroxyapatite scaffolds functionalized with a phage-displayed peptide (SC1) selected via biopanning for its similarity to bone matrix proteins. The peptide, identified through sequence alignment as a mimotope of osteonectin (SPARC), was used to functionalize scaffolds. Results from SC1 were gathered at different time points (14, 28 and 46 days) and compared with those from nonfunctionalized hydroxyapatite (HA) scaffolds. In vitro experiments, by seeding human adipose-derived stem cells (hASCs), indicated satisfactory biocompatibility for both types of scaffolds. Histochemical observations showed that SC1, better than HA scaffolds, was able to improve hASC osteogenic differentiation, as evaluated through Alizarin Red staining (showing on average a darker staining of 100%). An increase was also observed, especially at early stages (14 days), for osterix (up to 60% increase) and osteonectin immunoexpression (up to 50% increase). In in vivo experiments, cell-free scaffolds of both types were subcutaneously implanted into the backs of mice and analyzed after 2, 4, 8 and 16 weeks. Also, in this case, SC1 more effectively promoted the osteogenic differentiation of infiltrated resident cells. In particular, increased immunoexpression of osterix and osteonectin (+30% and 35%, respectively) was found already at 2 weeks. It can be concluded that SC1 scaffolds may represent a valuable tool to address critical-sized bone defects. Full article

The aim of this in vitro study is to investigate the adhesion, proliferation, and differentiation of human adipose-derived mesenchymal stem cells (hASC) on Trabecular Titanium scaffolds manufactured with different manufacturing processes (EBM and SLM). The in vitro adhesion and proliferation of hASC on titanium scaffolds with WST assays have been carried out. The comparison of the gene expression profiles of typical bone genes (Alp, Bglap, Col1a1, and Osx) through real-time PCR assays and the evaluation of extracellular matrix composition with immunofluorescence and SEM analysis have been performed. In addition, the possible osteoinductive properties of the two scaffolds have been investigated through real-time PCR and ALP assays. Data showed that Trabecular Titanium supports human adipose-derived mesenchymal stem cell colonization and induces differentiation in bone with the deposition of the abundant extracellular mineralized matrix regardless of the manufacturing process, proving that the micro- and macro-design features are the key factors responsible for the osteoinduction behavior. These features can only be achieved through tailored 3D printing process parameters. Full article

Nanotechnology is transforming contemporary medicine by providing cutting-edge tools for the treatment and diagnosis of complex disorders. Advanced techniques such as bioimaging and photodynamic therapy (PDT) combine early diagnosis and targeted therapy, offering a more precise approach than conventional treatments. However, a significant obstacle for PDT is the need to selectively deliver photosensitizers to disease sites while minimizing systemic side effects. In this context, mesenchymal stem cells have emerged as promising biological carriers due to their natural tropism towards tumors, low immunogenicity, and their ability to overcome biological barriers. In this study, two push–pull compounds, NPI-PTZ and BTZ-PTZ, phenothiazine derivatives featuring aggregation-induced emission (AIE) abilities, were analyzed. These molecules proved to be excellent fluorescent probes and photosensitizing agents. When administered to human bone marrow-derived multipotent stromal cells (hBM-MSCs) and human adipose multipotent stem cells (hASCs), the compounds were efficiently internalized, maintained a stable fluorescent emission for several days, and showed phototoxicity after irradiation, without inducing major cytotoxic effects under normal conditions. These results highlight the potential of NPI-PTZ and BTZ-PTZ combined with mesenchymal stem cells as theranostic tools, bridging bioimaging and PDT, and suggest new possibilities for advanced therapeutic approaches in clinical applications. Full article

A platform of two-component cross-linked hydrogel (cGEL) based on gelatinous peptides and anhydride-containing cross-linkers (oPNMA, oPDMA) is extended for use in peripheral nerve regeneration. Hybrid composites with bio-/chemical cues for enhanced biophysical and biochemical properties were fabricated by covalently grafting small molecular, heterobifunctional amines including the nerve growth factor mimetic LM11A-31 to the oligomeric cross-linkers prior to hydrogel formation. The cytocompatibility and growth-supportive conditions within the matrix are confirmed for pristine and modified hydrogels using L929 mouse fibroblasts and human adipose-derived stem cells (hASCs). For hASCs, cell behavior depends on the type of cross-linker and integrated amine. In a subsequent step, neonatal rat Schwann cells (SCs) are seeded on pristine and functionalized cGEL to investigate the materials’ capabilities to support SC growth and morphology. Within all formulations, cell viability, adherence, and cell extension are maintained though the cell elongation and orientation vary compared to the two-dimensional control. It is possible to merge adjustable two-component hydrogels with amines as biochemical signals, leading to improved nervous cell proliferation and activity. This indicates the potential of tunable bioactive cGEL as biomaterials in nerve implants, suggesting their use as a foundational component for nerve conduits. Full article

Mesenchymal stem cells isolated from human adipose tissue (hASCs) are a promising tool for tissue repair due to their ability to differentiate into specific cell lineages. The possibility of modulating the adipogenic differentiation of hASCs is crucial in improving their therapeutic potential. This study aimed to investigate the effects of cannabidiol (CBD), a phytocannabinoid isolated from Cannabis sativa L., on hASCs. Few studies have evaluated its role in stem cell (SC) properties and their differentiation potential. hASCs were first treated with different concentrations of CBD (ranging from 0.1 to 10 μM) to assess its effects on viability, demonstrating that this molecule is non-toxic, except at the concentration of 10 μM. Subsequently, the role of CBD in the proliferation, metabolism and adipogenic potential of hASCs was analyzed. CBD promoted adipogenesis in a dose-dependent manner, even in the absence of differentiation medium. This result was evidenced by the presence of lipid vacuoles, the expression of adipogenic markers, cytoskeletal actin rearrangement and modulation in the expression of osteogenic genes. Although the results indicated a role of CBD in promoting hASC adipogenesis, further research will be needed to explore the mechanism of action of CBD in SC differentiation and to deepen its utility in SC-based approaches. Full article

Wound healing progresses through four phases: hemostasis, inflammation, proliferation, and remodeling. Wounds may become chronic if this process is disrupted. The use of small extracellular vesicle (sEV; EVs < 200 nm) exosomes (exo; ~40–120 nm) derived from human adipose stem cells (hASCs) as a treatment for wounds is well studied. The cargo of these exosomes is of great interest as this accelerates wound healing. Our previous studies identified lncRNAs GAS5 and MALAT1 as packaged and enriched in hASC exosomes. In this study, we use a rat model to examine the effects on wound healing when hASC exosomes are depleted of GAS5 and MALAT1. Rats were wounded and wounds were treated with 100 μg hASC_exo or hASC_exo-G-M every 2 days for 1 week. qPCR was completed to evaluate the molecular effects of depletion of GAS5 and MALAT1 from hASC_exo. RNAseq was performed on wound tissue to evaluate the molecular mechanisms changed by hASC_exo-G-M in wound healing. While hASC_exo-G-M significantly improved wound healing rate compared to control wounds, healing occurred slower than in wounds treated with hASC_exo that were not depleted of GAS5 and MALAT1. Overall, this study reveals that molecular functions associated with healing are reduced in the absence of GAS5 and MALAT1, highlighting the importance of these lncRNAs. Full article

Innovations in spinal cord injury (SCI) models are crucial for developing effective therapies. This study introduces a novel in vitro SCI model using cultures of primary mixed spinal cord cells from rat pups, featuring key spinal cord cell types. This model offers distinct advantages in terms of feasibility, reproducibility, and cost-effectiveness, requiring only basic cell culture equipment. Following hyperosmotic stress via sorbitol treatment, the model recapitulated SCI pathophysiological hallmarks, with a 65% reduction in cell viability and gradual cell death over 48 h, making it ideal for evaluating neuroprotective agents. Notably, the human adipose tissue stem cell (hASC) secretome provided significant protection: it preserved metabolic viability, reduced β amyloid precursor protein (β-APP) expression in surviving neurons, and modulated the shift in the astrocytic morphotype. A transcriptomic profile of the effect of the hASC secretome treatment showed significant functional enrichments related to cell proliferation and cycle progression pathways. In addition to supporting the use of the hASC secretome as a therapy for SCI, this study is the first to use sorbitol as a hyperosmolar stressor to recapitulate key aspects of SCI pathophysiology. Thereby, this model can be used as a promising platform for evaluating therapeutic agents targeting neuroprotection and neuroregeneration, offering outputs related to cell death, neuronal stress, and protection, as well as induction of glial reactivity. Full article

To mimic the important features of progressing adiposity, in vitro adipose cell culture models must allow gradual intracellular fat accumulation in the three-dimensional (3D) arrangement of adipose-derived stem cells (ASCs) over a long-term culture period. Previously, elastin-like polypeptide (ELP) and polyethyleneimine (PEI) have been used to culture human adipose-derived stem cells (hASCs) as 3D spheroids and to differentiate them to adipocytes over a relatively long culture period of up to 5 weeks. In this study, to further enhance the spheroid adhesion properties, ELP was fused with Arginine–Glycine–Aspartic Acid (RGD) residues, known for their role as cell-attachment sites. This study aimed to assess whether the addition of RGD to the C-or N-terminus of ELP would impact the spheroid-forming ability of ELP-PEI coatings. ELP-RGD conjugates were produced using genetically modified Escherichia coli to express ELP-(RGD)₃ and (RGD)₃-ELP, followed by chemical conjugation with PEI. SDS gel electrophoresis, FTIR spectroscopy, and turbidimetry analyses revealed that ELP was conjugated with RGD without much alteration in the molecular weight, functional groups present, and transition temperature of ELP. The addition of RGD to ELP also did not affect the chemical conjugation capacity of ELP to PEI. We observed that the ELP-PEI coating formed slightly larger spheroids (61.8 ± 3.2 µm) compared to the ELP-(RGD)₃-PEI and (RGD)₃-ELP-PEI coatings (56.6 ± 3.0 and 53.4 ± 2.4 µm, respectively). Despite the size difference, ELP-(RGD)₃-PEI coatings exhibited superior spheroid retention during media changes, with minimal spheroid loss. DNA assay results confirmed a significant decrease in the DNA concentration (p < 0.05) after the 20 media changes for spheroids cultured on the ELP-PEI coating, indicating spheroid loss. However, there was no significant difference in DNA concentration before and after 20 media changes for spheroids cultured on the ELP-(RGD)₃-PEI and (RGD)₃-ELP-PEI coatings (p > 0.05). These findings suggest that RGD incorporation does not hinder the initial spheroid formation ability of the ELP-PEI coating and enhances spheroid retention under dynamic culture conditions. Full article

Nanocomposite hydrogels are gaining significant attention for biomedical applications in soft tissue engineering due to the increasing demand for highly flexible and durable soft polymer materials. This research paper focused on investigating and optimizing a procedure for the development of novel nanocomposite hydrogels based on poly(2-hydroxyethyl methacrylate)-co-(2-acrylamido-2-methylpropane sulfonic acid) (HEMA/AMPSA) copolymers. These hydrogels were synthesized through a grafting-through process, where the polymer network was formed using a modified clay crosslinker. The layered double hydroxide (LDH) clay modified with 3-(trimethoxysilyl)propyl methacrylate (ATPM) was synthesized using a novel recipe through a two-step procedure. The nanocomposite hydrogel compositions were optimized to achieve soft hydrogels with high flexibility. The developed materials were analyzed for their mechanical and morphological properties using tensile and compressive tests, transmission electron microscopy (TEM), scanning electron microscopy (SEM), and micro-computed tomography (micro-CT). The swelling behavior, network density, and kinetic diffusion mechanism demonstrated the specific characteristics of the materials. The modified LDH-ATPM was further characterized using Thermogravimetry (TGA), FTIR-ATR and X-ray diffraction (XRD). Biological assessments on human adipose-derived stem cells (hASCs) were essential to evaluate the biocompatibility of the nanocomposite hydrogels and their potential for soft tissue applications. Full article

Background: Adipose-derived mesenchymal stem cell conditioned medium (ASC-CM) improved the viability and wound closure of human tenocytes (HTCN) exposed to high glucose (HG) by activating the transforming growth factor beta 1 (TGF-β1) pathway. Objectives: Since ASC-CM can also modulate microRNAs (miRNAs) in recipient cells, this study investigated the effects of ASC-CM on the miRNAs regulating tendon repair (miR-29a-3p, miR-210-3p and miR-21-5p) in HG-HTNC. Methods: ASC-CM was obtained by ASCs isolated from the abdominal fat tissue of seven non-diabetic patients. HTNC were cultured in HG for 20 days, then scratched and exposed for 24 h to ASC-CM. qRT-PCR and ELISAs assessed miRNA and target levels. Results: HG-HTNC exhibited a significant downregulation of miRNAs. ASC-CM restored the levels of miRNAs and their related targets involved in tendon repair. Conclusions: The epigenetic modulation observed in HG-HTNC exposed to ASC-CM could be an innovative option in the management of diabetic tendinopathy. Full article

Background: Traumatic brain injury (TBI) occurs in individuals of all ages, predominantly during sports, accidents, and in active military service members. Chronic consequences of TBI include declined cognitive and motor function, dementia, and emotional distress. Small extracellular vesicles (sEVs), previously referred to as exosomes, are nano-sized lipid vesicles that play a role in intercellular communication. Our prior research established the efficacy of sEVs derived from human adipose stem cells (hASC sEVs) in accelerating the healing of brain injuries. The hASC sEVs are a biologic therapeutic and need to be stored at −20 °C or −80 °C. This limits their use in translating to everyday use in clinics or their inclusion in first-aid kits for application immediately after injury. To address this, here we demonstrate that hASC sEVs can be stored at room temperature (RT) for two months post lyophilization. Methods: A transmission electron microscope (TEM) and nanoparticle tracking analysis (NTA) were used to validate the morphology of lyophilized RT sEVs. Using in vitro models of neuronal injury mimicking physical injury, inflammation, and oxidative stress, we demonstrate that lyophilized RT hASC sEVs are viable and promote the healing of neuronal injuries. Results: The lyophilized sEVs maintain their purity, size, and morphology upon rehydration. Lyophilized, RT stored sEVs showed better efficacy after two months compared with −80 °C stored sEVs. Conclusions: RT storage of lyophilized hASC sEVs maintains their efficacy to accelerate the healing of injuries in neuronal cells. This will advance the use of hASC sEVs, bringing them closer to use in clinics, home first-aid kits, and on battlefields by active service members. Full article

Background: Chronic low-grade inflammation in obesity is linked to white adipose tissue (WAT) dysfunction. Plasma lipopolysaccharide (LPS) activates Toll-like receptor 4 (TLR4), triggering NF-κB and worsening these disturbances. Previously, we showed that histone H3 lysine 27 (H3K27) epigenetic modifications affect WAT gene expression in high-fat-diet mice, identifying key pathways in adipose-derived stem cells (ASCs). This study explores whether NF-κB influences H3K27 modifiers in human ASCs and evaluates fish oil (FO) as a modulator. Methods: Human visceral WAT ASCs were stimulated with LPS and treated with FO enriched with eicosapentaenoic acid (EPA). Flow cytometry, PCR array, RT-PCR, and Western blot assays were used. Results: LPS increased NF-κB activity, elevating KDM6B demethylase levels and H3K27 acetylation. These epigenetic modifications in LPS-stimulated ASCs were associated with persistent changes in the expression of genes involved in adipogenesis, metabolic regulation, and inflammation, even after LPS removal and cell differentiation. FO mitigated these effects, reducing H3K27 acetylation and promoting methylation. Conclusions: FO demonstrates potential in modulating inflammation-induced epigenetic changes and preserving adipocyte function. Full article

Osteochondral lesions may be due to trauma or congenital conditions. In both cases, therapy is limited because of the difficulty of tissue repair. Tissue engineering is a promising approach that relies on designed scaffolds with variable mechanical attributes to favor cell attachment and differentiation. Human adipose-derived stem cells (hASCs) are a very promising cell source in regenerative medicine with osteochondrogenic potential. Based on the assumption that stiffness influences cell commitment, we investigated three different scaffolds: a semisynthetic animal-derived GelMA hydrogel, a combined scaffold made of rigid PEGDA coated with a thin GelMA layer and a decellularized plant-based scaffold. We investigated the role of different biomechanical stimulations in the scaffold-induced osteochondral differentiation of hASCs. We demonstrated that all scaffolds support cell viability and spontaneous osteochondral differentiation without any exogenous factors. In particular, we observed mainly osteogenic commitment in higher stiffness microenvironments, as in the plant-based one, whereas in a dense and softer matrix, such as in GelMA hydrogel or GelMA-coated-PEGDA scaffold, chondrogenesis prevailed. We can induce a specific cell commitment by combining hASCs and scaffolds with particular mechanical attributes. However, in vivo studies are needed to fully elucidate the regenerative process and to eventually suggest it as a potential approach for regenerative medicine. Full article

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the progressive degeneration of upper and lower motor neurons (MNs) in the brain and spinal cord, leading to progressive paralysis and death. Increasing evidence indicates that neuroinflammation plays an important role in ALS’s pathogenesis and disease progression. Neuroinflammatory responses, primarily driven by activated microglia and astrocytes, and followed by infiltrating peripheral immune cells, contribute to exacerbate/accelerate MN death. In particular, the role of the microglia in ALS remains unclear, partly due to the lack of experimental models that can fully recapitulate the complexity of ALS’s pathology. In this study, we developed and characterized a microglial cell line, SIM-A9-expressing human mutant protein Cu⁺/Zn⁺ superoxide dismutase_1 (SIM-A9hSOD1(G93A)), as a suitable model in vitro mimicking the microglia activity in ALS. The expression of hSOD1(G93A) in SIM-A9 cells induced a change in their metabolic activity, causing polarization into a pro-inflammatory phenotype and enhancing reactive oxygen species production, which is known to activate cell death processes and apoptosis. Afterward, we used our microglial model as an experimental set-up to investigate the therapeutic action of extracellular vesicles isolated from adipose mesenchymal stem cells (ASC-EVs). ASC-EVs represent a promising therapeutic treatment for ALS due to their neuroprotective and immunomodulatory properties. Here, we demonstrated that treatment with ASC-EVs is able to modulate activated ALS microglia, reducing their metabolic activity and polarizing their phenotype toward an anti-inflammatory one through a mechanism of reduction of reactive oxygen species. Full article