Enhancing high-quality fat survival: A novel strategy using cell-free fat extract.

High-quality fat (HQF) improves the survival rate of fat and volumetric filling compared to traditional Coleman fat. However, this HQF strategy inevitably leads to a significant amount of unused fat being wasted. "CEFFE" (cell-free fat extract) is an acellular aqueous-phase liquid, rich in bioactive proteins. The remaining fat from preparing HQF can be further processed into CEFFE to promote the survival of HQF. HQF was obtained and the remaining fat was processed into CEFFE, then HQF was transplanted subcutaneously in nude mice. Animal studies showed that CEFFE significantly improved the survival rate of HQF. Histological analysis revealed that CEFFE improved the survival rate of HQF, by enhancing cell proliferation activity, reducing apoptosis, increasing angiogenesis, and improving the inflammatory state. Under simulated anaerobic conditions, CEFFE also improved the viability of HQF. In vitro, studies demonstrated that CEFFE enhanced the survival rate of HQF through multiple mechanisms. Transcriptomic analysis and qPCR showed that CEFFE increased the expression of angiogenesis-related genes in ADSCs while enhancing their proliferation-related gene expression and suppressing the expression of three differentiation-related genes. Moreover, functional experiments demonstrated that CEFFE-induced ADSCs exhibited stronger proliferation and adipogenic differentiation abilities. Tube formation and migration assays revealed that CEFFE promoted tube formation and migration of HUVECs, indicating its inherent pro-angiogenic properties. CEFFE facilitated the development of M0 to M2 macrophages, suggesting its role in improving the inflammatory state. This innovative clinical strategy optimizes HQF transplantation strategy, minimizing fat wastage and enhancing the efficiency of fat utilization.

Adipose-derived stem cells promote glycolysis and peritoneal metastasis via TGF-ß1/SMAD3/ANGPTL4 axis in colorectal cancer.

Peritoneal metastasis, the third most common metastasis in colorectal cancer (CRC), has a poor prognosis for the rapid progression and limited therapeutic strategy. However, the molecular characteristics and pathogenesis of CRC peritoneal metastasis are poorly understood. Here, we aimed to elucidate the action and mechanism of adipose-derived stem cells (ADSCs), a prominent component of the peritoneal microenvironment, in CRC peritoneal metastasis formation. Database analysis indicated that ADSCs infiltration was increased in CRC peritoneal metastases, and high expression levels of ADSCs marker genes predicted a poor prognosis. Then we investigated the effect of ADSCs on CRC cells in vitro and in vivo. The results revealed that CRC cells co-cultured with ADSCs exhibited stronger metastatic property and anoikis resistance, and ADSCs boosted the intraperitoneal seeding of CRC cells. Furthermore, RNA sequencing was carried out to identify the key target gene, angiopoietin like 4 (ANGPTL4), which was upregulated in CRC specimens, especially in peritoneal metastases. Mechanistically, TGF-ß1 secreted by ADSCs activated SMAD3 in CRC cells, and chromatin immunoprecipitation assay showed that SMAD3 facilitated ANGPTL4 transcription by directly binding to ANGPTL4 promoter. The ANGPTL4 upregulation was essential for ADSCs to promote glycolysis and anoikis resistance in CRC. Importantly, simultaneously targeting TGF-ß signaling and ANGPTL4 efficiently reduced intraperitoneal seeding in vivo. In conclusion, this study indicates that tumor-infiltrating ADSCs promote glycolysis and anoikis resistance in CRC cells and ultimately facilitate peritoneal metastasis via the TGF-ß1/SMAD3/ANGPTL4 axis. The dual-targeting of TGF-ß signaling and ANGPTL4 may be a feasible therapeutic strategy for CRC peritoneal metastasis.

Pulsed Electromagnetic Fields Combined With Adipose-Derived Stem Cells Protect Ischemic Myocardium by Regulating miR-20a-5p/E2F1/p73 Signaling.

Myocardial infarction (MI) is a serious threat to human health. Although monotherapy with pulsed electromagnetic fields (PEMFs) or adipose-derived stem cells (ADSCs) has been reported to have positive effect on the treatment of MI, a satisfactory outcome has not yet been achieved. In recent years, combination therapy has attracted widespread interest. Herein, we explored the synergistic therapeutic effect of combination therapy with PEMFs and ADSCs on MI and found that the combination of PEMFs and ADSCs effectively reduced infarct size, inhibited cardiomyocyte apoptosis and protected the cardiac function in mice with MI. In addition, bioinformatics analysis and RT-qPCR showed that the combination therapy could affect apoptosis by regulating the expression of miR-20a-5p. A dual-luciferase reporter gene assay also confirmed that the miR-20a-5p could target E2F transcription factor 1 (E2F1) and inhibit cardiomyocyte apoptosis by regulating the E2F1/p73 signaling pathway. Therefore, our study systematically demonstrated the effectiveness of combination therapy on the inhibition of cardiomyocyte apoptosis by regulating the miR-20a-5p/E2F1/p73 signaling pathway in mice with MI. Thus, our study underscored the effectiveness of the combination of PEMFs and ADSCs and identified miR-20a-5p as a promising therapeutic target for the treatment of MI in the future.

Safety and efficacy of adipose-derived mesenchymal stem cell therapy in elderly Parkinson's disease patients: an intermediate-size expanded access program.

OBJECTIVE: This intermediate-size expanded access program aimed to evaluate safety and clinical efficacy of multiple intravenous infusions of autologous, Hope Biosciences adipose-derived mesenchymal stem cell (HB-adMSC) therapy in elderly patients with Parkinson's disease (PD). METHODS: Ten eligible participants (aged 76-95 years) received six intravenous infusions each with 200MM autologous HB-adMSCs over 18 weeks, with the end of study (EOS) at week 26. Safety was assessed through adverse events (AEs) and serious adverse events (SAEs). Efficacy was measured through improvements in both motor and non-motor symptoms, utilizing scales including Movement Disorder Society Unified Parkinson's Disease Rating Scale (MDS-UPDRS) parts I-IV, Parkinson's Disease Questionnaire-39 (PDQ-39), Parkinson's disease Fatigue Scale (PFS-16), Patient Health Questionnaire-9 (PHQ-9), and Visual Analog Scale (VAS). Analysis employed paired t-tests and Minimal Clinically Important Difference (MCID) thresholds for the patient-reported outcomes. RESULTS: Most AEs (37 out of 46) were mild in severity, with 5 SAEs reported, none attributed to the drug. No deaths occurred. Despite lack of statistical significance across the efficacy endpoints, modest yet clinically meaningful improvements with effect size > 0.3 were observed in several secondary efficacy endpoints (MDS-UPDRS part I & III, PDQ-39, and PHQ-9) at the EOS, nearing or surpassing the established MCID values. CONCLUSIONS: The administration of autologous 200MM HB-adMSCs was found to be safe and well-tolerated in the elderly PD population. Although not achieving statistical significance, modest clinical improvements were noted across multiple secondary endpoints. These findings underscore the safety profile of the treatment in elderly patients and highlight the importance of evaluating clinical relevance alongside statistical measures for meaningful patient outcomes. Further investigation with a larger, randomized, placebo-controlled design is warranted to validate these observations.

Efficacy of adipose-derived mesenchymal stem cell therapy in the treatment of chronic micro- and macrovascular complications of diabetes.

Diabetes mellitus is a highly prevalent disease characterized by hyperglycaemia that damages the vascular system, leading to micro- (retinopathy, neuropathy, nephropathy) and macrovascular diseases (cardiovascular disease). There are also secondary complications of diabetes (cardiomyopathy, erectile dysfunction or diabetic foot ulcers). Stem cell-based therapies have become a promising tool targeting diabetes symptoms and its chronic complications. Among all stem cells, adipose-derived mesenchymal stem cells (ADMSCs) are of great importance because of their abundance, non-invasive isolation and no ethical limitations. Characteristics that make ADMSCs good candidates for cell-based therapy are their wide immunomodulatory properties and paracrine activities through the secretion of an array of growth factors, chemokines, cytokines, angiogenic factors and anti-apoptotic molecules. Besides, after transplantation, ADMSCs show great ex vivo expansion capacity and differentiation to other cell types, including insulin-producing cells, cardiomyocytes, chondrocytes, hepatocyte-like cells, neurons, endothelial cells, photoreceptor-like cells, or astrocytes. Preclinical studies have shown that ADMSC-based therapy effectively improved visual acuity, ameliorated polyneuropathy and foot ulceration, arrested the development and progression of diabetic kidney disease, or alleviated the diabetes-induced cardiomyocyte hypertrophy. However, despite the positive results obtained in animal models, there are still several challenges that need to be overcome before the results of preclinical studies can be translated into clinical applications. To date, there are several clinical trials or ongoing trials using ADMSCs in the treatment of diabetic complications, most of them in the treatment of diabetic foot ulcers. This narrative review summarizes the most recent outcomes on the usage of ADMSCs in the treatment of long-term complications of diabetes in both animal models and clinical trials.

Oxaloacetate as new inducer for osteogenic differentiation of human adipose tissue-derived mesenchymal stem cells in vitro.

BACKGROUND: Mitochondrial organelles play a crucial role in cellular metabolism so different cell types exhibit diverse metabolic and energy demands. Therefore, alternations in the intracellular distribution, quantity, function, and structure of mitochondria are required for stem cell differentiation. Finding an effective inducer capable of modulating mitochondrial activity is critical for the differentiation of specific stem cells into osteo-like cells for addressing issues related to osteogenic disorders. This study aimed to investigate the effect of oxaloacetate (OAA) on the osteogenic differentiation of human adipose-derived mesenchymal stem cells (hADSCs) in vitro. METHODS AND RESULTS: First, the most favorable OAA concentration was measured through MTT assay and subsequently confirmed using acridine orange staining. Human ADSCs were cultured in osteogenic medium supplemented with OAA and analyzed on days 7 and 14 of differentiation. Various assays including alkaline phosphatase assay (ALP), cellular calcium content assay, mineralized matrix staining with alizarin red, catalase (CAT) and superoxide dismutase (SOD) activity, and real-time RT-PCR analysis of three bone-specific markers (ALP, osteocalcin, and collagen type I) were conducted to characterize the differentiated cells. Following viability assessment, OAA at a concentration of 1 µM was considered the optimal dosage for further studies. The results of osteogenic differentiation assays showed that OAA at a concentration of 1 × 10- 6 M significantly increased ALP enzyme activity, mineralization, CAT and SOD activity and the expression of bone-specific genes in differentiated cells compared to control groups in vitro. CONCLUSIONS: In conclusion, the fundings from this study suggest that OAA possesses favorable properties that make it a potential candidate for application in medical bone regeneration.

Enhanced adipose-derived stem cells with IGF-1-modified mRNA promote wound healing following corneal injury.

The cornea serves as an important barrier structure to the eyeball and is vulnerable to injuries, which may lead to scarring and blindness if not treated promptly. To explore an effective treatment that could achieve multi-dimensional repair of the injured cornea, the study herein innovatively combined modified mRNA (modRNA) technologies with adipose-derived mesenchymal stem cells (ADSCs) therapy, and applied IGF-1 modRNA (modIGF1)-engineered ADSCs (ADSCmodIGF1) to alkali-burned corneas in mice. The therapeutic results showed that ADSCmodIGF1 treatment could achieve the most extensive recovery of corneal morphology and function when compared not only with simple ADSCs but also IGF-1 protein eyedrops, which was reflected by the healing of corneal epithelium and limbus, the inhibition of corneal stromal fibrosis, angiogenesis and lymphangiogenesis, and also the repair of corneal nerves. In vitro experiments further proved that ADSCmodIGF1 could more significantly promote the activity of trigeminal ganglion cells and maintain the stemness of limbal stem cells than simple ADSCs, which were also essential for reconstructing corneal homeostasis. Through a combinatorial treatment regimen of cell-based therapy with mRNA technology, this study highlighted comprehensive repair in the damaged cornea and showed the outstanding application prospect in the treatment of corneal injury.

Evaluation of canine adipose-derived mesenchymal stem cells for neurological functional recovery in a rat model of traumatic brain injury.

BACKGROUND: Traumatic brain injury (TBI) is a common condition in veterinary medicine that is difficult to manage.Veterinary regenerative therapy based on adipose mesenchymal stem cells seem to be an effective strategy for the treatment of traumatic brain injury. In this study, we evaluated therapeutic efficacy of canine Adipose-derived mesenchymal stem cells (AD-MSCs)in a rat TBI model, in terms of improved nerve function and anti-neuroinflammation. RESULTS: Canine AD-MSCs promoted neural functional recovery, reduced neuronal apoptosis, and inhibited the activation of microglia and astrocytes in TBI rats. According to the results in vivo, we further investigated the regulatory mechanism of AD-MSCs on activated microglia by co-culture in vitro. Finally, we found that canine AD-MSCs promoted their polarization to the M2 phenotype, and inhibited their polarization to the M1 phenotype. What's more, AD-MSCs could reduce the migration, proliferation and Inflammatory cytokines of activated microglia, which is able to inhibit inflammation in the central system. CONCLUSIONS: Collectively, the present study demonstrates that transplantation of canine AD-MSCs can promote functional recovery in TBI rats via inhibition of neuronal apoptosis, glial cell activation and central system inflammation, thus providing a theoretical basis for canine AD-MSCs therapy for TBI in veterinary clinic.

Evaluating the pro-survival potential of apoptotic bodies derived from 2D- and 3D- cultured adipose stem cells in ischaemic flaps.

In the realm of large-area trauma flap transplantation, averting ischaemic necrosis emerges as a pivotal concern. Several key mechanisms, including the promotion of angiogenesis, the inhibition of oxidative stress, the suppression of cell death, and the mitigation of inflammation, are crucial for enhancing skin flap survival. Apoptotic bodies (ABs), arising from cell apoptosis, have recently emerged as significant contributors to these functions. This study engineered three-dimensional (3D)-ABs using tissue-like mouse adipose-derived stem cells (mADSCs) cultured in a 3D environment to compare their superior biological effects against 2D-ABs in bolstering skin flap survival. The findings reveal that 3D-ABs (85.74 ± 4.51) % outperform 2D-ABs (76.48 ± 5.04) % in enhancing the survival rate of ischaemic skin flaps (60.45 ± 8.95) % (all p < 0.05). Mechanistically, they stimulated angiogenesis, mitigated oxidative stress, suppressed apoptosis, and facilitated the transition of macrophages from M1 to M2 polarization (all p < 0.05). A comparative analysis of microRNA (miRNA) profiles in 3D- and 2D-ABs identified several specific miRNAs (miR-423-5p-up, miR30b-5p-down, etc.) with pertinent roles. In summary, ABs derived from mADSCs cultured in a 3D spheroid-like arrangement exhibit heightened biological activity compared to those from 2D-cultured mADSCs and are more effective in promoting ischaemic skin flap survival. These effects are attributed to their influence on specific miRNAs.

A quick and innovative pipeline for producing chondrocyte-homing peptide-modified extracellular vesicles by three-dimensional dynamic culture of hADSCs spheroids to modulate the fate of remaining ear chondrocytes in the M1 macrophage-infiltrated microenvironment.

BACKGROUND: Extracellular vesicles (EVs) derived from human adipose-derived mesenchymal stem cells (hADSCs) have shown great therapeutic potential in plastic and reconstructive surgery. However, the limited production and functional molecule loading of EVs hinder their clinical translation. Traditional two-dimensional culture of hADSCs results in stemness loss and cellular senescence, which is unfavorable for the production and functional molecule loading of EVs. Recent advances in regenerative medicine advocate for the use of three-dimensional culture of hADSCs to produce EVs, as it more accurately simulates their physiological state. Moreover, the successful application of EVs in tissue engineering relies on the targeted delivery of EVs to cells within biomaterial scaffolds. METHODS AND RESULTS: The hADSCs spheroids and hADSCs gelatin methacrylate (GelMA) microspheres are utilized to produce three-dimensional cultured EVs, corresponding to hADSCs spheroids-EVs and hADSCs microspheres-EVs respectively. hADSCs spheroids-EVs demonstrate excellent production and functional molecule loading compared with hADSCs microspheres-EVs. The upregulation of eight miRNAs (i.e. hsa-miR-486-5p, hsa-miR-423-5p, hsa-miR-92a-3p, hsa-miR-122-5p, hsa-miR-223-3p, hsa-miR-320a, hsa-miR-126-3p, and hsa-miR-25-3p) and the downregulation of hsa-miR-146b-5p within hADSCs spheroids-EVs show the potential of improving the fate of remaining ear chondrocytes and promoting cartilage formation probably through integrated regulatory mechanisms. Additionally, a quick and innovative pipeline is developed for isolating chondrocyte homing peptide-modified EVs (CHP-EVs) from three-dimensional dynamic cultures of hADSCs spheroids. CHP-EVs are produced by genetically fusing a CHP at the N-terminus of the exosomal surface protein LAMP2B. The CHP + LAMP2B-transfected hADSCs spheroids were cultured with wave motion to promote the secretion of CHP-EVs. A harvesting method is used to enable the time-dependent collection of CHP-EVs. The pipeline is easy to set up and quick to use for the isolation of CHP-EVs. Compared with nontagged EVs, CHP-EVs penetrate the biomaterial scaffolds and specifically deliver the therapeutic miRNAs to the remaining ear chondrocytes. Functionally, CHP-EVs show a major effect on promoting cell proliferation, reducing cell apoptosis and enhancing cartilage formation in remaining ear chondrocytes in the M1 macrophage-infiltrated microenvironment. CONCLUSIONS: In summary, an innovative pipeline is developed to obtain CHP-EVs from three-dimensional dynamic culture of hADSCs spheroids. This pipeline can be customized to increase EVs production and functional molecule loading, which meets the requirements for regulating remaining ear chondrocyte fate in the M1 macrophage-infiltrated microenvironment.

Photobiomodulation effects on neuronal transdifferentiation of immortalized adipose-derived mesenchymal stem cells.

Adipose-derived mesenchymal stem cells (ADMSCs) possess the ability to transform into various cell types, including neurons. It has been proposed that the optimization of this transformation can be achieved by using photobiomodulation (PBM). The objective of this laboratory-based investigation was to induce the transformation of immortalized ADMSCs (iADMSCs) into neurons with chemical triggers and then evaluate the supportive effects of PBM at two different wavelengths, 525 nm and 825 nm, each administered at a dose of 5 J/cm2, as well as the combined application of these wavelengths. The results revealed that the treated cells retained their stem cell characteristics, although the cells exposed to the green laser exhibited a reduction in the CD44 marker. Furthermore, early, and late neuronal markers were identified using flow cytometry analysis. The biochemical analysis included the assessment of cell morphology, viability, cell proliferation, potential cytotoxicity, and the generation of reactive oxygen species (ROS). The findings of this study indicate that PBM does not harm the differentiation process and may even enhance it, but it necessitates a longer incubation period in the induction medium. These research findings contribute to the validation of stem cell technology for potential applications in in vivo, pre-clinical, and clinical research environments.

Isolation of the Stromal Vascular Fraction Using a New Protocol with All Clinical-Grade Drugs: From Basic Study to Clinical Application.

OBJECTIVE: The purpose of this study was to evaluate the yield, viability, clinical safety, and efficacy of the stromal vascular fraction (SVF) separated with a new protocol with all clinical-grade drugs. MATERIALS AND METHODS: SVF cells were isolated from lipoaspirate obtained from 13 participants aged from 30 to 56 years by using a new clinical protocol and the laboratory protocol. The cell yield, viability, morphology, mesenchymal stem cell (MSC) surface marker expression, and differentiation abilities of the SVF cells harvested from the two protocols were compared. Furthermore, three related clinical trials were conducted to verify the safety and efficiency of SVF cells isolated by the new clinical protocol. RESULTS: There were no significant differences in the yield, viability, morphology, and differentiation potential of the SVFs isolated with the clinical protocol and laboratory protocol. Adipose-derived mesenchymal stem cell (ASC) surface marker expression, including that of CD14, CD31, CD44, CD90, CD105, and CD133, was consistent between the two protocols. Clinical trials have demonstrated the effectiveness of the SVF isolated with the new clinical protocol in improving skin grafting, promoting mechanical stretch-induced skin regeneration and improving facial skin texture. No complications occurred. CONCLUSION: SVF isolated by the new clinical protocol had a noninferior yield and viability to that of the SVF separated by the laboratory protocol. SVFs obtained by the new protocol can be safely and effectively applied to improve skin grafting, promote mechanical stretch-induced skin regeneration, and improve facial skin texture. TRIAL REGISTRATION: The trials were registered with the ClinicalTrials.gov (NCT03189628), the Chinese Clinical Trial Registry (ChiCTR2000039317), and the ClinicalTrials.gov (NCT02546882). All the three trials were not patient-funded trials. NO LEVEL ASSIGNED: This journal requires that authors assign a level of evidence to each submission to which Evidence-Based Medicine rankings are applicable. This excludes Review Articles, Book Reviews, and manuscripts that concern Basic Science, Animal Studies, Cadaver Studies, and Experimental Studies. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Mechanical Purification of Lipofilling: The Relationship Between Cell Yield, Cell Growth, and Fat Volume Maintenance.

BACKGROUND: The mechanical manipulations of fat tissue represented from centrifugation, filtration, washing, and fragmentation were considered the most effective strategies aiming to obtain purified lipofilling with different impacts both in terms of adipose-derived stem cells amount contained in stromal vascular fraction, and fat volume maintenance. OBJECTIVES: The present work aimed to report results in fat volume maintenance obtained by lipofilling purification based on the combined use of washing and filtration, in a clinical study, and to deeply investigate the adipose-derived stem cells yield and growth capacity of the different stromal vascular fraction extraction techniques with an in vitro approach. METHODS: A preliminary prospective, case-control study was conducted. 20 patients affected by face and breast soft tissue defects were treated with lipofilling and divided into two groups: n = 10 patients (study group) were treated with lipofilling obtained by washing and filtration procedures, while n = 10 (control group) were treated with lipofilling obtained by centrifugation according to the Coleman technique. 6 months after the lipofilling, the volume maintenance percentage was analyzed by clinical picture and magnetic resonance imaging comparisons. Additionally, extracted stromal vascular fraction cells were also in vitro analyzed in terms of adipose-derived stem cell yield and growth capacity. RESULTS: A 69% ± 5.0% maintenance of fat volume after 6 months was observed in the study group, compared with 44% ± 5.5% in the control group. Moreover, the cellular yield of the control group resulted in 267,000 ± 94,107 adipose-derived stem cells/mL, while the study group resulted in 528,895 ± 115,853 adipose-derived stem cells /mL, with a p-value = 0.1805. Interestingly, the study group showed a fold increase in cell growth of 6758 ± 0.7122, while the control group resulted in 3888 ± 0.3078, with a p < 0.05 (p = 0.0122). CONCLUSIONS: The comparison of both groups indicated that washing and filtration were a better efficient system in lipofilling preparation, compared to centrifugation, both in terms of volume maintenance and adipose-derived stem cell growth ability. LEVEL OF EVIDENCE III: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors http://www.springer.com/00266 .

Exploring the Potential Effects of Cryopreservation on the Biological Characteristics and Cardiomyogenic Differentiation of Rat Adipose-Derived Mesenchymal Stem Cells.

Cryopreservation is essential for the broad clinical application of mesenchymal stem cells (MSCs), yet its impact on their cellular characteristics and cardiomyogenic differentiation potential remains a critical concern in translational medicine. This study aimed to evaluate the effects of cryopreservation on the biological properties and cardiomyogenic capacity of rat adipose-derived MSCs (AD-MSCs). We examined their cellular morphology, surface marker expression (CD29, CD90, CD45), trilineage differentiation potential (adipogenic, osteogenic, chondrogenic), and gene expression profiles for the pluripotency marker REX1 and immunomodulatory markers TGFß1 and IL-6. After inducing cardiomyocyte differentiation, we assessed cardiac-specific gene expressions (Troponin I, MEF2c, GSK-3ß) using quantitative RT-qPCR, along with live/dead cell staining and immunofluorescence for cardiac-specific proteins (Troponin T, α-actinin, Myosin Heavy Chain). Cryopreserved AD-MSCs preserved their morphology, surface markers, and differentiation potential, but exhibited a reduced expression of REX1, TGFß1, and IL-6. Additionally, cryopreservation diminished cardiomyogenic differentiation, as indicated by the lower levels of Troponin I, MEF2c, and GSK-3ß seen compared to non-cryopreserved cells. Despite this, high cell viability (>90%) and maintained cardiac protein expression were observed post-cryopreservation. These findings highlight the necessity of optimizing cryopreservation protocols to ensure the full therapeutic potential of AD-MSCs, particularly in applications related to cardiac regenerative medicine.

Melatonin Enhances Neural Differentiation of Adipose-Derived Mesenchymal Stem Cells.

Adipose-derived mesenchymal stem cells (ASCs) are adult multipotent stem cells, able to differentiate toward neural elements other than cells of mesodermal lineage. The aim of this research was to test ASC neural differentiation using melatonin combined with conditioned media (CM) from glial cells. Isolated from the lipoaspirate of healthy donors, ASCs were expanded in a basal growth medium before undergoing neural differentiation procedures. For this purpose, CM obtained from olfactory ensheathing cells and from Schwann cells were used. In some samples, 1 µM of melatonin was added. After 1 and 7 days of culture, cells were studied using immunocytochemistry and flow cytometry to evaluate neural marker expression (Nestin, MAP2, Synapsin I, GFAP) under different conditions. The results confirmed that a successful neural differentiation was achieved by glial CM, whereas the addition of melatonin alone did not induce appreciable changes. When melatonin was combined with CM, ASC neural differentiation was enhanced, as demonstrated by a further improvement of neuronal marker expression, whereas glial differentiation was attenuated. A dynamic modulation was also observed, testing the expression of melatonin receptors. In conclusion, our data suggest that melatonin's neurogenic differentiation ability can be usefully exploited to obtain neuronal-like differentiated ASCs for potential therapeutic strategies.

Photobiomodulation Dose-Response on Adipose-Derived Stem Cell Osteogenesis in 3D Cultures.

Osteoporosis and other degenerative bone diseases pose significant challenges to global healthcare systems due to their prevalence and impact on quality of life. Current treatments often alleviate symptoms without fully restoring damaged bone tissue, highlighting the need for innovative approaches like stem cell therapy. Adipose-derived mesenchymal stem cells (ADMSCs) are particularly promising due to their accessibility, abundant supply, and strong differentiation potential. However, ADMSCs tend to favor adipogenic pathways, necessitating the use of differentiation inducers (DIs), three-dimensional (3D) hydrogel environments, and photobiomodulation (PBM) to achieve targeted osteogenic differentiation. This study investigated the combined effects of osteogenic DIs, a fast-dextran hydrogel matrix, and PBM at specific wavelengths and fluences on the proliferation and differentiation of immortalized ADMSCs into osteoblasts. Near-infrared (NIR) and green (G) light, as well as their combination, were used with fluences of 3 J/cm2, 5 J/cm2, and 7 J/cm2. The results showed statistically significant increases in alkaline phosphatase levels, a marker of osteogenic differentiation, with G light at 7 J/cm2 demonstrating the most substantial impact on ADMSC differentiation. Calcium deposits, visualized by Alizarin red S staining, appeared as early as 24 h post-treatment in PBM groups, suggesting accelerated osteogenic differentiation. ATP luminescence assays indicated increased proliferation in all experimental groups, particularly with NIR and NIR-G light at 3 J/cm2 and 5 J/cm2. MTT viability and LDH membrane permeability assays confirmed enhanced cell viability and stable cell health, respectively. In conclusion, PBM significantly influences the differentiation and proliferation of hydrogel-embedded immortalized ADMSCs into osteoblast-like cells, with G light at 7 J/cm2 being particularly effective. These findings support the combined use of 3D hydrogel matrices and PBM as a promising approach in regenerative medicine, potentially leading to innovative treatments for degenerative bone diseases.

Adipose-derived mesenchymal stem cells overexpressing prion improve outcomes via the NLRP3 inflammasome/DAMP signalling after spinal cord injury in rat.

Traumatic spinal cord injury (SCI) is a highly destructive disease in human neurological functions. Adipose-derived mesenchymal stem cells (ADMSCs) have tissue regenerations and anti-inflammations, especially with prion protein overexpression (PrPcOE ). Therefore, this study tested whether PrPcOE -ADMSCs therapy offered benefits in improving outcomes via regulating nod-like-receptor-protein-3 (NLRP3) inflammasome/DAMP signalling after acute SCI in rats. Compared with ADMSCs only, the capabilities of PrPcOE -ADMSCs were significantly enhanced in cellular viability, anti-oxidative stress and migration against H2 O2 and lipopolysaccharide damages. Similarly, PrPcOE -ADMSCs significantly inhibited the inflammatory patterns of Raw264.7 cells. The SD rats (n = 32) were categorized into group 1 (Sham-operated-control), group 2 (SCI), group 3 (SCI + ADMSCs) and group 4 (SCI + PrPcOE -ADMSCs). Compared with SCI group 2, both ADMSCs and PrPcOE -ADMSCs significantly improved neurological functions. Additionally, the circulatory inflammatory cytokines levels (TNF-α/IL-6) and inflammatory cells (CD11b/c+/MPO+/Ly6G+) were highest in group 2, lowest in group 1, and significantly higher in group 3 than in group 4. By Day 3 after SCI induction, the protein expressions of inflammasome signalling (HGMB1/TLR4/MyD88/TRIF/c-caspase8/FADD/p-NF-κB/NEK7/NRLP3/ASC/c-caspase1/IL-ß) and by Day 42 the protein expressions of DAMP-inflammatory signalling (HGMB1/TLR-4/MyD88/TRIF/TRAF6/p-NF-κB/TNF-α/IL-1ß) in spinal cord tissues displayed an identical pattern as the inflammatory patterns. In conclusion, PrPcOE -ADMSCs significantly attenuated SCI in rodents that could be through suppressing the inflammatory signalling.

Effects of hepatocyte growth factor-transfected mesenchymal stem cell transplantation in canine injured vocal folds.

This study aimed to assess the efficacy of hepatocyte growth factor (HGF)-transfected adipose-derived mesenchymal stem cell (ADSC) transplantation in the injured vocal folds (VFs) of canines. A lentiviral vector encoding HGF was successfully produced via Gateway cloning, which was used to infect ADSCs. Four weeks after transoral laser microsurgery (type II) with CO2 laser, the beagles of each group were injected with HGF-transfected ADSCs or uninfected ADSCs into VFs. The results showed that the retention of HGF-transfected ADSCs in the VFs persisted about three months post-injection. The VFs in the HGF-transfected ADSCs group exhibited a closer-to-normal structure with less collagen deposition and higher amounts of hyaluronic acid (HA) in the third month. The short microvilli in the HGF-transfected ADSCs group showed a dense and uniform distribution. These results revealed that HGF-transfected ADSC is a potential treatment option for injured VFs.

Exosomal microRNA-342-5p secreted from adipose-derived mesenchymal stem cells mitigates acute kidney injury in sepsis mice by inhibiting TLR9.

BACKGROUND: Sepsis-related acute kidney injury (AKI) is an inflammatory disease associated with extremely high mortality and health burden. This study explored the possibility of exosomes secreted by adipose-derived mesenchymal stem cells (AMSCs) serving as a carrier for microRNA (miR)-342-5p to alleviate sepsis-related AKI and investigated the possible mechanism. METHODS: Serum was obtained from 30 patients with sepsis-associated AKI and 30 healthy volunteers for the measurement of miR-342-5p, blood urea nitrogen (BUN), and serum creatinine (SCr) levels. For in vitro experiments, AMSCs were transfected with LV-miR-342-5p or LV-miR-67 to acquire miR-342-5p-modified AMSCs and miR-67-modified AMSCs, from which the exosomes (AMSC-Exo-342 and AMSC-Exo-67) were isolated. The human renal proximal tubular epithelial cell line HK-2 was induced by lipopolysaccharide (LPS) to construct a cellular model of sepsis. The expression of Toll-like receptor 9 (TLR9) was also detected in AKI cells and mouse models. The interaction between miR-342-5p and TLR9 was predicted by dual luciferase reporter gene assay. RESULTS: Detection on clinical serum samples showed that BUN, SCr, and TLR9 were elevated and miR-342-5p level was suppressed in the serum of patients with sepsis-associated AKI. Transfection with LV-miR-342-5p reinforced miR-342-5p expression in AMSCs and AMSC-secreted exosomes. miR-342-5p negatively targeted TLR9. LPS treatment enhanced TLR9 expression, reduced miR-342-5p levels, suppressed autophagy, and increased inflammation in HK-2 cells, while the opposite trends were observed in LPS-induced HK-2 cells exposed to AMSC-Exo-342, Rapa, miR-342-5p mimic, or si-TLR9. Additionally, the effects of AMSC-Exo-342 on autophagy and inflammation in LPS-induced cells could be weakened by 3-MA or pcDNA3.1-TLR9 treatment. Injection of AMSC-Exo-342 enhanced autophagy, mitigated kidney injury, suppressed inflammation, and reduced BUN and SCr levels in sepsis-related AKI mouse models. CONCLUSION: miR-342-5p transferred by exosomes from miR-342-5p-modified AMSCs ameliorated AKI by inhibiting TLR9 to accelerate autophagy.

The HIFIA/LINC02913/IGF1R axis promotes the cell function of adipose-derived mesenchymal stem cells under hypoxia via activating the PI3K/AKT pathway.

OBJECTIVE: Promoting angiogenesis is crucial for tissue repair. Adipose-derived mesenchymal stem cells (ADSCs) are endowed with the ability of paracrine secretion of various angiogenic cytokines and the differentiation potential into endothelium-like cells to directly participate in angiogenesis. ADSCs are key seed cells for promoting angiogenesis in regenerative medicine and tissue engineering. This study aimed to explore the role and mechanism of C9orf106 (LINC02913) in the angiogenesis of ADSCs. METHODS: The microarray dataset GSE12884 was analyzed to identify the differentially expressed lncRNAs in ADSCs under normoxia and hypoxia. The expression of the key genes was detected using qRT-PCR, western blot assay (western blot), and immunofluorescence (IF) staining. The adipogenic ability and tube formation ability of ADSCs was detected using oil red O staining and tube formation assay, respectively. The regulatory relationship between hypoxia-inducible factor-1alpha (HIF1A) and LINC02913 was verified using chromatin immunoprecipitation (ChIP) assay and dual-luciferase reporter gene assay. A skin wound healing nude mice model was established. Hematoxylin and eosin (H&E) staining was applied to detect pathological skin damage. Immunohistochemistry (IHC) staining was used to determine the level of CD31 in skin tissues. RESULTS: LINC02913 expression was decreased in ADSCs under hypoxia; LINC02913 overexpression inhibited the proliferation, adipogenic ability, endothelial differentiation ability, and tube formation ability of ADSCs. ChIP assay and dual-luciferase reporter gene assay results showed that HIF1A could directly bind to the LINC02913 promoter region to inhibit its transcription. Through RNAact prediction and analysis of the correlation with LINC02913 expression, it was found that IGF1R may directly interact with LINCO02913. The HIF1A/LINC02913/IGF1R axis could activate the PI3K/AKT pathway to promote the biological function of ADSCs. Hypoxia-ADSCs significantly promoted vascularization in the wounded skin. The regulatory effect of LINC02913/IGF1R axis on hypoxia-ADSCs treated skin wound healing were verified. CONCLUSION: The HIF1A/LINC02913/IGF1R axis promoted the proliferation, adipogenic ability, and tube formation ability of ADSCs under hypoxia via activating the PI3K/AKT pathway.