Adipose Tissue-derived Stem Cells Research Articles

Collagen Isolated from Human Adipose Tissue and Its Cellular Affinity.

The use of collagen in cell cultures promotes cell proliferation and differentiation, and it has been commercialized. In this study, we separated and purified collagen from adipose tissue discarded during liposuction and prepared collagen-coated dishes. After collagen was identified from human adipose tissue, type identification and quantification were performed using SDS-PAGE and FPLC. Collagen type I was used to coat culture dishes. Human skin fibroblasts and human adipose tissue-derived stem cells were seeded at a density of 2.5×105 cells/mL on prepared dishes at a collagen concentration of 3 mg/mL and cultured for 7 days. Cell viability was then measured and analyzed. The WST-1 assay was used to evaluate the results. The amount of collagen in 300 g of adipose tissue was 25.5 mg for type I, 41.4 mg for type III, 10.6 mg for type IV, 6.5 mg for type V, and 15 mg for type VI. The highest rates were observed for adipose stem cells cultured on human adipose tissue-derived collagen-coated dishes. In cell cultures, cell affinity was higher when cells and the substrate used were of the same origin, and affinity was stronger when the tissue of origin was the same.

7-Ketocholesterol Effects on Osteogenic Differentiation of Adipose Tissue-Derived Mesenchymal Stem Cells

Some oxysterols were shown to promote osteogenic differentiation of mesenchymal stem cells (MSCs). Little is known about the effects of 7-ketocholesterol (7-KC) in this process. We describe its impact on human adipose tissue-derived MSC (ATMSC) osteogenic differentiation. ATMSCs were incubated with 7-KC in osteogenic or adipogenic media. Osteogenic and adipogenic differentiation was evaluated by Alizarin red and Oil Red O staining, respectively. Osteogenic (ALPL, RUNX2, BGLAP) and adipogenic markers (PPARƔ, C/EBPα) were determined by RT-PCR. Differentiation signaling pathways (SHh, Smo, Gli-3, β-catenin) were determined by indirect immunofluorescence. ATMSCs treated with 7-KC in osteogenic media stained positively for Alizarin Red. 7-KC in adipogenic media decreased the number of adipocytes. 7-KC increased ALPL and RUNX2 but not BGLAP expressions. 7-KC decreased expression of PPARƔ and C/EBPα, did not change SHh, Smo, and Gli-3 expression, and increased the expression of β-catenin. In conclusion, 7-KC favors osteogenic differentiation of ATMSCs through the expression of early osteogenic genes (matrix maturation phase) by activating the Wnt/β-catenin signaling pathway, while inhibiting adipogenic differentiation. This knowledge can be potentially useful in regenerative medicine, in treatments for bone diseases.

Anti-tumor effects of ellagic acid and mesenchymal stem cell-conditioned medium on breast cancer cells on the 3D printed PCL/agarose scaffolds

Numerous investigations have demonstrated that natural bioactive compounds, such as flavonoids, exhibit synergistic effects with anticancer agents utilized in clinical practice. Stem cell therapies play a critical role in the domain of oncology; nevertheless, conflicting results have surfaced regarding the potential pro-cancer or anti-cancer characteristics of mesenchymal stem cells. The primary aim of the current investigation was to examine the anticancer efficacy of ellagic acid (EA) alone or in combination with adipose tissue stem cells derived conditioned medium (ADSCs-CM) on human breast cancer cells. In order to overcome the potential effects of two-dimensional (2D) culture, a three-dimensional (3D) printed polycaprolactone (PCL)/agarose scaffold is utilized to evaluate cancer cell behavior. More recently it was reported this scaffold had open and interconnected pores, as well as good water absorption and mechanical properties. Assessment of MCF-7 cancer cell viability was conducted through MTT assay, flow cytometry, cell cycle assay, and quantitative real-time PCR within a 3D cell culture framework. The outcomes revealed a reduction in cancer cell viability across all treatment cohorts in comparison to the control group. Particularly noteworthy was the significantly enhanced effect of the EA and CM combination relative to each component administered individually. Flow cytometry assessment using annexin V/PI staining indicated a decrease in cancer cells, particularly evident in the combination-treated group. Furthermore, the mRNA expression levels of BAX, BCL2, vascular endothelial growth factor (VEGF), and caspase 3 genes were consistent with pathways associated with apoptosis. Consequently, the synergistic anti-proliferative impact of EA in conjunction with ADSCs-CM on MCF-7 cells has been substantiated. These findings have two important implications for breast cancer research; highlight the utility of the PCL/agarose scaffold as a cancer model and suggest new avenues for the development of adjuvant anticancer therapeutic approaches.

Acquisition of durable insulin-producing cells from human adipose tissue-derived mesenchymal stem cells as a foundation for cell- based therapy of diabetes mellitus

This study aimed to identify the suitable induction protocol to produce highly qualified insulin producing cells (IPCs) from human adipose tissue derived stem cells (ADSCs) and evaluate the efficacy of the most functionally IPCs in management of diabetes mellitus (DM) in rats. The ADSCs were isolated and characterized according to the standard guidelines. ADSCs were further induced to be IPCs in vitro using three different protocols. The success of trans-differentiation was assessed in vitro through analysis of pancreatic endocrine genes expression, and insulin release in response to glucose stimulation. Then, the functionalization of the generated IPCs was evaluated in vivo. The in vitro findings revealed that the laminin-coated plates in combination with insulin-transferrin-selenium, B27, N2, and nicotinamide could efficiently up-regulate the expression of pancreatic endocrine genes. The in vivo study indicated effectual homing of the PKH-26-labelled IPCs in the pancreas of treated animals. Moreover, IPCs infusion in diabetic rats induced significant improvement in the metabolic parameters and prompted considerable up-regulation in the expression of the pancreatic related genes. The regenerative effect of infused IPCs was determined through histological examination of pancreatic tissue. Conclusively, the utilization of laminin–coated plates in concomitant with extrinsic factors promoting proliferation and differentiation of ADSCs could efficiently generate functional IPCs.

Administration of Adipose Tissue Derived Stem Cells before the Onset of the Disease Lowers the Levels of Inflammatory Cytokines IL-1 and IL-6 in the Rat Model of Necrotizing Enterocolitis

There is little research concerning the role of stem cells in necrotizing enterocolitis (NEC). Bone marrow-derived mesenchymal stem cells (BMDSC) and amniotic fluid-derived stem cells significantly reduced the amount and severity of NEC in the animal models. ADSCs share similar surface markers and differentiation potential with BMDSCs. Their potential role in the setting of NEC has not been researched before. The hypothesis of the study was that prophylactic intraperitoneal administration of ADSCs before the onset of the disease will result in limiting the inflammatory response, effecting a lower incidence of NEC. On a molecular level, this should result in lowering the levels of inflammatory cytokines IL-1 and IL-6. The local ethical committee for animal experiments approval was acquired (WAW2/093/2021). We utilized a self-modified rat NEC model based on single exposure to hypothermia, hypoxia, and formula feeding. One hundred and twenty-eight rat puppies were divided into two groups—prophylaxis (ADSC-NEC, n = 66) and control group (NEC-PLCB, n = 62)—to measure the influence of ADSCs administration on the inflammatory changes in NEC, the level of cell engraftment, and the histopathology of the disease. The analysis did not show a significant effect on histopathology between groups, H(2) = 2.12; p = 0.347; η²H = 0.00. The intensity of the NEC variable results was similar across the analyzed groups (NEC-PLCB and ADSC-NEC). For IL-1 and IL-6, the difference between the NEC-PLCB group and the ADSC-NEC group was statistically significant, p = 0.002 and p < 0.001, respectively. To conclude, administration of adipose tissue-derived stem cells before the onset of the disease lowers the levels of inflammatory cytokines IL-1 and IL-6 but does not affect the histopathological results in the rat model of NEC.

Incorporation of Superparamagnetic Magnetic–Fluorescent Iron Oxide Nanoparticles Increases Proliferation of Human Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) have significant therapeutic potential and their use requires in-depth studies to better understand their effects. Labeling cells with superparamagnetic iron oxide nanoparticles allows real-time monitoring of their location, migration, and fate post-transplantation. This study aimed to investigate the efficacy and cytotoxicity of magnetic–fluorescent nanoparticles in human adipose tissue-derived mesenchymal stem cells (hADSCs). The efficacy of Molday ION rhodamine B (MIRB) labeling in hADSCs was evaluated and their biocompatibility was assessed using various techniques and differentiation assays. Prussian blue and fluorescence staining confirmed that 100% of the cells were labeled with MIRB and this labeling persisted for at least 3 days. Transmission electron microscopy revealed the internalization and clustering of the nanoparticles on the outer surface of the cell membrane. The viability assay showed increased cell viability 3 days after nanoparticle exposure. Cell counts were higher in the MIRB-treated group compared to the control group at 3 and 5 days and an increased cell proliferation rate was observed at 3 days post-exposure. Adipogenic, osteogenic, and chondrogenic differentiation was successfully achieved in all groups, with MIRB-treated cells showing an enhanced differentiation rate into adipocytes and osteocytes. MIRB was efficiently internalized by hADSCs but induced changes in cellular behavior due to the increased cell proliferation rate.

Donor variability in adipose tissue-derived stem cells: implications for the clinical efficacy of autologous fat grafting

Donor variability in adipose tissue-derived stem cells: implications for the clinical efficacy of autologous fat grafting

Protective Effect of Mesenchymal Stem Cells and Melatonin on Testicular Torsion-Induced Infertility

We aimed to explore the effects of systemic melatonin and intratesticular Adipose tissue-derived mesenchymal stem cells (AdMSCs) administration on rats with acute unilateral testicular torsion. Rats were randomized into Sham group (S) (n=8), torsion/detorsion group (T/D by torsion of right testis with rotated 720° counter clockwise for 3 h, then detorsion) (n=8), Melatonin group given 25 mg/kg after torsion/detorsion (M) (n=8), Adipose tissue-derived mesenchymal stem cell-treated group after torsion/detorsion (MSC) (n=8), Adipose tissue-derived mesenchymal stem cell-treated group with melatonin after torsion/detorsion (MSC+M) (n=8). We measured MDA, Testosterone, FSH and LH levels, performed histopathological analyses in testicles, and identified SOX, VASA and Caspas-3 reactions immunohistochemically. Testosterone, FSH, LH values did not yield any significant difference between the groups. While the Johnson score in the right testis remained the lowest in T/D, the highest score was noted in the S. The T/D manifested some degenerative seminiferous tubules, abnormal spermatogenesis and maturation arrest. The degenerative appearance monitored in M, MSC and MSC+M groups persisted in some tubules, while markedly reduced degeneration was observed in some other tubules. The highest Caspase-3 expression in T/D, whereas SOX-9 expression remained significantly higher in the treatment groups. Another aspect deserving attention is that MSC were characterized by low VASA expression. Our experimental trial suggests that the torsion-induced degeneration in testicular tissue was ameliorated in all the treatment groups. Although MSC, MSC+M and M administrations decreased the torsion-induced degeneration in the testicular tissue, these treatments did not prove to be superior to each other.

Melatonin enhances therapeutic outcomes of adipose tissue-derived mesenchymal stem cell therapy in rat osteoarthritis by reducing TNF-α and IL-1β-induced injuries.

Although adipose tissue-derived mesenchymal stem cell (ADSC) transplantation has been effectively used to treat osteoarthritis (OA), the low cell survival rate induced by the inflammatory and oxidative stress, severely affects the therapeutic efficiency of ADSC transplantation in OA. This study was designed to evaluate whether melatonin pretreatment could improve ADSC survival and its therapeutic efficacy in OA. The papain-induced OA rats were pretreated with melatonin via intra-articular injection and then intra-articular injected with indocyanine green (ICG)-labeled ADSCs (3 × 106/rat). Afterward, ADSC retention was evaluated by NIR-II fluorescence imaging. The tibia and synovial fluid were collected for histopathological examination and ELISA assay, respectively. To confirm the anti-inflammatory effect of melatonin, a TNF-α and IL-1β-induced cell model was used to evaluate the protective effects of melatonin on ADSC viability, cell apoptosis, and migration. Our results showed that melatonin pretreatment enhanced ADSC survival and improved the therapeutic effects of ADSC transplantation on cartilage repair, and anti-inflammation by reducing TNF-α, IL-6, IL-1β, and IL-12 in vivo. In particular, we also found that melatonin promoted ADSC viability and migration, and reduced cell apoptosis in vitro. In conclusion, this study supports that melatonin pretreatment can effectively improve ADSC survival and therapeutic efficiency in OA by reducing inflammatory injuries, which provides a novel strategy for enhancing ADSC therapy.

Adipose tissue-derived stem cells: a potent tool in regenerative medicine?

Adipose tissue-derived stem cells: a potent tool in regenerative medicine?

Exploring herbal preconditioning strategies to improve adipose tissue stem cell therapy efficacy

The application of mesenchymal stem cells (MSCs), specifically those sourced from adipose tissue, has become a focal point in regenerative medicine, showing potential for effective cell therapy. Adipose-derived stem cells (ASCs) offer hope in treating various conditions, yet their viability and integration rates are hindered by the harsh post-transplantation environment marked by inflammation and oxidative stress at the injury site. Lawsonia inermis (henna), Zizyphus spina-christi (Christ's Thorn), and Glycyrrhiza glabra (licorice) are three plants known for their potent antioxidant properties primarily due to their rich content of phenolic compounds and flavonoids. Their potential health benefits make them valuable in both traditional medicine and modern therapeutic applications.This research delves into investigating and contrasting the effects of hydroalcoholic extracts from Lawsonia inermis, Zizyphus spina-christi, and Glycyrrhiza glabra on human adipose tissue stem cells as a pre-conditioning method to alleviate oxidative stress in cell therapy. ASCs were isolated from the pelvic cavity and abdomen, characterized using flow cytometry, and prompted to differentiate into adipogenic and osteogenic lineages. They were then subjected to different concentrations of the aforementioned herbal extracts at varying time frames, followed by MIC and MTT analysis. The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay and the Minimum Inhibitory Concentration (MIC) assay are both used to evaluate the effects of substances on cell viability and cell growth receptively. RNA extraction from ASCs and evaluation of antioxidant gene expression were conducted via Real-time PCR. Flow cytometry data confirmed the presence of MSC markers in the isolated cells from ASCs. Analysis of the herbal extract concentrations revealed that 50 ng/ml had a toxic effect on ASCs, establishing a toxicity reference point for adipose-derived MSCs. Real-time PCR results showcased a notable increase in the expression of antioxidant genes post-preconditioning with hydroalcoholic extracts. This study underscores, for the first time, the safety and lack of toxicity of preconditioning with these extracts for h.ASCs, enhancing their resilience and acclimatization under oxidative stress conditions, potentially boosting the therapeutic potential of h.ASCs. The antioxidant properties of Lawsonia inermis, Zizyphus spina-christi, and Glycyrrhiza glabra may enhance the expression of antioxidant genes (Sod, Gpx and Cata) through several mechanisms, including the activation of transcription factors like Nrf2 (Nuclear factor erythroid 2-related factor 2), which regulates antioxidant gene expression; modulation of signaling pathways such as MAPK (Mitogen-Activated Protein Kinase) and PI3K/Akt (Phosphoinositide 3-kinase/Protein Kinase B); and influencing epigenetic modifications like histone changes or DNA methylation that can alter gene expression. However, further research is necessary to clarify the specific mechanisms by which these plant extracts enhance antioxidant gene expression and to explore their potential therapeutic applications in combating oxidative stress-related diseases. Future proposals should consider more gene investigation and in vivo applications, as well as the long-term effects of pre-conditioning.

In vitro Chondrogenic Induction Promotes the Expression Level of IL-10 via the TGF-β/SMAD and Canonical Wnt/β-catenin Signaling Pathways in Exosomes Secreted by Human Adipose Tissue-derived Mesenchymal Stem Cells.

Current treatment approaches cannot exactly regenerate cartilage tissue. Regarding some problems encountered with cell therapy, exosomes are advantageous because of their "cell-free" nature. This study examines the relationship between IL-10 and TGF-β and Canonical Wnt/β-catenin signal pathways in human adipose tissue-derived MSCs exosomes (hAT-MSCs-Exos) after in vitro chondrogenic differentiation. Human adipose tissue-derived mesenchymal stem cells (hAT-MSCs) and, as a control group, human fetal chondroblast cells (hfCCs) were differentiated chondrogenically in vitro. Exosome isolation and characterization analyses were performed. Chondrogenic differentiation was shown by Alcian Blue and Safranin O stainings. The expression levels of IL-10, TGF-β/SMAD signaling pathway genes, and Canonical Wnt/β-catenin signaling pathway genes, which play an essential role in chondrogenesis, were analyzed by RT-qPCR. Conditioned media cytokine levels were measured by using the TGF-β and IL-10 ELISA kits. IL-10 expression was upregulated in both chondrogenic differentiated hAT-MSC-Exos (dhAT-MSC-Exos) (p < 0.0001). In the TGF-β signaling pathway, TGF-β (p < 0.0001), SMAD2 (p < 0.0001), SMAD4 (p < 0.001), ACAN (p < 0.0001), SOX9 (p < 0.05) and COL1A2 (p < 0.0001) expressions were upregulated in dhAT-MSC-Exos. SMAD3 expression was upregulated in non-differentiated hAT-MSC-Exos. In the Canonical Wnt/β-catenin signaling pathway, WNT (p < 0.0001) and CTNNB1(p < 0.0001) expressions were upregulated in dhAT-MSC-Exos. AXIN (p < 0.0001) expression was upregulated in non-differentiated hAT-MSC-Exos. TGF-β and IL-10 levels were higher in dhAT-MSCs) (p < 0.0001). Related to these results, IL-10 may induce TGF-β/SMAD and Canonical Wnt/β-catenin signaling pathways in hAT-MSC exosomes obtained after chondrogenic differentiation. Therefore, using these exosomes for cartilage regeneration can lead to the development of treatment methods.

Effect of Stromal Vascular Fraction in the Rat Model of Pharyngocutaneous Fistulas.

A pharyngocutaneous fistula is one of the complications after laryngeal and pharyngeal surgery. It may also contribute to wound healing due to adipose tissue-derived stem cells and the growth factors in the stromal vascular fraction. The aim of this study was to investigate the effects of the adipose tissue-derived stromal vascular fraction on wound healing in the pharyngocutaneous fistula model induced in rats. Approval was received from the Animal Experiments Local Ethics Committee before starting the study (29.01.2016-2016/09). Eleven male Sprague-Dawley rats weighing approximately 300 g were included in the study. The animals were randomly divided into the study and control groups so that each group would include five animals. An animal was assigned as a donor for the removal of omental adipose tissue. Among the animals in which the pharyngocutaneous fistula model was created under general anesthesia, 1 ml of the stromal vascular fraction was injected into the study group on postoperative day 1. In postoperative week 2, all the animals were sacrificed and examined histologically (epithelialization-cell infiltration - mucosal injury). IBM SPSS Statistics for Windows, version 15, was used in the analyses. A p-value <0.05 was considered significant. Epithelialization was higher in the study group compared to the control group. However, no statistically significant difference was found between the two groups (p = 0.08). The cell infiltration was found to be statistically higher in the control group compared to the study group (p = 0.03). The mucosal injury was found to be significantly higher in the control group compared to the study group (p = 0.03). According to the Pearson correlation test, a negative correlation was found between epithelialization and cell infiltration and mucosal injury (p = 0.019 and p = 0.001). A positive correlation was found between cell infiltration and mucosal injury (p = 0.009). It was shown that stromal vascular fraction had a positive effect on wound healing in the pharyngocutaneous fistula model. According to the data we have obtained, we think that it can be effective in the treatment of pharyngocutaneous fistulas after more extensive preclinical and clinical studies are carried out.

Comparison between USPIOs and SPIOs for Multimodal Imaging of Extracellular Vesicles Extracted from Adipose Tissue-Derived Adult Stem Cells.

Adipose tissue-derived adult stem (ADAS) cells and extracellular vesicle (EV) therapy offer promising avenues for treating neurodegenerative diseases due to their accessibility and potential for autologous cell transplantation. However, the clinical application of ADAS cells or EVs is limited by the challenge of precisely identifying them in specific regions of interest. This study compares two superparamagnetic iron oxide nanoparticles, differing mainly in size, to determine their efficacy for allowing non-invasive ADAS tracking via MRI/MPI and indirect labeling of EVs. We compared a USPIO (about 5 nm) with an SPIO (Resovist®, about 70 nm). A physicochemical characterization of nanoparticles was conducted using DLS, TEM, MRI, and MPI. ADAS cells were labeled with the two nanoparticles, and their viability was assessed via MTT assay. MRI detected labeled cells, while TEM and Prussian Blue staining were employed to confirm cell uptake. The results revealed that Resovist® exhibited higher transversal relaxivity value than USPIO and, consequently, allows for detection with higher sensitivity by MRI. A 200 µgFe/mL concentration was identified as optimal for ADAS labeling. MPI detected only Resovist®. The findings suggest that Resovist® may offer enhanced detection of ADAS cells and EVs, making it suitable for multimodal imaging. Preliminary results obtained by extracting EVs from ADAS cells labeled with Resovist® indicate that EVs retain the nanoparticles, paving the way to an efficient and multimodal detection of EVs.

Sox2-mediated transdifferentiation of hAT-MSCs into induced neural progenitor-like cells for remyelination therapies

Mesenchymal stem cells (MSCs) are converted to neural cells using growth factors and chemicals. Although these neural cells are effective at modulating disease symptoms, they are less effective at replacing lost neural cells. Direct transdifferentiation seems to be a promising method for generating the required cells for regenerative medicine applications. Sox2 is a key transcription factor in neural progenitor (NP) fate determination and has been frequently used for transdifferentiating different cell types to NPs. Here, we demonstrated that the overexpression of a single transcription factor, Sox2, in human adipose tissue-derived mesenchymal stem cells (hAT-MSCs) led to the generation of induced NPs-like cells that were clonogenic, proliferative and passageable, and showed the potential to differentiate into three neural lineages. NPs are known as progenitors with the potential to differentiate into oligodendrocytes. In vivo, following transplantation into demyelinated adult mouse brains, they survived, differentiated and integrated into the adult brain while participating in the remyelination process and behavioral improvement. This report introduces a beneficial, low-cost and effective approach for generating NPs from an accessible adult source for autologous applications in treating neurodegenerative diseases, including remyelination therapies for multiple sclerosis and other demyelinating diseases.

A Dopamine-Modified Hyaluronic Acid-Based Mucus Carrying Phytoestrogen and Urinary Exosome for Thin Endometrium Repair.

Thin endometrium (TE) is closely associated with infertility in reproductive medicine. Estrogen therapy gains unsatisfactory outcomes. In this study, an artificial mucus based on dopamine (L-DOPA)-modified hyaluronic acid combining phytoestrogen cajaninstilbene acid and rat urinary exosomes (CUEHD) is constructed for TE treatment using a rat TE model. In the rat TE model, the dominant elastic behavior and adhesive properties of CUEHD guarantee adequate retention, rendering superior synergistic treatment efficacy and favorable biosafety characteristics. CUEHD treatment significantly increases endometrial thickness and promotes receptivity and fertility. Mechanistically, estrogen homeostasis, inflammation inhibition, and endometrial regeneration are achieved through the crosstalk between ER-NLRP3-IL1β and Wnt-β catenin-TGFβ-smad signaling pathways. Moreover, the therapeutic potential of exosomes from human urine and adipose tissue-derived stem cells (ADSCs) and rat ADSCs are also demonstrated, indicating extensive use of the artificial mucus system. Thus, this study illustrates a platform combining phytoestrogen and exosomes with promising implications for TE treatment.

The Adrenal Pheochromocytoma Cell Line PC12 Efficiently Promotes the Regeneration Capability of Adipose Tissue-Derived Mesenchymal Stem Cells in Myogenesis: A Particular Approach to Improving Skeletal Muscle Cell Regeneration.

Researchers are looking for a way to improve the myogenic differentiation of stem cells. Adipose-derived stem cells (ADSCs), known for their multipotency and regenerative capabilities, have been extensively studied for their therapeutic potential. Meanwhile, PC12 cells, derived from rat pheochromocytoma, have been found pivotal in neuroscience research, particularly as a neuronal model system. The current study investigated the effect of the PC12 adrenal pheochromocytoma cell line on the myogenic differentiation of ADSCs. This experimental study was conducted during 2019-2022 (Ahvaz, Iran). Differentiation of ADSCs was induced by using 3 μg/mL 5-azacytidine for 24 hours. Then, the culture media was changed with Dulbecco's Modified Eagle-High Glucose (DMEM-HG) containing 5% horse serum (HS) and kept for 7 days. Different percentages of differentiated ADSCs and PC12 (100:0, 70:30, 50:50, 30:70) were cocultured for 7 days in DMEM-HG containing 5% HS. PC12 was labeled with cell tracker C7000. The real-time polymerase chain reaction and Western blotting techniques were utilized to assess gene and protein expression. All experiments were repeated three times. Data were analyzed using GraphPad Prism 8.0.2 software with a one-way analysis of variance. P<0.05 was considered statistically significant. PC12 visualization confirmed the accuracy of the co-culture process. The differentiated cells showed an aligned, multinucleated shape. The differentiated ADSCs revealed significantly elevated levels of Myh1, Myh2, and Chrn-α1 gene expression compared with undifferentiated ADSCs (P<0.0001). The ADSCs cocultured with PC12 cells showed significantly higher Myh1, Myh2, and Chrn-α1 gene expression than differentiated ADSCs (P<0.001). ADSCs cocultured with 50% PC12 revealed significantly higher MYH and nAchR protein expression than the differentiated group (P<0.01 and P<0.001). Coculturing PC12 cells and ADSCs improves the efficiency of myogenic differentiation. However, the effectiveness of myogenic differentiation depends on the proportions of administered PC12 cells.

Exploring the impact of acetylsalicylic acid and conditioned medium obtained from mesenchymal cells, individually and in combination, on cognitive function, histological changes, and oxidant-antioxidant balance in male rats with hippocampal injury.

The hippocampus is susceptible to damage, leading to negative impacts on cognition. Conditioned medium (CM) obtained from adipose tissue-derived mesenchymal stem cells (MSCs) and acetylsalicylic acid (ASA) have shown neuroprotective effects independently. This study explored the synergistic potential of ASA and CM from adipose-derived MSCs against hippocampal injury. Adult male Wistar rats received bilateral hippocampal ethidium bromide (EB) injections to induce hippocampal damage. Rats were treated with ASA and/or CM derived from adipose tissue MSCs every 48h for 16 days. Behavioral tests (open field test, Morris water maze, novel object recognition, and passive avoidance), oxidative stress, Western blot analysis of brain-derived neurotrophic factor (BDNF) and cerebral dopamine neurotrophic factor (CDNF) expression, and hippocampal histological investigation were conducted. Administration of EB caused impairments in spatial, recognition, and passive avoidance memory, as well as heightened oxidative stress, reduced BDNF/CDNF expression, and pyramidal cell loss in the hippocampal CA1 region. Administration of ASA, CM, or a combination of both mitigated these hippocampal damages and cognitive deficits, elevated BDNF and CDNF levels, and alleviated the CA1 necrosis caused by EB. Moreover, co-administering ASA and CM resulted in greater improvements in spatial memory compared to administering ASA alone, suggesting possible synergistic interactions. The ability of ASA, CM obtained from adipose tissue-derived MSCs, and their combination therapy to alleviate hippocampal injuries highlights their promising therapeutic potential as a neuroprotection strategy against brain damage. Our findings provide preliminary evidence of the potential synergistic effects of ASA and CM, which warrants further investigations.

Anti-inflammatory effects of polydeoxyribonucleotide and adipose tissue-derived mesenchymal stem cells in a canine cell model of osteoarthritis.

A relatively new therapeutic agent for osteoarthritis (OA), polydeoxyribonucleotide (PDRN), shows potential in treating human OA due to its regenerative and anti-inflammatory effects. However, studies on PDRN for canine OA are limited, and no study has investigated their use with mesenchymal stem cells (MSCs) conventionally used for OA treatment. This study aimed to evaluate the potential of PDRN and explore its combined effect with adipose tissue-derived MSCs (AdMSCs) in treating canine OA. To study the impact of PDRN, canine chondrocytes, synoviocytes, and AdMSCs were exposed to various PDRN concentrations, and viability was assessed using cell counting kit-8. The OA model was created by treating chondrocytes and synoviocytes with lipopolysaccharide, followed by treatment under three different conditions: PDRN alone, AdMSCs alone, and a combination of PDRN and AdMSCs. Using real-time quantitative polymerase chain reaction, the anti-inflammatory effects and mechanisms were investigated by quantitatively assessing pro-inflammatory cytokines, collagen degradation markers, adenosine A2a receptor (ADORA2A), and nuclear factor-kappa B. PDRN alone and combined with AdMSCs significantly reduced the expression of pro-inflammatory cytokines and collagen degradation markers in an OA model. PDRN promoted AdMSC proliferation and upregulated ADORA2A expression. AdMSCs exhibited comprehensive anti-inflammatory effects through paracrine effects, and both substances reduced inflammatory gene expression through different mechanisms, potentially enhancing therapeutic effects. The results indicate that PDRN is a safe and effective anti-inflammatory material that can be used independently or as an adjuvant for AdMSCs. Although additional research is necessary, this study is significant because it provides a foundation for future research at the cellular level.

Mesenchymal stem cells from adipose tissue prone to lose their stemness associated markers in obesity related stress conditions

Obesity is a health problem characterized by large expansion of adipose tissue. During this expansion, genotoxic stressors can be accumulated and negatively affect the mesenchymal stem cells (MSCs) of adipose tissue. Due to the oxidative stress generated by these genotoxic stressors, senescence phenotype might be observed in adipose tissue MSCs. Senescent MSCs lose their proliferations and differentiation properties and secrete senescence-associated molecules to their niche thus triggering senescence for the rest of the tissue. Accumulation of senescent cells in adipose tissue results in decreased tissue regeneration and functional impairment not only in the close vicinity but also in the other tissues. Here we hypothesized that declined tissue regeneration might be associated with loss of stemness markers in MSCs population. We analyzed the expression of several stemness-associated genes of in vitro cultured MSCs originated from adipose tissue of high-fat diet and normal diet mice models. Since the heterogenous MSCs population covers a small percentage of the pluripotent stem cells, which have roles in proliferation and tissue regeneration, we measured the percentage of these cells via TRA-1-60 pluripotent state antigen. Additionally, by conducting a shotgun proteomic approach using LC-MS/MS, whole cell proteome of the adipose tissue MSCs of high-fat diet and normal diet mice were analyzed and identified proteins were evaluated via gene ontology and PPI network analysis. MSCs of obese mice showed senescent phenotype and altered cell cycle distribution due to a hostile environment with oxidative stress in adipose tissue where they reside. Additionally, the number of pluripotent markers expressing cells declined in the MSC population of the high-fat diet mice. Gene expression analysis evidenced the loss of stemness with a decrease in the expression of stemness-associated genes. Of the proteomic comparison of the normal and the high-fat diet group, MSCs revealed that stemness-associated molecules were decreased while inflammation and senescence-associated phenotypes emerged in obese mice MSCs. Our results showed us that the MSCs of adipose tissue may lose their stemness properties due to obesity-associated stress conditions.