Human Adipose Tissue-derived Stem Cells Research Articles

Bioreactor expansion reconfigures metabolism and extracellular vesicle biogenesis of human adipose-derived stem cells in vitro

Human mesenchymal stem cells (hMSCs), including human adipose tissue-derived stem cells (hASCs), as well as the secreted extracellular vesicles (EVs), are promising therapeutics in treating inflammatory and neural degenerative diseases. However, prolonged expansion can lead to cellular senescence characterized by a gradual loss of self-renewal ability while altering secretome composition and EV generation. Additionally, hMSCs are highly sensitive to biophysical microenvironment in bioreactor systems utilized in scaling production. In this study, hASCs grown on Plastic Plus or Synthemax II microcarriers in a spinner flask bioreactor (SFB) system were compared to traditional 2D culture. The SFB microenvironment was found to increase the expression of genes associated with hASC stemness, nicotinamide adenine dinucleotide (NAD+) metabolism, glycolysis, and the pentose phosphate pathway as well as alter cytokine secretion (e.g., PGE2 and CXCL10). Elevated reactive oxidative species levels in hASCs of SFB culture were observed without increasing rates of cellular senescence. Expression levels of Sirtuins responsible for preventing cellular senescence through anti-oxidant and DNA repair mechanisms were also elevated in SFB cultures. In particular, the EV biogenesis genes were significantly upregulated (3–10 fold) and the EV production increased 40 % per cell in SFB cultures of hASCs. This study provides advanced understanding of hASC sensitivity to the bioreactor microenvironment for EV production and bio-manufacturing towards the applications in treating inflammatory and neural degenerative diseases.

ODP045 Implantation of Steroidogenic Cells Derived from Human Adipose-derived Stem Cells Extends Survival in a Mouse Model of Adrenal Insufficiency

Abstract Cell therapy has an advantage of compensating hormone in response to physiological stimuli. We have previously shown that mouse mesenchymal stem cells (MSCs) were transformed into steroidogenic cells by forced expression of NR5A1 (a master regulator of steroidogenesis, also known as SF-1/Ad4BP) and that the syngeneic implantation of NR5A1-induced steroidogenic cells extended survival of bilateral adrenoectomised (bAdx) mice. However, ACTH receptor is not induced by NR5A1 in mouse MSCs, and ACTH responsiveness was not detected in recipient mice. In contrast to mice, in human MSCs, ACTH receptor is induced by NR5A1 and ACTH enhances steroid production in NR5A1 induced-steroidogenic cells in vitro studies. In the present study, we implanted human ADSCs-derived steroidogenic cells into immunodeficient mice with adrenal insufficiency. Human adipose tissue-derived stem cells (ADSCs) were inoculated with recombinant adenovirus containing human NR5A1 or LacZ cDNA (MOI=50) and cultured for 7 days. NR5A1 induced-steroidogenic cells secreted both adrenal and gonadal steroids and responded to ACTH. Before xenotransplantation studies, adrenal glands of SCID/Beige mice were surgically removed and implanted into under capsule of the kidney. bAdx mice were dead within 9 days (N=11), bAdx mice (N=12) transplanted with adrenal glands survived to the endpoint of 30 days, and died within 9 days after removal of the kidney containing the graft. We next implanted NR5A1-induced steroidogenic cells or control cells into bAdx mice. The overall survival rate (endpoint of 30 days) of NR5A1 implanted mice was 16.7%, (N=12) while control cell implanted mice died within 13 days (N=10). Median survival time of bAdx mice implanted with NR5A1 induced-steroidogenic cells and control ADSCs was 16. 0 and 11. 0 days, respectively (log-rank test, p < 0. 0001). In the NR5A1 induced-steroidogenic cells implanted mice, serum aldosterone was undetectable, whereas serum corticosterone and cortisol were detectable. Although we performed ACTH loading test on bAdx mice implanted with NR5A1-induced steroidogenic cells, no ACTH responsiveness was detected. The expression of Pomc mRNA in the pituitary gland was increased by bAdx, whereas this increase was not suppressed by implantation of steroid-producing cells (N=3), suggesting that the amount of steroids in the graft complemented survival but was insufficient for negative feedback. These results indicate that human ADSCs are transformed into steroidogenic cells by NR5A1 and are responsive to ACTH in vitro but not in vivo. Xenotransplantation of the induced steroidogenic cells into immunodeficient bAdx mice prolonged the survival, which was supported by the significant detection of basal serum corticosterone and cortisol levels. In particular, serum cortisol indicated hormone secretion from the grafts, suggesting that human ADSCs may also be useful as a regenerative source of steroid-producing cells. Presentation: No date and time listed

Assessing the Efficacy of Whole-Body Titanium Dental Implant Surface Modifications in Inducing Adhesion, Proliferation, and Osteogenesis in Human Adipose Tissue Stem Cells.

Although the influence of titanium implants' micro-surface properties on titanium discs has been extensively investigated, the research has not taken into consideration their whole-body effect, which may be considered possible using a combinatorial approach. Five titanium dental implants with a similar moderate roughness and different surface textures were thoroughly characterized. The cell adhesion and proliferation were assessed after adipose-tissue-derived stem cells (ADSCs) were seeded on whole-body implants. The implants' inductive properties were assessed by evaluating the osteoblastic gene expression. The surface micro-topography was analyzed, showing that hydroxyapatite (HA)-blasted and bland acid etching implants had the highest roughness and a lower number of surface particles. Cell adhesion was observed after 24 h on all the implants, with the highest score registered for the HA-blasted and bland acid etching implants. Cell proliferation was observed only on the laser-treated and double-acid-etched surfaces. The ADSCs expressed collagen type I, osteonectin, and alkaline phosphatase on all the implant surfaces, with high levels on the HA-treated surfaces, which also triggered osteocalcin expression on day seven. The findings of this study show that the morphology and treatment of whole titanium dental implants, primarily HA-treated and bland acid etching implants, impact the adherence and activity of ADSCs in osteogenic differentiation in the absence of specific osteo-inductive signals.

Small extracellular vesicles derived from human adipose-derived stem cells regulate energetic metabolism through the activation of YAP/TAZ pathway facilitating angiogenesis.

Recent studies have found small extracellular vesicles (sEVs) that are secreted from human adipose tissue-derived stem cells (hADSCs-sEVs) and contribute to angiogenesis. Glycolysis, the primary energetic pathway of vascular endothelial cells, plays a key role in the process of angiogenesis. However, hADSCs-sEVs'effects on energy metabolism within endothelial cells remain unclear. In our study, we found that hADSCs-sEVs restored glycolytic metabolism suppressed by 3-(pyridinyl)-1-(4-pyridinyl)-2-propen-1-one(3PO), a unique glycolytic inhibitor increasing the extracellular acidification rate (ECAR), oxygen consumption rate (OCR), glycolytic gene expression as well as pyruvate, lactate, and ATP production in HUVEC cells. In contrast, hADSCs-sEVs decreased PDH-E1α expression and acetyl-CoA production. The above results indicate that hADSCs-sEVs promote HUVEC angiogenesis via enhancing glycolysis and suppressing mitochondrial oxidative phosphorylation. Furthermore, we found that the YAP/TAZ pathway may play a key role in the effects hADSCs-sEVs have on HUVECs, thus, providing a promising approach for pro-angiogenesis-related regeneration.

Stem cell-derived extracellular vesicles reduce the expression of molecules involved in cardiac hypertrophy-In a model of human-induced pluripotent stem cell-derived cardiomyocytes.

Cardiac pathological hypertrophy is the major risk factor that usually progresses to heart failure. We hypothesized that extracellular vesicles (EVs), known to act as important mediators in regulating physiological and pathological functions, could have the potential to reduce the cardiac hypertrophy and the ensuing cardiovascular diseases. Herein, the effects of mesenchymal stem cell-derived extracellular vesicles (EV-MSCs) on cardiac hypertrophy were investigated. EVs were isolated from the secretome of human adipose tissue-derived stem cells (EV-ADSCs) or bone marrow-derived stem cells (EV-BMMSCs). Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were stimulated with AngII and TGF-β1, in absence or presence of EVs. The results showed that exposure of hiPSC-CMs to AngII and TGF-β1 generated in vitro model of hypertrophic cardiomyocytes characterized by increases in surface area, reactive oxygen species production, protein expression of cardiac-specific biomarkers atrial natriuretic factor, migration inhibitory factor, cTnI, COL1A1, Cx43, α-SMA and signalling molecules SMAD2 and NF-kBp50. The presence of EV-ADSCs or EV-BMMSCs in the hiPSC-CM culture along with hypertrophic stimuli reduced the protein expressions of hypertrophic specific markers (ANF, MIF, cTnI, COL1A1) and the gene expressions of IL-6 molecule involved in inflammatory process associated with cardiac hypertrophy and transcription factors SMAD2, SMAD3, cJUN, cFOS with role in cardiomyocyte hypertrophic response induced by AngII and TGF-β1. The EV-ADSCs were more effective in reducing the protein expressions of hypertrophic and inflammatory markers, while EV-BMMSCs in reducing the gene expressions of transcription factors. Notably, neither EV-ADSCs nor EV-BMMSCs induced significant changes in cardiac biomarkers Cx43, α-SMA and fibronectin. These different effects of stem cell-derived EVs could be attributed to their miRNA content: some miRNAs (miR-126-3p, miR-222-3p, miR-30e-5p, miR-181b-5p, miR-124-3p, miR-155-5p, miR-210-3p hsa-miR-221-3p) were expressed in both types of EVs and others only in EV-ADSCs (miR-181a-5p, miR-185-5p, miR-21-5p) or in EV-BMMSCs (miR-143-3p, miR-146a-5p, miR-93-5p), some of these attenuating the cardiac hypertrophy while others enhance it. In conclusion, in hiPSC-CMs the stem cell-derived EVs through their cargo reduced the expression of hypertrophic specific markers and molecules involved in inflammatory process associated with cardiac hypertrophy. The data suggest the EV potential to act as therapeutic mediators to reduce cardiac hypertrophy and possibly the subsequent cardiovascular events.

Effects of Thymoquinone on Adipocyte Differentiation in Human Adipose-Derived Stem Cells.

Inhibition of adipocyte differentiation would be a key strategy to control obesity. Human adipose tissue-derived stem cells (ADSCs) are a promising tool for adipocyte differentiation research. Thymoquinone (TQ) as a potent antioxidant molecule may inhibit adipocyte differentiation. Herein, we aim to investigate the inhibitory effect of TQ on lipid differentiation in ADSCs. Quantification of cell surface markers was used by Flow-Cytometry and the effect of TQ on cell viability was assessed using the AlamarBlue test. ADSCs were subjected to induction of differentiation in the presence of non-cytotoxic concentrations of TQ (6.25, 12.5 and 25 μg/mL). Lipid accumulation was assessed using the Oil-Red O staining technique. Moreover, the expression of PPARγ (Peroxisome proliferator-activated receptor-γ) and FAS (Fatty Acid Synthetase) proteins was evaluated using Western blotting. Flow-cytometry demonstrated the expression of CD44, CD90, and CD73 as mesenchymal stem cell markers on the cell surface. At concentrations ≤100 μg/mL of TQ, no significant difference in cell viability was observed compared to the control. Lipid accumulation in ADSCs significantly decreased at 25 μg/mL (P < 0.001) and 12.5 μg/mL (P < 0.01) of TQ. The findings of the qualitative examination of Lipid Droplets also confirmed these results. Western-blot showed that TQ at 12.5 (p < 0.05) and 25 μg/mL (p < 0.01) reduced FAS/β-actin ratio compared to the positive group. TQ also decreased the expression of PPARγ at 6.25 μg/mL but not at higher concentrations. In conclusion, TQ may reduce differentiation of fat stem cells into fat cells through inhibition of the expression of PPARγ and FAS proteins and might be a potential anti-obesity compound.

Use of collagenase to isolate adipose tissue-derived stem cells – substantial manipulation or not?

Background: Collagenase is commonly used to isolate the stromal vascular fraction (SVF) or adipose tissue-derived stem cells (ADSCs) from human adipose tissue. Enzymatic breakdowns may be a substantial manipulation according to the classifications of medical regulatory authorities. This study investigates the possible effects of human adipose tissue dissociation with collagenase on in vitro function and behavior of ADSCs. Methods and results: Adipose tissue from nine donors was divided into two equal fractions. SVF was then isolated either mechanically or with collagenase, respectively. The resulting cells were analyzed for their surface markers directly after isolation and at passage five. Proliferation, tri-lineage differentiation, and secretome markers were measured after passage four. Using collagenase compared to mechanical isolation did not alter the expression of typical surface markers of ADSCs. ADSCs isolated with collagenase showed a significantly shorter population doubling time (p &lt; 0.001), a significantly higher mean specific GPDH-activity, a stronger intensity in perilipin staining (p = 0.005), and a significantly higher extracellular calcium deposition (p = 0.006) than mechanically isolated ADSCs. The expression of adipogenic and osteogenic marker genes was not different in mechanically versus enzymatically isolated ADSCs. There were no significant differences in proteoglcyan production (p &gt; 0.05) and the concentration of type 2 collagen. Except for an increased CCL2 concentration in mechanically isolated ASDCs (p = 0.01), there were no significant differences in the concentration of secreted proteins between both isolation methods. Conclusions: The use of collagenase does not substantially impair central in vitro characteristics and functions of human adipose tissue-derived stem cells.

Nicotinamide-riboside shifts the differentiation of human primary white adipocytes to beige adipocytes impacting substrate preference and uncoupling respiration through SIRT1 activation and mitochondria-derived reactive species production.

Beige adipocytes play key roles in organismal energy and metabolic balance. In this study, we assessed whether the supplementation of human white adipocytes, differentiated from human adipose tissue-derived stem cells, with nicotinamide riboside (NR), a potent NAD + precursor, can shift differentiation to beige adipocytes (beiging). NR induced mitochondrial biogenesis and the expression of beige markers (TBX1 and UCP1) in white adipocytes demonstrating that NR can declutch beiging. NR did not induce PARP activity but supported SIRT1 induction, which plays a key role in beiging. NR induced etomoxir-resistant respiration, suggesting increases in the oxidation of carbohydrates, carbohydrate breakdown products, or amino acids. Furthermore, NR boosted oligomycin-resistant respiration corresponding to uncoupled respiration. Enhanced etomoxir and oligomycin-resistant respiration were dependent on mitochondrial reactive-species production. Taken together, NR supplementation can induce beiging and uncoupled respiration, which are beneficial for combatting metabolic diseases.

Senescence recovering by dual drug-encapsulated liposomal nanoparticles for large-scale human cell expansion.

Although regenerative therapy and bioartificial tissues and organs require a sufficient number of human cells, current cell expansion processes are accompanied by accumulation of senescent cells that are related to deterioration of cellular functions and induction of senescence-associated secretory phenotype (SASP). Therefore, suppression of replicative senescence during expansion is one of the crucial issues for dissemination of regenerative medicine. We herein developed dual drug-encapsulated liposomal nanoparticles (LNPs) to suppress cellular senescence in human adipose tissue-derived mesenchymal stem cells (hAT-MSCs) and natural killer (NK) cells by removal of dysfunctional mitochondria from the senescent cells. We found that LNP treatment reduced senescent makers; downregulation of p21 expression and reduction of SA-β-Gal activity in both cells provably due to mitophagy reactivation in the cells. Moreover, SASP secretion in hAT-MSCs and tumor cytotoxicity in NK cells were also improved upon LNP treatments. These findings may contribute to the production of highly effective expanded cells for regenerative medicine and bioartificial tissues and organs.

Transplantation of insulin-producing cells derived from human mesenchymal stromal/stem cells into diabetic humanized mice

BackgroundThe purpose of this study was to investigate allogenic immune responses following the transplantation of insulin-producing cells (IPCs) differentiated from human adipose tissue-derived stem cells (hAT-MSCs) into humanized mice.MethodshAT-MSCs were isolated from liposuction aspirates obtained from HLA-A2-negative healthy donors. These cells were expanded and differentiated into IPCs. HLA-A2-positive humanized mice (NOG-EXL) were divided into 4 groups: diabetic mice transplanted with IPCs, diabetic but nontransplanted mice, nondiabetic mice transplanted with IPCs and normal untreated mice. Three million differentiated cells were transplanted under the renal capsule. Animals were followed-up to determine their weight, glucose levels (2-h postprandial), and human and mouse insulin levels. The mice were euthanized 6–8 weeks posttransplant. The kidneys were explanted for immunohistochemical studies. Blood, spleen and bone marrow samples were obtained to determine the proportion of immune cell subsets (CD4+, CD8+, CD16+, CD19+ and CD69+), and the expression levels of HLA-ABC and HLA-DR.ResultsFollowing STZ induction, blood glucose levels increased sharply and were then normalized within 2 weeks after cell transplantation. In these animals, human insulin levels were measurable while mouse insulin levels were negligible throughout the observation period. Immunostaining of cell-bearing kidneys revealed sparse CD45+ cells. Immunolabeling and flow cytometry of blood, bone marrow and splenic samples obtained from the 3 groups of animals did not reveal a significant difference in the proportions of immune cell subsets or in the expression levels of HLA-ABC and HLA-DR.ConclusionTransplantation of IPCs derived from allogenic hAT-MSCs into humanized mice was followed by a muted allogenic immune response that did not interfere with the functionality of the engrafted cells. Our findings suggest that such allogenic cells could offer an opportunity for cell therapy for insulin-dependent diabetes without immunosuppression, encapsulation or gene manipulations.Graphical

Enhanced osteogenic differentiation of stem cells by 3D printed PCL scaffolds coated with collagen and hydroxyapatite

Bone tissue engineering uses various methods and materials to find suitable scaffolds that regenerate lost bone due to disease or injury. Poly(ε-caprolactone) (PCL) can be used in 3D printing for producing biodegradable scaffolds by fused deposition modeling (FDM). However, the hydrophobic surfaces of PCL and its non-osteogenic nature reduces adhesion and cell bioactivity at the time of implantation. This work aims to enhance bone formation, osteogenic differentiation, and in vitro biocompatibility via PCL scaffolds modification with Hydroxyapatite (HA) and Collagen type I (COL). This study evaluated the osteosupportive capacity, biological behavior, and physicochemical properties of 3D-printed PCL, PCL/HA, PCL/COL, and PCL/HA/COL scaffolds. Biocompatibility and cells proliferation were investigated by seeding human adipose tissue-derived mesenchymal stem cells (hADSCs) onto the scaffolds, which were analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, and 6-diamidino-2-phenylindole (DAPI) staining. In addition, the bone differentiation potential of the hADSCs was assessed using calcium deposition, alkaline phosphatase (ALP) activity, and bone-related protein and genes. Although all constructed scaffolds support hADSCs proliferation and differentiation, the results showed that scaffold coating with HA and COL can boost these capacities in a synergistic manner. According to the findings, the tricomponent 3D-printed scaffold can be considered as a promising choice for bone tissue regeneration and rebuilding.

Biophysical evaluation of treating adipose tissue-derived stem cells using non-thermal atmospheric pressure plasma

Non-thermal atmospheric pressure plasma (NTAPP) is a partially ionized gas containing fast electrons and relatively slow ions. This study aims to investigate the influences of NTAPP on human adipose tissue-derived stem cells (ADSCs) and examine the feasibility of using optical spectroscopy as a non-destructive method for cell analysis. A plasma jet is used as the source of low-temperature plasma in which pure helium gas is ionized by a high voltage (8 kV) and frequency (6 kHz). ADSCs were exposed to the NTAPP for 30 s, 60 s, 90 s, and 120 s. The efficiency of the plasma treatment was investigated using flow cytometry and optical spectroscopy methods. This study compared surface markers of NTAPP treated and untreated ADSCs using CD90 and CD105 as positive markers. The result proved that NTAPP-exposed ADSCs maintain their stemming. Measuring ADSCS apoptosis by labeling Annexin V-Propidium Iodide showed that the plasma at short exposure time is relatively non-toxic. However, a longer exposure time can lead to apoptosis and necrosis. Moreover, Cell cycle analysis revealed that NTAPP accelerates the cell cycle in very low doses and can cause proliferation. In this experiment, flow cytometry measurements have been used to determine oxidative stress. The results showed that with increasing plasma dose, intracellular ROS levels reduced. This data also suggests that intracellular ROS are not responsible for the cells' viability. Furthermore, we used reflectance spectroscopy as a non-destructive method for evaluating treatment response and comparing this method with cell analysis techniques. The results indicate spectroscopy's efficiency as a method of cell analysis. This study suggests that NTAPP would be an efficient tool to improve ADSCs culture's efficiency in vitro; thus, we support the potential applications of NTAPP in the field of stem cell therapy and regenerative medicine.

An encapsulated fibrin-based bioartificial tissue construct with integrated macrovessels, microchannels, and capillary tubes.

Facilitating sufficient nutrient and oxygen supply in large-scale bioartificial constructs is a critical step in organ bioengineering. Immediate perfusion not only depends on a dense capillary network, but also requires integrated large-diameter vessels that allow vascular anastomoses during implantation. These requirements set high demands for matrix generation as well as for in vitro cultivation techniques and remain mostly unsolved challenges up until today. Additionally, bioartificial constructs must have sufficient biomechanical stability to withstand mechanical stresses during and after implantation. We developed a bioartificial tissue construct with a fibrin matrix containing human umbilical vein endothelial cells and adipose tissue-derived stem cells facilitating capillary-like network formation. This core matrix was surrounded by a dense acellular fibrin capsule providing biomechanical stability. Two fibrin-based macrovessels were integrated on each side of the construct and interconnected via four 1.2 mm thick microchannels penetrating the cellularized core matrix. After 4 days of perfusion in a custom-built bioreactor, homogeneous capillary-like network formation throughout the core matrix was observed. The fibrin capsule stabilized the core matrix and facilitated the generation of a self-supporting construct. Thus, the encapsulated fibrin tissue construct could provide a universal prevascularized matrix for seeding with different cell types in various tissue engineering approaches.

Internalization of FITC-albumin in Human Adipose-derived Stem Cells: Involvement of Clathrin and Caveolin.

Adipose tissue-derived stem cells (AdSCs) are one of the most promising cell types for cell-based therapies. In addition, AdSCs systematically injected into the body have been reported to localize to damaged tissues and certain types of tumor. As an important part of establishing a potent drug delivery system with AdSCs, the mechanism and efficiency of uptake into AdSCs has drawn much research attention. However, this remains to be fully clarified. The aim of this study was to examine the characteristics of endocytosis-mediated uptake in human AdSCs. We used fluorescein isothiocyanate-labeled albumin (FITC-albumin) as a potent marker of endocytosis. FITC-albumin uptake was time- and temperature-dependent. Confocal microscopy showed punctate localization of fluorescence in the cytoplasm. FITC-albumin uptake was inhibited by human serum albumin in a concentration-dependent manner. FITC-albumin uptake was inhibited by a metabolic inhibitor (2,4-dinitrophenol), a microtubule polymerization inhibitor (colchicine), an actin polymerization inhibitor (cytochalasin D), endosomal acidification inhibitors (chloroquine and bafilomycin A1), clathrin-dependent endocytosis inhibitors (chloropromazine, phenylarsine oxide, and Pitstop2), and caveolin-dependent endocytosis inhibitors (nystatin and methyl-β-cyclodextrin). Furthermore, the knockdown of the clathrin heavy chain and caveolin-1 significantly reduced FITC-albumin uptake. These findings suggest that AdSCs take up albumin via endocytic pathways in which clathrin and caveolin are involved.

Endo- and Exometabolome Crosstalk in Mesenchymal Stem Cells Undergoing Osteogenic Differentiation.

This paper describes, for the first time to our knowledge, a lipidome and exometabolome characterization of osteogenic differentiation for human adipose tissue stem cells (hAMSCs) using nuclear magnetic resonance (NMR) spectroscopy. The holistic nature of NMR enabled the time-course evolution of cholesterol, mono- and polyunsaturated fatty acids (including ω-6 and ω-3 fatty acids), several phospholipids (phosphatidylcholine, phosphatidylethanolamine, sphingomyelins, and plasmalogens), and mono- and triglycerides to be followed. Lipid changes occurred almost exclusively between days 1 and 7, followed by a tendency for lipidome stabilization after day 7. On average, phospholipids and longer and more unsaturated fatty acids increased up to day 7, probably related to plasma membrane fluidity. Articulation of lipidome changes with previously reported polar endometabolome profiling and with exometabolome changes reported here in the same cells, enabled important correlations to be established during hAMSC osteogenic differentiation. Our results supported hypotheses related to the dynamics of membrane remodelling, anti-oxidative mechanisms, protein synthesis, and energy metabolism. Importantly, the observation of specific up-taken or excreted metabolites paves the way for the identification of potential osteoinductive metabolites useful for optimized osteogenic protocols.

The impairment of osteogenic differentiation of human adipose tissue-derived mesenchymal stem cells under high D-glucose concentrations

Type 2 diabetic patients have an increased risk of developing serious long-term complications such as cardiovascular disease, retinopathy, nephropathy, neuropathy, diabetic foot, and osteoporosis. However, the correlation between hyperglycaemia and osteoporosis has not yet been fully clarified. In this research, we investigated the effect of different high D-glucose concentrations (25, 50, and 100 mM) on osteogenic differentiation of human non-diabetic and diabetic adipose tissue-derived mesenchymal stem cells (nAT-MSCs and dAT-MSCs). The differentiated cells were qualified by Alizarin Red S staining and quantified by measuring the absorbance at 482 nm. The expressions of osteogenic master genes were examined by quantitative reverse transcription polymerase chain reaction (qRT-PCR) methods. Interestingly, the osteogenic differentiation and the expression of osteogenic-specific genes (Runx-2 and ALP) decreased with an increase of D-glucose concentration. Our work contributes to the understanding of the relationship between hyperglycaemia and osteoporosis and support the role of stem cells in developing new medicine or therapeutic treatment for preventing diabetic complications.

Potential of Conditioned Medium of hATMSCs in Aging Cells Model

Skin aging is caused by the exposure cumulative of ultraviolet radiation, it leads reactive oxygen species (ROS) production in the skin. The conditioned medium of human Adipose Tissue-derived Mesenchymal Stem Cells (hATMSCs) can scavenge free radicals and increase the survival rate of skin cells under oxidative stress. This study examined the protective effects of Conditioned Medium (CM) of hATMSCs in H2O2-induced human skin fibroblast cell line (BJ). The aging cells model using H2O2-induced BJ cells were added CM-hATMSCs in concentrations (0, 10, 30%) and incubated in various time, furthermore BJ cells induced by various H2O2 concentrations (0, 50, 100, 200 µM) incubated for 1 h. The anti-aging potential were measured including viability, ROS and collagen levels in BJ cells which treated CM-hATMSCs. The median inhibitory concentration (IC50) of H2O2 on BJ cells for 1 h incubation was 107.87 μM and 91.25 μM for 10 min incubation. CM-hATMSCs increased the viability on aging model cells. CM-hATMSCs concentration 30% increased the viability of H2O2 50, 100, 200 µM-induced BJ cells. CM-hATMSCs concentration 25% decreased ROS, increased collagen level in H2O2 50, 100, 200 µM-induced BJ cells. CM-hATMSCs increase the viability cells, collagen level and decrease ROS level in aging model cells.

Effects of obesity on reparative function of human adipose tissue-derived mesenchymal stem cells on ischemic murine kidneys.

Introduction:Obesity is a health burden that impairs cellular processes. Mesenchymal stem/stromal cells (MSCs) are endowed with reparative properties and can ameliorate renal injury. Obesity impairs human MSC function in-vitro, but its effect on their in-vivo reparative potency remains unknown.Subjects and Methods:Abdominal adipose tissue-derived MSC were harvested from patients without (‘lean’) or with obesity (‘obese’) (body mass index<30 or ≥30kg/m2, respectively) during kidney donation or bariatric surgery, respectively. MSC (5x105/200μL) or vehicle were then injected into 129S1 mice 2 weeks after renal artery stenosis (RAS) or sham surgery (n=8/group). Two weeks later, mice underwent magnetic resonance imaging to assess renal perfusion and oxygenation in-vivo, and kidneys then harvested for ex-vivo studies.Results:Similar numbers of lean and obese-MSCs engrafted in stenotic mouse kidneys. Vehicle-treated RAS mice had reduced stenotic-kidney cortical and medullary perfusion and oxygenation. Lean (but not obese) MSC normalized ischemic kidney cortical perfusion, whereas both effectively mitigated renal hypoxia. Serum creatinine and blood pressure were elevated in RAS mice and lowered only by lean-MSC. Both types of MSCs alleviated stenotic-kidney fibrosis, but lean-MSC more effectively than obese-MSC. MSC senescence-associated beta-gal activity, and gene expression of p16, p21, and vascular endothelial growth factor correlated with recipient kidney perfusion and tissue injury, linking MSC characteristics with their in-vivo reparative capacity.Discussion:Human obesity impairs the reparative properties of adipose-tissue-derived MSCs, possibly by inducing cellular senescence. Dysfunction and senescence of the endogenous MSC repair system in patients with obesity may warrant targeting interventions to restore MSC vitality.

Reprogramming by Cytosolic Extract of Human Embryonic Stem Cells to Improve Dopaminergic Differentiation Potential of Human Adipose Tissue-derived Stem Cells.

The extract of pluripotent stem cells induces dedifferentiation of somatic cells with restricted plasticity. In this study, we used the extract of human embryonic stem cells (hESC) to dedifferentiate adipose tissue-derived stem cells (ADSCs) and examined the impact of this reprogramming event on the dopaminergic differentiation of the cells. For this purpose, cytoplasmic extract of ESCs was prepared by repeated freezing and thawing cycles. The plasma membrane of hADSCs was reversibly permeabilized by streptolysin O (SLO), exposed to hESC extract, and resealed by a CaCl2-containing medium. As revealed by qPCR analysis, expression of OCT4, SOX2, NANOG, LIN28A, and KLF4 mRNAs were downregulated in the ADSCs one week after extract incubation, while all mRNAs except for KLF4 were upregulated at the end of the second week. For dopaminergic differentiation, control and reprogrammed ADSCs were induced by a serum-free neurobasal medium containing B27 and a cocktail of sonic hedgehog (SHH), basic fibroblast growth factor (bFGF), fibroblastic growth factor 8 (FGF8), and brain-derived neurotrophic factor (BDNF) for 12 days. After differentiation, the expression levels of some neuronal and dopaminergic-related genes, including PAX6, NESTIN, NEFL, GLI1, LMXB1, EN1, NURR1, and TH, significantly increased in the reprogrammed ADSCs compared to the control group. On the whole, two weeks after reprogramming by ESC extract, ADSCs showed an improved dopaminergic differentiation potential. These findings suggest that the cytoplasmic extract of hESCs contains some regulatory factors which induce the expression of pluripotency-associated markers in somatic cells and that the exposure to ESC extract may serve as a simple and rapid strategy to enhance the plasticity of somatic stem cells for cell replacement therapy purposes. hADSCs have emerged as a valuable candidate for transplantation therapy of neurodegenerative diseases.Several studies have documented dopaminergic dedifferentiation of hADSCs.Implementing ADSCs towards a more pluripotent state using different strategies like somatic cell nuclear transfer. The extract of pluripotent stem cells induces dedifferentiation of somatic cells with restricted plasticity. In this study, we used the extract of hESC to dedifferentiate ADSCs and examined the impact of this reprogramming event on the dopaminergic differentiation of the cells. Cytoplasmic extract of ESCs was prepared by repeated freezing and thawing cycles. These cells express several neuron-specific genes, secrete several factors associated with neuroprotection, and exhibit differentiation into neural and glial cells in vitro. In recent years, several studies have documented dopaminergic differentiation of hADSCs.

Differential Lung Protective Capacity of Exosomes Derived from Human Adipose Tissue, Bone Marrow, and Umbilical Cord Mesenchymal Stem Cells in Sepsis-Induced Acute Lung Injury.

Exosomes derived from human mesenchymal stem cells (hMSCs) have the capacity to regulate various biological events associated with sepsis-induced acute respiratory distress syndrome (ARDS), including cellular immunometabolism, the production of proinflammatory cytokines, allowing them to exert therapeutic effects. However, little is known about which type of hMSC-derived exosomes (hMSC-exo) is more effective and suitable for the treatment of sepsis-induced ARDS. The purpose of this study is to compare the efficacy of hMSC-derived exosomes from human adipose tissue (hADMSC-exo), human bone marrow (hBMMSC-exo), and human umbilical cord (hUCMSC-exo) in the treatment of sepsis-induced ARDS. We cocultured lipopolysaccharide- (LPS-) stimulated RAW264.7 macrophage cells with the three kinds of hMSCs and found that all hMSCs reduced the glycolysis level and the content of lactic acid in macrophages. Accordingly, the expression of proinflammatory cytokines also decreased. Notably, the protective effects of hMSCs from adipose tissue were more obvious than those of bone marrow and umbilical cord hMSCs. However, this protective effect was eliminated when an exosome inhibitor, GW4869, was added. Subsequently, we extracted and cocultured hMSC-derived exosomes with LPS-stimulated RAW264.7 cells and found that all three kinds of exosomes exerted a similar protective effect as their parental cells, with exosomes from adipose hMSCs showing the strongest protective effect. Finally, an experimental sepsis model in mice was established, and we found that all three types of hMSCs have obvious lung-protective effects, in reducing lung injury scores, lactic acid, and proinflammatory cytokine levels in the lung tissues and decreasing the total protein content and inflammatory cell count in the bronchoalveolar lavage fluid (BALF), and also can attenuate the systemic inflammatory response and improve the survival rate of mice. Intravenous injection of three types of hMSC-exo, in particular those derived from adipose hADMSCs, also showed lung-protective effects in mice. These findings revealed that exosomes derived from different sources of hMSCs can effectively downregulate sepsis-induced glycolysis and inflammation in macrophages, ameliorate the lung pathological damage, and improve the survival rate of mice with sepsis. It is worth noting that the protective effect of hADMSC-exo is better than that of hBMMSC-exo and hUCMSC-exo.