Differentiation Of Adipose Tissue-derived Stem Cells Research Articles

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.

Aspirin Stimulates the Osteogenic Differentiation of Human Adipose Tissue-Derived Stem Cells In Vitro.

This study investigates the impact of acetylsalicylic acid (ASA), also known as aspirin, on adipose tissue-derived stem cells (ASCs), aiming to elucidate its dose-dependent effects on morphology, viability, proliferation, and osteogenic differentiation. Isolated and characterized human ASCs were exposed to 0 µM, 100 µM, 200 µM, 400 µM, 800 µM, 1000 µM, 10,000 µM, and 16,000 µM of ASA in vitro. Cell morphology, viability, and proliferation were evaluated with fluorescent live/dead staining, alamarBlue viability reagent, and CyQUANT® cell proliferation assay, respectively. Osteogenic differentiation under stimulation with 400 µM or 1000 µM of ASA was assessed with alizarin red staining and qPCR of selected osteogenic differentiation markers (RUNX2, SPP1, ALPL, BGLAP) over a 3- and 21-day-period. ASA doses ≤ 1000 µM showed no significant impact on cell viability and proliferation. Live/dead staining revealed a visible reduction in viable cell confluency for ASA concentrations ≥ 1000 µM. Doses of 10,000 µM and 16,000 µM of ASA exhibited a strong cytotoxic and anti-proliferative effect in ASCs. Alizarin red staining revealed enhanced calcium accretion under the influence of ASA, which was macro- and microscopically visible and significant for 1000 µM of ASA (p = 0.0092) in quantification if compared to osteogenic differentiation without ASA addition over a 21-day-period. This enhancement correlated with a more pronounced upregulation of osteogenic markers under ASA exposure (ns). Our results indicate a stimulatory effect of 1000 µM of ASA on the osteogenic differentiation of ASCs. Further research is needed to elucidate the precise molecular mechanisms underlying this effect; however, this discovery suggests promising opportunities for enhancing bone tissue engineering with ASCs as cell source.

A hybrid construct of decellularized matrix and fibrin for differentiating adipose stem cells into insulin-producing cells, an optimized in vitro assessment.

The generation of insulin-producing cells (IPCs) is an attractive approach for replacing damaged β cells in diabetic patients. In the present work, we introduced a hybrid platform of decellularized amniotic membrane (dAM) and fibrin encapsulation for differentiating adipose tissue-derived stem cells (ASCs) into IPCs. ASCs were isolated from healthy donors and characterized. Human AM was decellularized, and its morphology, DNA, collagen, glycosaminoglycan (GAG) contents, and biocompatibility were evaluated. ASCs were subjected to four IPC differentiation methods, and the most efficient method was selected for the experiment. ASCs were seeded onto dAM, alone or encapsulated in fibrin gel with various thrombin concentrations, and differentiated into IPCs according to a method applying serum-free media containing 2-mercaptoethanol, nicotinamide, and exendin-4. PDX-1, GLUT-2 and insulin expression were evaluated in differentiated cells using real-time PCR. Structural integrity and collagen and GAG contents of AM were preserved after decellularization, while DNA content was minimized. Cultivating ASCs on dAM augmented their attachment, proliferation, and viability and enhanced the expression of PDX-1, GLUT-2, and insulin in differentiated cells. Encapsulating ASCs in fibrin gel containing 2 mg/ml fibrinogen and 10 units/ml thrombin increased their differentiation into IPCs. dAM and fibrin gel synergistically enhanced the differentiation of ASCs into IPCs, which could be considered an appropriate strategy for replacing damaged β cells.

Differentiation of Adipose Tissue Mesenchymal Stem Cells into Endothelial Cells Depends on Fat Depot Conditions: Regulation by miRNA.

The development of obesity is associated with substantial modulation of adipose tissue (AT) structure. The plasticity of the AT is reflected by its remarkable ability to expand or reduce in size throughout the adult lifespan, which is linked to the development of its vasculature. This increase in AT vasculature could be mediated by the differentiation of adipose tissue-derived stem cells (ASCs) into endothelial cells (ECs) and form new microvasculature. We have already shown that microRNA (miRNA)-145 regulates the differentiation of ASCs into EC-like (ECL) cells. Here, we investigated whether ASCs-differentiation into ECs is governed by a miRNAs signature that depends on fat depot location and /or the metabolic condition produced by obesity. Human ASCs, which were obtained from white AT by surgical procedures from lean and obese patients, were induced to differentiate into ECL cells. We have identified that miRNA-29b-3p in both subcutaneous (s)ASCs and visceral ASCs and miRNA-424-5p and miRNA-378a-3p in subcutaneous (s)ASCs are involved in differentiation into EC-like cells. These miRNAs modulate their pro-angiogenic effects on ASCs by targeting FGFR1, NRP2, MAPK1, and TGF-β2, and the MAPK signaling pathway. We show for the first time that miRNA-29b-3p upregulation contributes to ASCs' differentiation into ECL cells by directly targeting TGFB2 in both sASCs and visceral ASCs. Moreover, our results reveal that, independent of sASCs' origin (obese/lean), the upregulation of miRNA-378a-3p and the downregulation of miRNA-424-5p inhibit MAPK1 and overexpress FGFR1 and NRP2, respectively. In summary, both the adipose depot location and obesity affect the differentiation of resident ASCs through the expression of specific miRNAs.

GDNF facilitates the differentiation of ADSCs to Schwann cells and enhances nerve regeneration through GDNF/MTA1/Hes1 axis

Adipose tissue-derived stem cells (ADSCs) are a kind of stem cells with multi-directional differentiation potential, which mainly restore tissue repair function and promote cell regeneration. It can be directionally differentiated into Schwann-like cells to promote the repair of peripheral nerve injury. Glial cell line-derived neurotrophic factor (GDNF) plays an important role in the repair of nerve injury, but the underlying mechanism remains unclear, which seriously limits its further application.The study aimed to identify the molecular mechanism by which overexpression of glial cell line–derived neurotrophic factor (GDNF) facilitates the differentiation of ADSCs into Schwann cells, enhancing nerve regeneration after injury. In vitro, ADSCs overexpressing GDNF for 48 h exhibited changes in their morphology, with 80% of the cells having two or more prominences. Compared with that of ADSCs, GDNF-ADSCs exhibited increased expression of the Schwann cell marker S100, nerve damage repair-related factors.ADSC cells in normal culture and ADSC cells were overexpressing GDNF(GDNF-ADSCs) were analysed using TMT-Based Proteomic Analysis and revealed a significantly higher expression of MTA1 in GDNF-ADSCs than in control ADSCs. Hes1 expression was significantly higher in GDNF-ADSCs than in ADSCs and decreased by MTA1 silencing, along with a simultaneous decrease in the expression of S100 and nerve damage repair factors. These findings indicate that GDNF promotes the differentiation of ADSCs into Schwann cells and induces factors that accelerate peripheral nerve damage repair.

Nd:YAG-photobiomodulation enhanced ADSCs multilineage differentiation and immunomodulation potentials

To investigate the effects of Nd: YAG (1064 nm) photobiomodulation on multilineage differentiation and immunomodulation potentials of adipose tissue-derived stem cells (ADSCs) in vitro and in vivo. For in vitro experiments, cells were divided into the control group (non-irradiated control ADSCs) and photobiomodulation groups. 0.5 J/cm2, 1 J/cm2, 2 J/cm2, and 4 J/cm2 were used for proliferation assays; for ADSCs adipogenic differentiation assays, 0.5 J/cm2, 1 J/cm2 were applied; 1 J/cm2 was used for migration and immunomodulation assays. The differentiation abilities were assessed by qPCR, Oil Red O staining, and Alizarin Red staining. The immunomodulation potential was assessed by qPCR and human cytokine array. DSS-induced colitis model. was used to test the effect of photobiomodulation on ADSCs immunomodulation potentials in vivo. Nd:YAG-based photobiomodulation dose-dependently promoted ADSCs proliferation and migration; 1 J/cm2 showed the best promotion effect on proliferation. Moreover, Nd:YAG photobiomodulation promoted ADSCs osteogenic differentiation and brown adipose adipogenic differentiation. The potential immunomodulation assays showed Nd:YAG photobiomodulation improved Anti-inflammation capacity of ADSCs and photobiomodulation irradiated ADSCs effectively alleviated DSS-induced colitis severity in vivo. Our study suggests Nd:YAG photobiomodulation might enhance the ADSCs multilineage differentiation and immunomodulation potentials. These results might help to enhance ADSCs therapeutic effects for clinical application. However, further studies are needed to explore the mechanisms of Nd:YAG photobiomodulation promoting multilineage differentiation and immunomodulation potentials of ADSCs.

Conversion of Unmodified Stem Cells to Pacemaker Cells by Overexpression of Key Developmental Genes.

Arrhythmias of the heart are currently treated by implanting electronic pacemakers and defibrillators. Unmodified adipose tissue-derived stem cells (ASCs) have the potential to differentiate into all three germ layers but have not yet been tested for the generation of pacemaker and Purkinje cells. We investigated if-based on overexpression of dominant conduction cell-specific genes in ASCs-biological pacemaker cells could be induced. Here we show that by overexpression of certain genes that are active during the natural development of the conduction system, the differentiation of ASCs to pacemaker and Purkinje-like cells is feasible. Our study revealed that the most effective procedure consisted of short-term upregulation of gene combinations SHOX2-TBX5-HCN2, and to a lesser extent SHOX2-TBX3-HCN2. Single-gene expression protocols were ineffective. Future clinical implantation of such pacemaker and Purkinje cells, derived from unmodified ASCs of the same patient, could open up new horizons for the treatment of arrythmias.

Hydrogen sulfide potently promotes neuronal differentiation of adipose tissue-derived stem cells involving nitric oxide-mediated signaling cascade with the aid of cAMP-elevating agents

Neuronal differentiation of adipose tissue-derived stem cells (ASCs) is potently promoted by valproic acid (VPA) through a gaseous signaling molecule, nitric oxide (NO). Here, we investigated the involvement of hydrogen sulfide (H2S), another gaseous signaling molecule, in neuronal differentiation of ASCs. VPA-promoted neuronal differentiation of ASCs was accompanied by increased intracellular H2S and sulfane sulfur with increased mRNA expression of enzymes synthesizing sulfane sulfur including cystathionine β-synthase (CBS), of which inhibition reduced the differentiation efficiency. H2S donors, GYY4137 (GYY) or NaHS, potently promoted neuronal differentiation of ASCs when cAMP-elevating agents, dibutyryl cyclic adenosine monophosphate and isobutyl methyl-xanthine, were added as neuronal induction medium (NIM). Neuronal differentiation of ASCs promoted by NaHS or GYY was accompanied by Ca2+ entry and increased mRNA expression of voltage-gated Ca2+ channels. NaHS or GYY also increased mRNA expression of enzymes of the NO-citrulline cycle including inducible NO synthase (iNOS). It was concluded from these results that H2S potently promoted differentiation of ASCs into neuronal cells expressing functional voltage-gated Ca2+ channels with the aid of cAMP-elevating agents, involving NO-mediated signaling cascade. These effects of H2S were also considered as a partial mechanism for the VPA-promoted neuronal differentiation of ASCs.

Triiodotironina não influencia na diferenciação condrogênica de células tronco do tecido adipose de ratas jovens

ABSTRACT: The objective of this study was to investigate the in vitro action of triiodothyronine (T3) on the chondrogenic differentiation of adipose tissue-derived stem cells (ASCs) of female rats, with different time periods and doses. ASCs were extracted from female Wistar rats and were cultured in chondrogenic medium with and without the presence of T3. Five groups were established: 1) ASCs without T3; and 2,3,4,5) ASCs with 0.01, 1, 100 and 1,000 nM T3, respectively). After 7, 14 and 21 days, cell morphology, chondrogenic matrix formation, and expression of Sox9, aggrecan, collagen II, and collagen X were evaluated. The Student-Newman-Keuls test was used. ASCs showed CD54, CD73, and CD90 before chondrogenic differentiation. The hormone treatment did not alter chondrogenic matrix formation, Sox9 expression at 14 or 21 days, or expression of collagen II or collagen X at any time. However, the 0.01, 1, and 1000 nM T3 doses decreased Sox9 expression at 7 days. In conclusion, chondrogenic differentiation of ASCs of female rats is not influenced by T3.

Valproic acid promotes differentiation of adipose tissue-derived stem cells to neuronal cells selectively expressing functional N-type voltage-gated Ca2+ channels

The differentiation of adipose tissue-derived stem cells (ASCs) to neuronal cells is greatly promoted by valproic acid (VPA), and is synergistically enhanced by the following treatment with neuronal induction medium (NIM) containing cAMP-elevating agents. In the present study, we investigated the synergism between VPA and NIM in neuronal differentiation of ASCs, assessed by the expression of neurofilament medium polypeptide (NeFM), with respect to Ca2+ entry. VPA (2 mM) treatment for 3 days followed by NIM for 2 h synergistically increased the incidence of neuronal cells differentiated from ASCs to an extent more than VPA alone treatment for 6 days, shortening the time required for the differentiation. VPA increased intracellular Ca2+ and the mRNAs of voltage-gated Ca2+ channels, Cacna1b (Cav2.2) and Cacna1h (Cav3.2), in ASCs. Inward currents through Ca2+ channels were evoked electrophysiologically at high voltage potential in ASCs treated with VPA. NIM reduced the mRNAs of NeFM and Cacna1b in VPA-promoted neuronal differentiation of ASCs. It was concluded that functional N-type voltage-gated Ca2+ channels (Cav2.2) are selectively expressed in VPA-promoted neuronal differentiation of ASCs. NIM seems to enhance the mRNA translation of molecules required for the differentiation. Neuronal cells obtained from ASCs by this protocol will be used as a cell source for regenerative therapy of neurological disorders associated with altered Cav2.2 activity.

Let-7a-5p regulated by lncRNA-MEG3 promotes functional differentiation to Schwann cells from adipose derived stem cells via directly inhibiting RBPJ-mediating Notch pathway.

Schwann cells (SCs) have important roles in supporting and repairing peripheral neurons, and thus have great potential for nerve injury treatment. Adipose tissue-derived stem cells (ADSCs) can be reliably induced to differentiate into SCs. However, the underlying molecular mechanisms are unclear. We explored the roles of MEG3/let-7a-5p/RBPJ axis in the differentiation into SCs from ADSCs. Primary ADSCs were induced to differentiate into SCs by appropriate reagents. ELISA, immunostaining, Western blotting, and qRT-PCR were employed to examine levels of SC-markers such as S100, GFAP, SOX10, p75NTR, GAP43, MPZ, β-NGF, BDNF, and NCAM and let-7 family, MEG3, RBPJ, and Notch signaling related proteins. Dual luciferase assay and RNA immunoprecipitation were performed to validate interactions of let-7a-5p/RBPJ mRNA and MEG3/let-7a-5p. Cultured ADSCs could be induced to differentiate into functional SCs. Let-7a-5p and let-7d-5p were elevated during the differentiation while MEG3 and RBPJ/Notch-signaling were suppressed. Let-7a-5p mimics promoted ADSC differentiation into SCs and up-regulated the levels of SC-related markers including S100, GFAP, SOX10, p75NTR, GAP43, MPZ, β-NGF, and NCAM, while RBPJ or MEG3 overexpression retarded the differentiation and reduced those levels. Let-7a-5p directly targeted RBPJ and MEG3 disinhibited Notch-RBPJ signaling via sponging let-7a-5p. RBPJ overexpression reversed the acceleration of let-7a-5p mimics on SC differentiation while let-7a-5p mimics blocked MEG3-mediated suppression on SC differentiation. Let-7a-5p sponged by MEG3 promotes differentiation of ADSCs into SCs via suppressing Notch signaling by targeting RBPJ. These findings shed light on mechanisms underlying the differentiation of ADSCs to SCs and provide avenues to accelerate the process.

Experimental approach to nasal septal cartilage regeneration with adipose tissue-derived stem cells and decellularized porcine septal cartilage.

Cartilage shortage is a major problem in facial reconstructive surgery. Prior studies have shown that decellularized porcine nasal septal cartilage (DPNC) seeded with primary human nasal chondrocytes enabled cartilage regeneration and showed potential as a replacement material for nasal cartilage. Since adipose tissue-derived stem cells (ASCs) are easily accessible and almost abundantly available, they appear to be a promising alternative to limited chondrocytes making the combination of DPNC and ASCs a feasible approach towards clinical translation. Thus, this study was intended to investigate the interactions between ASCs and DPNC in an in vitro model. DPNCs were seeded and 3D-cultured with primary human ASCs that were priorly characterized with trilineage differentiation and flow cytometry. Cell vitality and proliferation were evaluated by Live-Dead, alamarBlue, and PicoGreen assays. Chondrogenic differentiation was examined by DMMB assay and cryosectioning-based histology. Cell invasion within DPNC was visualized and quantified by fluorescent histology (DAPI, Phalloidin). ASCs showed good adherence to DPNC and Live-Dead assay proved their viability over 2weeks. AlamarBlueassay showed an increase in metabolic activity compared to 2D cultures, and PicoGreen assay demonstrated an increase of cell number within DPNC over time. Biochemical assays and histology added evidence of chondrogenic differentiation of 3D-cultured ASCs under the influence of chondrogenic induction medium. Fluorescent image analysis showed a significant increase of cell-occupied areas of scaffolds over time (P<.05). DPNC scaffolds provided a suitable environment for ASCs that allowed good cell vitality, high proliferation, and chondrogenic differentiation. Thus, the use of ASCs and DPNC yields a promising alternative to the use of primary human chondrocytes. For facial cartilage tissue engineering, we regard ASCs as an attractive alternative to human nasal chondrocytes due to their better accessibility and availability. Further research will be necessary to determine long-term effects and in vivo outcomes of ASCs and DPNC in cartilage regeneration of the face.

D-Mannose Inhibits Adipogenic Differentiation of Adipose Tissue-Derived Stem Cells via the miR669b/MAPK Pathway.

The adipogenic differentiation of adipose tissue-derived stem cells (ADSCs) plays an important role in the process of obesity and host metabolism. D-Mannose shows a potential regulating function for fat tissue expansion and glucose metabolism. To explore the mechanisms through which D-mannose affects the adipogenic differentiation of adipose-derived stem cells in vitro, we cultured the ADSCs with adipogenic medium inducement containing D-mannose or glucose as the control. The adipogenic differentiation specific markers Pparg and Fabp4 were determined by real-time PCR. The Oil Red O staining was applied to measure the lipid accumulation. To further explore the mechanisms, microarray analysis was performed to detect the differences between glucose-treated ADSCs (G-ADSCs) and D-mannose-treated ADSCs (M-ADSCs) in the gene expression level. The microarray data were further analyzed by a Venn diagram and Gene Set Enrichment Analysis (GSEA). MicroRNA inhibitor transfection was used to confirm the role of key microRNA. Results. D-Mannose intervention significantly inhibited the adipogenic differentiation of ADSCs, compared with the glucose intervention. Microarray showed that D-mannose increased the expression of miR669b, which was an inhibitor of adipogenesis. In addition, GSEA and western blot suggested that D-mannose suppressed the adipogenic differentiation via inhibiting the MAPK pathway and further inhibited the expression of proteins related to glucose metabolism and tumorigenesis. Conclusion. D-Mannose inhibits adipogenic differentiation of ADSCs via the miR669b/MAPK signaling pathway and may be further involved in the regulation of glucose metabolism and the inhibition of tumorigenesis.

DNA methyltransferase inhibitor 5-azacytidine in high dose promotes ultrastructural maturation of cardiomyocyte.

The adult human heart muscle cells, cardiomyocytes are not capable of regenerate after injury. Stem cells are a powerful means for future regenerative medicine because of their capacity for self-renewal and multipotency. Several studies have reported the cardiogenic potential in human adipose tissue-derived stem cells (ADSCs) differentiation, but there is still no efficient protocol for the induction of cardiac differentiation by 5-azacytidine (5-Aza). The present study involves characterization and mainly, the ultrastructure of ADSCs derived cardiomyocyte-like cells. The cultured ADSCs were treated with 50 µM 5-Aza for 24 hours, followed by a 10-week extension. At different time points, cardiomyocyte-like cells were assessed by qRT-PCR and were evaluated by transmission electron microscopy at 10th week. The expression of cardiac-specific markers entailing cardiac troponin I (cTnI), connexin 43, myosin light chain-2v (Mlc-2v), increased over 10 weeks and the highest expression was at 10th week. The expression of the β-myosin heavy chain (β-MHC) increased significantly over 5 weeks and then decreased. At the ultrastructural level myofibrils, transverse tubules (T-tubules), sarcoplasmic reticular membrane, and intercalated discs were present. These data suggest that treatment with 5-Aza in high dose could promote differentiation of ADSCs into cardiomyocyte-like cells. These differentiated cells could be used for regeneration of damaged cardiomyocytes with the 3D scaffold for delivery of the cells.

3D Spheroids Derived from Human Lipedema ASCs Demonstrated Similar Adipogenic Differentiation Potential and ECM Remodeling to Non-Lipedema ASCs In Vitro.

The growth and differentiation of adipose tissue-derived stem cells (ASCs) is stimulated and regulated by the adipose tissue (AT) microenvironment. In lipedema, both inflammation and hypoxia influence the expansion and differentiation of ASCs, resulting in hypertrophic adipocytes and deposition of collagen, a primary component of the extracellular matrix (ECM). The goal of this study was to characterize the adipogenic differentiation potential and assess the levels of expression of ECM-remodeling markers in 3D spheroids derived from ASCs isolated from both lipedema and healthy individuals. The data showed an increase in the expression of the adipogenic genes (ADIPOQ, LPL, PPAR-γ and Glut4), a decrease in matrix metalloproteinases (MMP2, 9 and 11), with no significant changes in the expression of ECM markers (collagen and fibronectin), or integrin A5 in 3D differentiated lipedema spheroids as compared to healthy spheroids. In addition, no statistically significant changes in the levels of expression of inflammatory genes were detected in any of the samples. However, immunofluorescence staining showed a decrease in fibronectin and increase in laminin and Collagen VI expression in the 3D differentiated spheroids in both groups. The use of 3D ASC spheroids provide a functional model to study the cellular and molecular characteristics of lipedema AT.

Dysmetabolic adipose tissue in obesity: morphological and functional characteristics of adipose stem cells and mature adipocytes in healthy and unhealthy obese subjects.

The way by which subcutaneous adipose tissue (SAT) expands and undergoes remodeling by storing excess lipids through expansion of adipocytes (hypertrophy) or recruitment of new precursor cells (hyperplasia) impacts the risk of developing cardiometabolic and respiratory diseases. In unhealthy obese subjects, insulin resistance, type 2 diabetes, hypertension, and obstructive sleep apnoea are typically associated with pathologic SAT remodeling characterized by adipocyte hypertrophy, as well aschronic inflammation, hypoxia, increased visceral adipose tissue (VAT), and fatty liver. In contrast, metabolically healthy obese individuals are generally associated with SAT development characterized by the presence of smaller and numerous mature adipocytes, and a lower degree of VATinflammation and ectopic fat accumulation. The remodeling of SAT and VAT is under genetic regulation and influenced by inherent depot-specific differences of adipose tissue-derived stem cells (ASCs). ASCs have multiple functions such as cell renewal, adipogenic capacity, and angiogenic properties, and secrete a variety of bioactive molecules involved in vascular and extracellular matrix remodeling. Understanding the mechanisms regulating the proliferative and adipogenic capacity of ASCs from SAT and VAT in response to excess calorie intake has become a focus of interest over recent decades. Here, we summarize current knowledge about the biological mechanisms able to foster or impair the recruitment and adipogenic differentiation of ASCs during SAT and VAT development, which regulate body fat distribution and favorableor unfavorable metabolic responses.

Valproic acid up-regulates the whole NO-citrulline cycle for potent iNOS-NO signaling to promote neuronal differentiation of adipose tissue-derived stem cells

Valproic acid (VPA) remarkably promotes the differentiation of adipose tissue-derived stem cells (ASCs) to mature neuronal cells through nitric oxide (NO) signaling due to up-regulated inducible NO synthase (iNOS) as early as within 3 days. Here, we investigated mechanisms of VPA-promoted neuronal differentiation of ASCs concerning the NO-citrulline cycle, the metabolic cycle producing NO. Cultured rat ASCs were differentiated to mature neuronal cells rich in dendrites and expressing a neuronal marker by treatments with VPA at 2 mM for 3 days and subsequently with the neuronal induction medium for 2 h. Inhibitor (α-methyl-d, l-aspartic acid, MDLA) of arginosuccinate synthase (ASS), a key enzyme of the NO-citrulline cycle, abolishes intracellular NO increase and VPA-promoted neuronal differentiation in ASCs. l-Arginine, the substrate of iNOS, restores the promotion effect of VPA, being against MDLA. Immunocytochemistry showed that ASS and iNOS were increased in ASCs expressing neurofilament medium polypeptide (NeFM), a neuronal marker, by VPA and NIM synergistically. Real-time RT-PCR analysis showed that mRNAs of Ass and arginosuccinate lyase (Asl) in the NO-citrulline cycle were increased by VPA. Chromatin immunoprecipitation assay indicated that Ass and Asl were up-regulated by VPA through the acetylation of their associated histone. From these results, it was considered that VPA up-regulated the whole NO-citrulline cycle, which enabled continuous NO production by iNOS in large amounts for potent iNOS-NO signaling to promote neuronal differentiation of ASCs. This may also indicate a mechanism enabling short-lived NO to function conveniently as a potent signaling molecule that can disappear quickly after its role.

The Effect of Various Surface Treatments of Ti6Al4V on the Growth and Osteogenic Differentiation of Adipose Tissue-Derived Stem Cells

The physical and chemical properties of the material surface, especially its roughness and wettability, have a crucial effect on the adhesion, proliferation, and differentiation of cells. The aim of this study is to select the most appropriate surface modifications of Ti6Al4V implants for pre-colonization of the implants with adipose tissue-derived stem cells (ASCs) in order to improve their osseointegration. We compared the adhesion, growth, and osteogenic differentiation of rat ASCs on Ti6Al4V samples modified by methods commonly used for preparing clinically used titanium-based implants, namely polishing (PL), coating with diamond-like carbon (DLC), brushing (BR), anodizing (AND), and blasting (BL). The material surface roughness, measured by the Ra and Rq parameters, increased in the following order: PL &lt; DLC ˂ BR ˂ AND ˂ BL. The water drop contact angle was in the range of 60–74°, with the exception of the DLC-coated samples, where it was only 38°. The cell number, morphology, mitochondrial activity, relative fluorescence intensity of osteogenic markers RUNX2, type 1 collagen, and osteopontin, the calcium consumption by the cells and the alkaline phosphatase activity depended on the surface roughness rather than on the surface wettability of the materials. Materials with a surface roughness of several tens of nanometers (Ra 60–70 nm), i.e., the BR and AND samples, supported a satisfactory level of cell proliferation. At the same time, they achieved the highest level of osteogenic cell differentiation. These surface modifications therefore seem to be most suitable for pre-colonization of Ti6Al4V implants with stem cells pre-differentiated toward osteoblasts, and then for implanting them into the bone tissue.

Hypoxia Pretreatment Promotes Chondrocyte Differentiation of Human Adipose-Derived Stem Cells via Vascular Endothelial Growth Factor.

Human adipose tissue-derived stem cells (ADSCs) are attractive multipotent stem cell sources with therapeutic potential in various fields requiring repair and regeneration, such as acute and chronically damaged tissues. ADSC is suitable for cell-based therapy, but its use has been hampered due to poor survival after administration. Potential therapeutic use of ADSC requires mass production of cells through in vitro expansion. Many studies have consistently observed the tendency of senescence by mesenchymal stem cell (MSC) proliferation upon expansion. Hypoxia has been reported to improve stem cell proliferation and survival. We investigated the effects of hypoxia pretreatment on ADCS proliferation, migration capacity, differentiation potential and cytokine production. We also analyzed the effects of vascular endothelial growth factor (VEGF) on osteogenic and chondrogenic differentiation of ADSCs by hypoxia pretreatment. Hypoxia pretreatment increased the proliferation of ADSCs by increasing VEGF levels. Interestingly, hypoxia pretreatment significantly increased chondrogenic differentiation but decreased osteogenic differentiation compared to normoxia. The osteogenic differentiation of ADSC was decreased by the addition of VEGF but increased by the depletion of VEGF. We have shown that hypoxia pretreatment increases the chondrogenic differentiation of ADSCs while reducing osteogenic differentiation in a VEGF-dependent manner. These results show that hypoxia pretreatment can provide useful information for studies that require selective inhibition of osteogenic differentiation, such as cartilage regeneration.

Cardiac Differentiation of Adipose Tissue-Derived Stem Cells Is Driven by BMP4 and bFGF but Counteracted by 5-Azacytidine and Valproic Acid.

Objective Bone morphogenetic protein 4 (BMP4) and basic fibroblast growth factor (bFGF) play important roles in embryonic heart development. Also, two epigenetic modifying molecules, 5ˊ-azacytidine (5ˊ-Aza) and valproic acid (VPA) induce cardiomyogenesis in the infarcted heart. In this study, we first evaluated the role of BMP4 and bFGF in cardiac trans-differentiation and then the effectiveness of 5´-Aza and VPA in reprogramming and cardiac differentiation of human adipose tissue-derived stem cells (ADSCs). Materials and Methods In this experimental study, human ADSCs were isolated by collagenase I digestion. For cardiac differentiation, third to fifth-passaged ADSCs were treated with BMP4 alone or a combination of BMP4 and bFGF with or without 5ˊ-Aza and VPA pre-treatment. After 21 days, the expression of cardiac-specific markers was evaluated by reverse transcription polymerase chain reaction (RT-PCR), quantitative real-time PCR, immunocytochemistry, flow cytometry and western blot analyses. Results BMP4 and more prominently a combination of BMP4 and bFGF induced cardiac differentiation of human ADSCs. Epigenetic modification of the ADSCs by 5ˊ-Aza and VPA significantly upregulated the expression of OCT4A, SOX2, NANOG, Brachyury/T and GATA4 but downregulated GSC and NES mRNAs. Furthermore, pre-treatment with 5ˊ-Aza and VPA upregulated the expression of TBX5, ANF, CX43 and CXCR4 mRNAs in three-week differentiated ADSCs but downregulated the expression of some cardiac-specific genes and decreased the population of cardiac troponin I-expressing cells. ConclusionOur findings demonstrated the inductive role of BMP4 and especially BMP4 and bFGF combination in cardiac trans-differentiation of human ADSCs. Treatment with 5ˊ-Aza and VPA reprogrammed ADSCs toward a more pluripotent state and increased tendency of the ADSCs for mesodermal differentiation. Although pre-treatment with 5ˊ-Aza and VPA counteracted the cardiogenic effects of BMP4 and bFGF, it may be in favor of migration, engraftment and survival of the ADSCs after transplantation.