Differentiation Of Adipose-derived Mesenchymal Cells Research Articles

Magnetic nanowires substrate increases adipose-derived mesenchymal cells osteogenesis

Magnetic nanomaterials are increasingly impacting the field of biology and medicine. Their versatility in terms of shape, structure, composition, coating, and magnetic responsivity make them attractive for drug delivery, cell targeting and imaging. Adipose derived-mesenchymal cells (ASCs) are intensely scrutinized for tissue engineering and regenerative medicine. However, differentiation into musculoskeletal lineages can be challenging. In this paper, we show that uncoated nickel nanowires (Ni NW) partially released from their alumina membrane offer a mechanically-responsive substrate with regular topography that can be used for the delivery of magneto-mechanical stimulation. We have used a tailored protocol for improving ASCs adherence to the substrate, and showed that cells retain their characteristic fibroblastic appearance, cytoskeletal fiber distribution and good viability. We report here for the first time significant increase in osteogenic but not adipogenic differentiation of ASCs on Ni NW exposed to 4 mT magnetic field compared to non-exposed. Moreover, magnetic actuation is shown to induce ASCs osteogenesis but not adipogenesis in the absence of external biochemical cues. While these findings need to be verified in vivo, the use of Ni NW substrate for inducing osteogenesis in the absence of specific differentiation factors is attractive for bone engineering. Implant coating with similar surfaces for orthopedic and dentistry could be as well envisaged as a modality to improve osteointegration.

Dynamic of miRNA-101a-3p and miRNA-200a during Induction of Osteoblast Differentiation in Adipose-derived Mesenchymal Stem Cells.

miRNAs are known as the cellular phenomena regulators that exert their effects in post-transcriptional level. Recent studies highlight the role of miRNAs in mesenchymal stem cells differentiation into osteoblasts. The purpose of this study was to recognize the pattern of miRNA-101a-3p and miRNA-200a expression during osteoblastic differentiation of human adipose tissue-derived mesenchymal stem cells. The cells were incubated in osteoblastic differentiation medium for a period of 21 days. Alizarin red S staining was performed to confirm the successful differentiation of adipose-derived mesenchymal stem cells into osteoblast cells. The expression levels of miRNA-101a-3p and miRNA-200a were analyzed by real-time PCR during 0, 7, 14, and 21 days after differentiation induction. Data exhibited the increase of extracellular red color deposition which was evident at the end of the incubation period. The expression of miRNA-101a-3p and miRNA-200a was up regulated during adipose-derived mesenchymal stem cells trans-differentiation into osteoblast-like cells. These miRNAs could be potential novel biomarkers for monitoring successful differentiation of mesenchymal stem cells toward osteoblasts.

The feasibility of human adipose-derived mesenchymal stem cells induced to endothelial-like cells and the safety of induced cells intraocular application in a short period

Objective To evaluate the feasibility of differentiation of human adipose mesenchymal stem cells (hADSCs) into endothelial-like cells and the safety of induced cells intraocular application. Methods HADSCs were induced to endothelial-like cells in vitro.The experimental group was added with streptomycin, vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF), and the control group was added with the same amount of phosphate buffered saline (PBS), the expression of von Willebrand factor (vWF), a marker of endothelial cells, was observed in the two groups.Six C57BL/6J mice were randomly divided into experimental group and control group by using the random number method, 3 mice for each group, the induced cells were injected intravitreally into C57BL/6J mice as experimental group, and the same amount of PBS buffer was injected intravitreally into C57BL/6J mice as control group.The changes of retinal cells were observed by electron microscopy.The use and care of the animals complied with Regulations for the Administration of Affair Concerning Experimental Animals by State Science and Technology Commission. Results After induction of hADSCs in vitro, the endothelial cell marker was expressed.VWF immunofluorescence of the experimental group showed strong green fluorescence, and the color rendering rate was 100%.The control group showed no coloration of vWF immunofluorescence, and the color positive rate was 0.After 1 month of intravitreal injection, the retinal ganglion cells and rod cells were not degraded and necrotic. Conclusions HADSCs can differentiate into endothelial-like cells in vitro, and there is no retinal toxicity in a short-term. Key words: Adipose derived mesenchymal stem cells; Endothelium-like cells; Induced differentiation; Toxicity

CNT-based nanocarrier loaded with pyrimethamine for adipose mesenchymal stem cells differentiation and cancer treatment: The computational and experimental methods

Pyrimethamine is an effective drug in the cancer cell treatment and is a dihydrofolate reductase inhibitor. In this work, the amount of drug loading up on CNT and its cytotoxicity effect upon MCF-7 cell lines was surveyed. The novel applications of some drugs and nanocarriers can induce the differentiation of adipose mesenchymal cells into nerve cells. Hence carbon nanotube-pyrimethamine was used to differentiate mesenchymal stem cells into the neural category, for the first time. The results of NSE and NFM gene expression level were evaluated using the real-time PCR. A detailed study on the interaction between pyrimethamine anticancer drug and (6, 0) zigzag single-walled carbon nanotube was performed by DFT/B3LYP and DFT/M06-2X with 6-31G* basis set calculations in gas phase and in solvent using the PCM. Different configurations of the adsorbed pyrimethamine onto the CNT surface were studied. Based on the results, the process of pyrimethamine adsorption on diff ;erent sites on the outer wall of the nanotube was exothermic and configurations were stable. The adsorption energy values indicated that the pyrimethamine molecule could be physically adsorbed on the external surface of the SWCNT. The QTAIM was used for characterizing the nature of the interactions between the pyrimethamine and the selected nanocarrier.

Small Molecule-Based Strategy Promotes Nucleus Pulposus Specific Differentiation of Adipose-Derived Mesenchymal Stem Cells.

Adipose tissue-derived mesenchymal stem cells (ADSCs) are promising for regenerating degenerated intervertebral discs (IVDs), but the low efficiency of nucleus pulposus (NP)-specific differentiation limits their clinical applications. The Sonic hedgehog (Shh) signaling pathway is important in NP-specific differentiation of ADSCs, and Smoothened Agonist (SAG) is a highly specific and effective agonist of Shh signaling. In this study, we proposed a new differentiation strategy with the use of the small molecule SAG. The NP-specific differentiation and extracellular matrix (ECM) synthesis of ADSCs were measured in vitro, and the regenerative effects of SAG pretreated ADSCs in degenerated IVDs were verified in vivo. The results showed that the combination of SAG and transforming growth factor-β3 (TGF-β3) is able to increase the ECM synthesis of ADSCs. In addition, the gene and protein expression levels of NP-specific markers were increased by treatment with SAG and TGF-β3. Furthermore, SAG pretreated ADSCs can also improve the disc height, water content, ECM content, and structure of degenerated IVDs in vivo. Our new differentiation scheme has high efficiency in inducing NP-specific differentiation of ADSCs and is promising for stem cell-based treatment of degenerated IVDs.

The transcription factor E2F1 modulates the proliferation and differentiation of adipose mesenchymal stem cells and its underlyingmechanism

Objective To investigate the effect of transcription factor E2F1 on the proliferation and differentiation of adipose mesenchymal stem cells (ADSCs) and its underlying mechanism. Methods The primary culture cells of ADSCs were isolated from WT or E2F1-/-mice (6-8 weeks) adipose tissues. After 3rd generation culture of ADSCs (P4), the cell morphologies were observed and the cell surface markers were identified by flow cytometry. The proliferation and differentiation were compared between the two groups by cell counting kit-8 (CCK-8) and Oil-Red O staining assay. The expression levels of peroxisome proliferator activated receptor γ (PPAR-γ), which is the key regulator for adipogenesis, were detected by Western blotting. Results Firstly, the obtained ADSCs could stably grew and passaged, and the cell morphology was not significantly different in the two groups. The cell surface markers CD29, CD44, CD90 expression were positive while CD31 expression was negative. The CCK-8 result showed that the cell proliferation ability of WT ADSCs was stronger than E2F1-/-ADSCs (1.85±0.03 vs. 1.32±0.20, t=16.641, P<0.05). The Oil-Red O staining assay showed that lipid droplets in WT ADSCs group were more than those of E2F1-/-ADSCs group (1 890±128 vs. 1 109±116, t=18.505, P<0.05). The PPAR-γ protein expression level was higher in WT ADSCs group than that of E2F1-/-ADSCs group (0.81±0.02 vs. 0.44±0.01, t=3.040, P<0.05). Conclusion Transcription factor E2F1 can enhance the proliferation and lipid differentiation of ADSCs by promoting the expression of PPAR-γ. Key words: Adipose-derived mesenchymal stem cells; E2F1; Cell proliferation; Adipogenic differentiation; Peroxisome proliferator activated receptor γ

MafA Overexpression: A New Efficient Protocol for In Vitro Differentiation of Adipose-Derived Mesenchymal Stem Cells into Functional Insulin-Producing Cells.

Objective We proposed a novel differentiation method for the efficient differentiation of adipose-derived mesenchymal stem cells (ADMSCs) into functional insulin-producing cells (IPCs) based on MafA overexpression. Materials and Methods In this experimental study, a eukaryotic expression vector containing MafA [MafA/pcDNA3.1(+)] was constructed and purified. ADMSCs were differentiated into IPCs. ADMSCs were assigned in two groups including control (C), and the MafA overexpressed (MafA+) groups. The ADMSCs were transfected by MafA/pcDNA 3.1(+) at day 10 of the differentiation. Differentiated cells were analyzed for the expression of multiple β cell specific genes (Nkx2.2, Ngn3, Isl-1, Pdx1, MafA, Nkx6.1, and Insulin) using real-time polymerase chain reaction (PCR). The insulin secretion potency of the differentiated cells in response to glucose exposure was also determined using an enzyme-linked immunosorbent assay (ELISA) method and Dithizone (DTZ) staining. The IPCs from the control manipulated group, and un-differentiated ADMSCs group were transplanted to streptozotocin (STZ)-diabetic rats. Rats were monitored for blood glucose and insulin concentration. Results The results revealed that ADMSCs were successfully differentiated into IPCs through the 14 day differentiation protocol. The expression of β-cell specific genes in MafA+ IPCs was higher than in control cells. Glucose-induced insulin secretion after the exposure of IPCs to glucose was higher in MafA+ group than the control group. The STZ- diabetic rats showed an ability to secrete insulin and apparent hyperglycemic condition adjustment after transplantation of the control IPCs. The mean insulin concentration of diabetic rats that were transplanted by manipulated IPCs was significantly higher than ADMSCs-transplanted rats; however, no effect was observed in the concentration of blood glucose. ConclusionThe overexpression of MafA can be used as a novel promising approach for the efficient production of IPCs from ADMSCs in vitro. However, the future therapeutic use of the MafA+ IPCs in diabetic animals needs further investigations.

In vitro induction of differentiation of human adipose-derived mesenchymal stem cells into melanocytes: an experimental study

Objective To establish a method for inducing human adipose-derived mesenchymal stem cells (hADSCs) to differentiate into melanocytes in vitro.Methods hADSCs were isolated from human subcutaneous fat tissue,and subjected to phenotypic analysis by flow cytometry.The differentiation potential of the hADSCs was evaluated by induction of osteogenic and adipogenic differentiation.Then,the fifth passage hADSCs were induced to differentiate into melanocytes by conditioned medium containing stem cell factor (SCF),endothelin 3,basic fibroblast growth factor (bFGF) for ten weeks.Those hADSCs receiving no induction treatment served as the normal control.Subsequently,real-time fluorescence-based quantitative PCR (RT-PCR) was performed to determine the mRNA expressions of melanin-associated genes including microophthalmia-associated transcription factor (MITF),ckit receptor tyrosine kinase (KIT),dopachrome tautomerase (DCT),tyrosinase,tyrosinase-related protein-1 (TYRP1),and sex determining region Y-box 10 (SOX10).Statistical analysis was done by one-factor analysis of variance and the least significant difference (LSD) test.Further more,the hADSC-derived melanocytes were identified by immunocytochemistry and immunofluorescence assay.Results As flow cytometry revealed,the isolated hADSCs showed high expressions of CD29,CD44,CD73,CD90,CD105 and CD166 with the positivity rates being more than 95％,but low or absent expressions of CD31,CD34,CD45 and human leukocyte antigen (HLA)-DR.Additionally,the osteogenic and adipogenic differentiation of hADSCs was successfully induced.The relative mRNA expression levels of MITF,KIT,DCT,tyrosinase,TYRP1 and SOX10 were 0.325 ± 0.012,0.042 ± 0.006,0.046 ± 0.013,0.036 ± 0.005,0.041 ± 0.003,and 0.225 ± 0.014 respectively in hADSCs induced for 10 weeks,which were significantly upregulated compared with the normal control hADSCs (all P ＜ 0.05).Moreover,the induced hADSCs showed positive immunocytochemical staining for MITF and HMB45,as well as positive cytoimmunofluorescence staining for TYRP1 and S100.Conclusion The hADSCs have been successfully induced to differentiate into cells with the biological characteristics of melanocytes. Key words: Human adipose-derived mesenchymal stem cells; Cell differentiation; Melanocytes

Effects of adenovirus-mediated co-transfection of bone morphogenetic protein-2 gene and bone morphogenetic protein-7 gene on osteogenic differentiation of rat adipose-derived mesenchymal stromal cells

Objective To investigate the effects of adenovirus-mediated co-transfection of bone morphogenetic protein-2 (BMP-2) gene and bone morphogenetic protein-7 (BMP-7) gene on osteogenic differentiation of adipose-derived mesenchymal stromal cells (ADSCs).Methods The expression adenovirus vectors with green fluorescent protein gene carrying BMP-2 and adenovirus vectors with red fluorescent protein gene carrying BMP-7 were packaged and produced respectively.Rat adipose-derived stromal cells were isolated and cultured in vitro,and were transfected with Ad-BMP-2 (Ad-BMp-2 group),Ad-BMP-7 (Ad-BMP-7 group),co-transfected with Ad-BMP-2 and Ad-BMP-7 (Ad-BMP-2 ± Ad-BMP-7 group),or transfected with no virus (control group).The expression of osteocalcin (OCN) and collagen Ⅰ protein at 14th day after transfection was detected by using Western blotting in each group.Alkaline phosphatase (ALP) activity in ADSCs was measured.Results The expression of OCN and collagen Ⅰ protein in cotransfected group was significantly higher than that in the other groups (P ＜ 0.05).The ALP activity in cotransfected group was significantly stronger than that in the other groups (P ＜ 0.01).Conclusion Adenovirus-mediated co-transfection of BMP-2 gene and BMP-7 gene may promote osteogenic differentiation of adipose-derived ADSCs. Key words: Bone morphogenetic protein; Adenovirus; Adipose-derived stromal cells; Osteogenic differentiation

Differentiation of adipose-derived mesenchymal stem cells to renal tubular epithelial cells

Objective To detect whether adipose-derived mesenchymal stem cells (ADMCSs)can differentiate into renal tubular epithelial cells and be used for treatment of acute kidney injury (AKI).Methods ADMSCs were separated and cultured in vitro,and were transfected with lentivirus carrying reporter genes of firefly luciferase and monomeric red fluorescent protein (fluc-mrfp).Transfected ADMCSs (2 × l06) were directly injected into renal parenchyma after establishment of AKI models induced by ischemic reperfusion injury.Distribution and differentiation of ADMSCs in rats were detected by bioluminescence imaging (BLI) and immunofluorescent staining,respectively.Results ADMSCs had positive expressions of CD29 and CD90,rather than expressions of CD34,CD45 and CD31.Lentivirus-transduced ADMSCs had stable expressions of mrfp and fluc reporter genes.BLI showed persistent fluorescence signal in vivo even at day 14 after ADMSCs transplantation.Immunofluorescence staining further indicated that the transplanted ADMSCs could differentiate into tubular epithelial cells.Conclusion ADMSCs in vivo can differentiate into renal tubular epithelial cells,which provides an experimental basis for more researches on ADMSCs transplantation for treatment of AKI. Key words: Stem cells; Kidney tubules; Epithelial cells

Adipose-Derived Stem Cells: Characterization and Current Application in Orthopaedic Tissue Repair

Orthopaedic tissues, such as bone, cartilage, intervertebral disc and tendon, contain cells that are difficult to culture and stimulate in vitro for repair of damaged tissue. Stem cells have the ability to self-renew and differentiate into many tissue types. Recent progress in stem cell research has led to an enthusiastic effort to utilize stem cells for orthopaedic tissue regeneration. Due to ease of harvest and abundance, adipose-derived mesenchymal cells (ASC) are an attractive, readily available adult stem cell that has become increasingly popular for use in many stem cell applications. Recent progress has been made in characterizing ASC and looking mechanistically at gene expression and cellular pathways involved in differentiation. This review focuses on (i) the characterization of ASC through expression of appropriate surface markers; (ii) modulation of in vitro differentiation of ASC through different scaffolds, growth factors, and media; and (iii) the use of ASC in orthopaedic tissue repair. Strategies for repair involve the use of differentiated or undifferentiated, fresh or passaged ASC, in conjunction with appropriate choice of media, growth factors and scaffolds. Recent in vivo studies utilizing ASC are discussed giving results on defect repair and potential for clinical orthopaedic tissue regeneration.

Adipose-Derived Stem Cells: Characterization and Current Application in Orthopaedic Tissue Repair

Orthopaedic tissues, such as bone, cartilage, intervertebral disc and tendon, contain cells that are difficult to culture and stimulate in vitro for repair of damaged tissue. Stem cells have the ability to self-renew and differentiate into many tissue types. Recent progress in stem cell research has led to an enthusiastic effort to utilize stem cells for orthopaedic tissue regeneration. Due to ease of harvest and abundance, adipose-derived mesenchymal cells (ASC) are an attractive, readily available adult stem cell that has become increasingly popular for use in many stem cell applications. Recent progress has been made in characterizing ASC and looking mechanistically at gene expression and cellular pathways involved in differentiation. This review focuses on (i) the characterization of ASC through expression of appropriate surface markers; (ii) modulation of in vitro differentiation of ASC through different scaffolds, growth factors, and media; and (iii) the use of ASC in orthopaedic tissue repair. Strategies for repair involve the use of differentiated or undifferentiated, fresh or passaged ASC, in conjunction with appropriate choice of media, growth factors and scaffolds. Recent in vivo studies utilizing ASC are discussed giving results on defect repair and potential for clinical orthopaedic tissue regeneration.

Adhesion, proliferation and differentiation of human adipose mesenchymal stem cells on PLGA

Objective To study the ability of adhesion, proliferation and differentiation of human adipose mesenchymal stern cells,which were cultured in PLGA,and thus to provide a basis for their in vivo implantation. Methods PLA was mixed with PGA in a ratio of 1 ： 1, and PLGA was obtained by insert moldling with organic solvents. In vitro, human adipose mesenchymal stem cells were adhered to PLGA. The attachment and the proliferation of human adipose mesenchymal stem cells in PLGA were analysed by scanning electron microscope, and the morphological change of the cells was investigated before and after induction. Results Human adipose mesenchymal stem cells were able to attach, proliferate and secrete extracellular matrix in porous PLGA （pore size： 100 -400 pm） with porosity of 85 % , and to differentiate into round lipoblast-like cells under the induction of adipose medium. After 14 days, the particulate of PLGA was overlaid by extracellular matrix. Conclusion Human adipose mesenchymal stem cells have the capacity to adhere to PLGA and proliferate, indicating that PLGA can be used as a carrier for human adipose mesenchymal stem cells in adipose tissue engineering. Key words: Human adipose mesenchymal stem cells; Scaffold material; Scanning electron microscope

Effect of reduced oxygen tension on chondrogenesis and osteogenesis in adipose-derived mesenchymal cells

Recent studies have demonstrated that adipose-derived mesenchymal cells (AMCs) offer great promise for cell-based therapies because of their ability to differentiate toward bone, cartilage, and fat. Given that cartilage is an avascular tissue and that mesenchymal cells experience hypoxia during prechondrogenic condensation in endochondral ossification, the goal of this study was to understand the influence of oxygen tension on AMC differentiation into bone and cartilage. In vitro chondrogenesis was induced using a three-dimensional micromass culture model supplemented with TGF-beta1. Collagen II production and extracellular matrix proteoglycans were assessed with immunohistochemistry and Alcian blue staining, respectively. Strikingly, micromasses differentiated in reduced oxygen tension (2% O(2)) showed markedly decreased chondrogenesis. Osteogenesis was induced using osteogenic medium supplemented with retinoic acid or vitamin D and was assessed with alkaline phosphatase activity and mineralization. AMCs differentiated in both 21 and 2% O(2) environments. However, osteogenesis was severely diminished in a low-oxygen environment. These data demonstrated that hypoxia strongly inhibits in vitro chondrogenesis and osteogenesis in AMCs.

Mouse adipose-derived mesenchymal stromal cells undergo osteogenic differentiation in vitro and in vivo

Abstract Introduction: Although the multilineage potential of human processed lipoaspirate has previously been demonstrated, few studies exist utilizing mouse adipose-derived mesenchymal cells (ADMSCs) in vitro or in vivo. Here, we develop a method for osteogenic differentiation of ADMSCs in vitro and utilize them to repair calvarial defects in vivo. Methods: ADMSCs were harvested from 3 week old female FVB mice. ADMSCs were cultured in standard osteogenic media with various concentrations of BMP-2 or retinoic acid (RA) for 3 weeks. Growth and differentiation were assessed via cell counting, Oil Red O (ORO), alkaline phosphatase (AP), and von Kossa (VK) staining. Apatite-coated poly-lactic co-glycolic acid (PLGA) scaffolds were then seeded with ADMSCs and implanted into critical sized (4mm) calvarial defects in adult male mice. Calvarial regeneration was assessed using histology and radiography. Results: Only slight differences in proliferation were seen in response to BMP2, RA, or BMP2 + RA. ORO staining was dramatically decreased in treated cells. AP and von Kossa staining were induced in a synergistic fashion by BMP2 + RA. In vivo experiments revealed successful healing of calvarial defects by 12 weeks after surgery. ADMSC-seeded scaffolds produced significant intramembranous bone formation by 2 weeks, and areas of complete bony bridging by 12 weeks as demonstrated by X-ray and histology. Conclusions: Our data demonstrate successful osteogenic differentiation of ADMSCs in vitro and in vivo. This novel mouse model potentiates further investigation into the mechanisms governing osteogenesis and the epigenetic manipulations that can be used to enhance this process.