BACKGROUNDPrevious studies have shown that resveratrol increases endothelial progenitor cell (EPC) numbers and functional activity. Increased EPC numbers and activity are associated with the inhibition of EPC senescence. In this study, we investigated the effect of resveratrol on the senescence of EPCs, leading to potentiation of cellular function.

METHODSEPCs were isolated from human peripheral blood and identified immunocytochemically. EPCs were incubated with resveratrol (1, 10, and 50 µmol/L) or control for specified times. After in vitro cultivation, acidic β-galactosidase staining revealed the extent of senescence in the cells. To gain further insight into the underlying mechanism of the effect of resveratrol, we measured telomerase activity using a polymerase chain reaction (PCR)-enzyme-linked immunosorbent assay (ELISA) technique. Furthermore, we measured the expression of human telomerase reverse transcriptase (hTERT) and the phosphorylation of Akt by immunoblotting.

RESULTSResveratrol dose-dependently inhibited the onset of EPC senescence in culture. Resveratrol also significantly increased telomerase activity. Interestingly, quantitative real-time PCR analysis demonstrated that resveratrol dose-dependently increased the expression of the catalytic subunit, hTERT, an effect that was significantly inhibited by pharmacological phosphatidylinositol 3-kinase (PI3-K) blockers (wortmannin). The expression of hTERT is regulated by the PI3-K/Akt pathway; therefore, we examined the effect of resveratrol on Akt activity in EPCs. Immunoblotting analysis revealed that resveratrol led to dose-dependent phosphorylation and activation of Akt in EPCs.

CONCLUSIONResveratrol delayed EPCs senescence in vitro, which may be dependent on telomerase activation.

Cells, Cultured ; Cellular Senescence ; drug effects ; Endothelial Cells ; cytology ; drug effects ; enzymology ; Humans ; Stem Cells ; cytology ; drug effects ; enzymology ; Stilbenes ; toxicity ; Telomerase ; metabolism

OBJECTIVETo study the effect of insulin in different concentrations on secretion function of growth factors of adipose-derived stem cells (ADSCs).

METHODSADSCs were isolated from human abdominal adipose tissue and cultured. The immunophenotype and adipose induced-differentiation were identified, and the third generation cells were collected. The collected cells were assigned to 1 x 10(-8), 1 x 10(-7), 1 x 10(-6) mol/L insulin groups according to the concentration of added insulin. When cells grew into 70% confluence in conventional medium, ADSCs were cultured further in serum-free DMEM containing insulin in different concentrations for 3 days. ADSCs cultured in medium without insulin were used as control group. Secretion amount of vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) of ADSCs were determined by enzyme-linked immunosorbent assay. The effects of the supernatant fluid of ADSCs' nutrient solution on the proliferation and collagen synthesis of the cultured fibroblast were detected by MTT chromatometry and hydroxyproline chromatometry.

RESULTSThe secretion amounts of VEGF and HGF of ADSCs in 1 x 10(-8) and 1 x 10(-7) mol/L insulin groups [(471 +/- 41, 762 +/- 66 ng/L), (643 +/- 64, 930 +/- 67 ng/L), respectively] were significantly higher as compared with those in control group (286 +/- 47, 577 +/- 84 ng/L) (P < 0.05 or P < 0.01). No change occurred in the secretion amount of VEGF and HGF of ADSCs in 1 x l0(-6) mol/L insulin group (P > 0.05). The supernatant fluid of ADSCs' nutrient medium of 1 x 10(-8), 1 x 10(-7) mol/L insulin groups showed obvious stimulative effect on the proliferation and collagen synthesis of fibroblasts, and it was most obvious in the 1 x 10(-7) mol/L group (P < 0.05 or P < 0.01).

CONCLUSIONSInsulin in the concentrations of 1 x 10(-8) and 1 x 10(-7) mol/L can notably promote ADSCs' function of secreting VEGF and HGF.

Adipocytes ; cytology ; drug effects ; secretion ; Cells, Cultured ; Fibroblasts ; cytology ; Hepatocyte Growth Factor ; metabolism ; Humans ; Insulin ; pharmacology ; Stem Cells ; cytology ; drug effects ; secretion ; Vascular Endothelial Growth Factor A ; metabolism

BACKGROUNDHepatocyte transplantation has been proposed as an alternative to whole-organ transplantation to support many forms of hepatic insufficiency. Unfortunately, the lack of donor livers makes it difficult to obtain enough viable human hepatocytes for hepatocyte-based therapies. Therefore, it is urgent to find new ways to provide ample hepatocytes. Induced pluripotent stem (iPS) cells, a breakthrough in stem cell research, may terminate these hinders for cell transplantation. For the promise of iPS cells to be realized in liver diseases, it is necessary to determine if and how efficient they can be differentiated into functional hepatocytes.

METHODSIn this study, we directly compared the hepatic-differentiation capacity of mouse iPS cells and embryonic stem (ES) cells with three different induction approaches: conditions via embryonic body (EB) formation plus cytokines, conditions by combination of dimethyl sulfoxide and sodium butyrate and chemically defined, serum free monolayer conditions. Among these three induction conditions, more homogenous populations can be promoted under chemically defined, serum free conditions. The cells generated under these conditions exhibited hepatic functions in vitro, including glycogen storage, indocynine green (ICG) uptake and release as well as urea secretion. Although efficient hepatocytes differentiation from mouse iPS cells were observed, mouse iPS cells showed relatively lower hepatic induction efficiency compared with mouse ES cells.

RESULTSMouse iPS cells would be efficiently differentiated into functional hepatocytes in vitro, which may be helpful in facilitating the development of hepatocytes for transplantation and for research on drug discovery.

CONCLUSIONWe demonstrate that mouse iPS cells retain full potential for fetal liver development and describe procedures that facilitates the efficient generation of highly differentiated human hepatocyte-like cells from iPS cells in vitro.

Animals ; Butyrates ; pharmacology ; Cell Differentiation ; drug effects ; Cells, Cultured ; Cytokines ; pharmacology ; Embryonic Stem Cells ; cytology ; drug effects ; Hepatocytes ; cytology ; drug effects ; metabolism ; Induced Pluripotent Stem Cells ; cytology ; drug effects ; Mice ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo investigate the effects of Salvianolic acid B preconditioned endothelial progenitor cells (EPCs) on the Nkx2.5 and GATA-4 gene expressions at the early stage of cell differentiation of bone mesenchymal stem cells (BMSc) transplanted into infarcted myocardium, in order to find out the best synergism for co-transplantation of the two kinds of cells.

METHODSBMSc and EPCs of rats were isolated and cultured, and rats were modeled into acute myocardial infarction (AMI) by left coronary artery ligation. Then the EPCs preconditioned with different concentrations of Salvianolic acid B and BMSc or DMEM medium were implanted into heart ischemia area. Expressions of Nkx2.5 and GATA-4 mRNA expressions in myocardium were detected by Real-time RT-PCR 4 weeks later.

RESULTSCompared with those in the non-implanted model rats' myocardium, the gene expression of Nkx2.5 and GATA-4 mRNA were significantly higher in all the transplantation receptive groups, comparisons between the implanted groups showed that the highest value of expressions (2. 654 +/- 0.606 of Nkx2.5 and 1.573 +/- 0.372 of GATA-4) displayed in the group contained more EPCs, for 8-fold to BMSc in volume.

CONCLUSIONBMSc can differentiate into cardiac muscle like cells, and condition of their differentiation is related with the degree of the internal environment improved.

Animals ; Benzofurans ; pharmacology ; Cells, Cultured ; Endothelial Cells ; cytology ; drug effects ; transplantation ; Gene Expression ; drug effects ; Male ; Mesenchymal Stem Cell Transplantation ; Myocardial Infarction ; metabolism ; therapy ; Myocardium ; metabolism ; Random Allocation ; Rats ; Rats, Wistar ; Stem Cell Transplantation ; Stem Cells ; cytology ; drug effects

OBJECTIVETo induce DNA oxidative damage in colorectal crypt cells by hydrogen peroxide in vitro.

METHODSHydrogen peroxide was diluted into 100, 50, 10, 5 and 1 micromol/L with RPMI 1640. Colorectal crypt cells were treated with peroxide for 10 min, 30 min, 1 h, 1.5 h, 12 h and 24 h respectively. The survival of colorectal crypt cell was measured by MTT method, and the DNA oxidative damage special product, 8-OhdG was detected with immunohistochemical staining. Liner regression was used to measure the time trend of survival rate with SPSS 10.0 software.

RESULTSurvival rate of colorectal crypt cell was 60% and 80% after 10 min of hydrogen peroxide treatment. The longer treatment of hydrogen peroxide, the lower survival rate; the survival rate was reduced to 30% in 24 h. After 10 or 30 min treatment of 100 or 50 micromol/L hydrogen peroxide, the survival rates of colorectal crypt cells were reduced by 20% compared with those of 10, 5 and 1 micromol/L hydrogen peroxide. However, while cells were treated with different concentrations of hydrogen peroxide for 1.0 h or above, there were no differences in cell survival rates. The time trend test results demonstrated that the survival rates of colorectal crypt cells treated with 10, 5 and 1 micromol/L hydrogen peroxide were significantly decreased with the time length of treatment. Colorectal crypt cells treated with different concentrations of hydrogen peroxide for 15 minutes were positively stained brown in cytoplasm and nuclear by immunohistochemistry with 8-OhdG monoclonal antibody.

CONCLUSIONHydrogen peroxide could induce DNA oxidative damage in colorectal crypt cells. And treated with 1 - 10 micromol/L hydrogen peroxide for 10 - 30 min, DNA oxidative damage is apt to be induced in colorectal crypt cell.

Carbazoles ; analysis ; Cells, Cultured ; Colon ; cytology ; drug effects ; metabolism ; Humans ; Hydrogen Peroxide ; Models, Biological ; Oxidative Stress ; drug effects ; Propanolamines ; analysis ; Stem Cells ; cytology ; drug effects

Tissue damage induces cells into reprogramming-like cellular state, which contributes to tissue regeneration. However, whether factors promoting the cell reprogramming favor tissue regeneration remains elusive. Here we identified combination of small chemical compounds including drug cocktails robustly promoting in vitro cell reprogramming. We then administrated the drug cocktails to mice with acute liver injuries induced by partial hepatectomy or toxic treatment. Our results demonstrated that the drug cocktails which promoted cell reprogramming in vitro improved liver regeneration and hepatic function in vivo after acute injuries. The underlying mechanism could be that expression of pluripotent genes activated after injury is further upregulated by drug cocktails. Thus our study offers proof-of-concept evidence that cocktail of clinical compounds improving cell reprogramming favors tissue recovery after acute damages, which is an attractive strategy for regenerative purpose.
Animals ; Cellular Reprogramming ; drug effects ; Cellular Reprogramming Techniques ; methods ; Induced Pluripotent Stem Cells ; cytology ; metabolism ; Mice

OBJECTIVECytomegalovirus (CMV) infection was greatly common in the world. CMV infection produces usually mild or asymptomatic infections in individuals with normal immune responses, whereas it may cause serious disease in immunosuppressive patients. Clinical manifestations include suppression of myelopoiesis, a mononucleosis like syndrome, hepatosplenomegaly, lymphadenopathy, thrombocytopenia, and hemolytic anemia. In patients undergoing bone marrow transplantation CMV remains the most common infectious causes of morbidity and mortality. But the treatment drugs with specific effect for CMV was fewer at the present. This study was to investigate the effect of CMV on proliferation of colony forming unit granulocyte-macrophage (CFU-GM), CFU-erythroid (CFU-E), brust forming unit-erythroid (BFU-E), CFU-multipotential (CFU-Mix) and CFU-megakaryocyte (CFU-Mk) progenitor cells of cord blood (CB) with the presence of ganciclovir (GCV) and astragalus membranaceus in vitro.

METHODSTwenty CB samples were collected from fetal umbilical vein of normal term spontaneous delivery neonates. The colony forming unit-assay was applied to observe the suppression effect of CMV-AD169 strain on CFU-GM, CFU-E, BFU-E, CFU-Mix and CFU-Mk of CB with the presence of GCV and astragalus membranaceus in vitro. The technique of PCR was used to demonstrate the existence of CMV-AD169 DNA in the colony cells of cultured CFU-GM, CFU-E, BFU-E, CFU-Mix and CFU-Mk.

RESULTS(1) The numbers of CFU-GM, CFU-E, BFU-E, CFU-Mix and CFU-Mk colonies in CMV infection groups were significantly less than those in blank and mock group, respectively. The last time of colonies in groups with CMV infection was significantly shorten compared with the blank and mock group. (2) CMV-DNA was positively detected in the colony cells of CMV infection groups by PCR, while negative in the control groups. (3) The lasting time of CFU-GM, CFU-E, BFU-E, CFU-Mix and CFU-Mk colonies infected with CMV extended significantly with the presence of astragalus membranaceus and GCV, and the numbers of those increased significantly compared with the CMV infection group, respectively. The increasing rate of colonies was 27.2%, 45.2%, 49.1%, 39.0% and 11.9% with astragalus membranaceus group, 37.4%, 74.2%, 71.7%, 67.4% and 38.9% with GCV group, 53.6%, 83.8%, 88.7%, 87.8% and 61.5% with astragalus membranaceus and GCV group, respectively.

CONCLUSIONSThe differentiation and proliferation of CFU-GM, CFU-E, BFU-E, CFU-Mix and CFU-Mk were significantly inhibited after infected with CMV-AD169 strain. The suppression effect of CMV-AD169 on CFU-GM, CFU-E, BFU-E, CFU-Mix and CFU-Mk was inhibited with the presence of GCV and astragalus membranaceus in vitro. This suggested that CMV-AD169 may be inhibited or killed by GCV and Astragalus Membranaceus in vitro.

Antiviral Agents ; pharmacology ; Astragalus membranaceus ; chemistry ; Cell Division ; drug effects ; Cytomegalovirus ; drug effects ; Cytomegalovirus Infections ; drug therapy ; Drugs, Chinese Herbal ; pharmacology ; Erythroid Precursor Cells ; cytology ; drug effects ; metabolism ; Fetal Blood ; cytology ; drug effects ; metabolism ; Ganciclovir ; pharmacology ; Hematopoietic Stem Cells ; cytology ; drug effects ; metabolism ; Humans ; Multipotent Stem Cells ; cytology ; drug effects ; metabolism

OBJECTIVETo study the effects of licorice on the proliferation of intestinal crypt stem cell line IEC-6 and the expression of p53.

METHODSInduced by difluoro-methylornithine (DFMO), polyamine-depleted IEC-6 cells under growth inhibition were used as the pathological cell model in this study. Cells were divided into four groups, i. e., the control group, the DFMO-treated group, the high dose licorice group, and the low dose licorice group. The control group consisted of IEC-6 cells cultured in normal condition. The other three groups were all treated with 5 mmol/L DFMO. The high dose and low dose licorice groups were supplemented with 40 and 80 microg/mL licorice granule respectively. All the groups were cultured for 6 successive days. The cell number and viability were determined using flow cytometry. The level of p53 protein was detected by Western blot. The p53 mRNA levels and stability were detected using fluorescent quantitative Real-time PCR.

RESULTSCompared with the control group, the cell growth of the DFMO group was obviously inhibited on the 4th day (P < 0.05). The cell number increased more obviously in the low dose licorice and the high dose licorice groups in a dose-dependent way on the 6th day when compared with the DFMO group (P < 0.05). When compared with the control group, significantly elevated expression levels of p53 protein and mRNA in cells of the DFMO group were detected after 6-day treatment (P < 0.05). When compared with the DFMO group, the expression levels of p53 protein and mRNA were significantly down-regulated in the low dose licorice and the high dose licorice groups (P < 0.05). The degradation of p53 mRNA was the fastest in the control group, while the degradation speed of cells in the DFMO group was the slowest.

CONCLUSIONOne of mechanisms for protective and healing effects of licorice on the intestinal mucosa was possibly through down-regulating the stability of p53 mRNA, lowering the expression of p53, thus promoting the proliferation of the intestinal crypt stem cells.

Animals ; Cell Line ; Cell Proliferation ; Glycyrrhiza ; Intestines ; cytology ; metabolism ; RNA Stability ; drug effects ; RNA, Messenger ; genetics ; Rats ; Stem Cells ; cytology ; drug effects ; metabolism ; Tumor Suppressor Protein p53 ; metabolism

OBJECTIVETo observe the effect of astragaloside on oxidative low-density lipoprotein (Ox-LDL) mediated oxidative damage of endothelial progenitor cells (EPCs).

METHODSPeripheral blood mononuclear cells(PBMCs) were isolated by Ficoll density gradient centrifugation, and EPCs were identified by flow cytometry. Adherent cells were collected after seven-day incubation and randomly divided into the normal control group, the Ox-LDL group (as the model group, at the dose of 100 microg/mL), the low, middle, and high astragaloside groups (with 100 microg/mL Ox-LDL plus 2, 10, and 50 microg/mL astragaloside). Twenty-four h later, the proliferation and adhesion capabilities of EPCs were observed using MTT colorimetry and the adhesion capability detection. Levels of superoxide dismutase (SOD) and malonaldehyde (MDA) in the cell supernate of each group were determined.

RESULTSAfter Ox-LDL damage, the proliferative and adhesive capacities of EPCs were significantly injured (53 +/- 8 vs 42 +/- 6, 0.49 +/- 0.12 vs 0.37 +/- 0.02, both P<0.05). The SOD content obviously decreased (21.95 +/- 1.43 vs 14.76 +/- 3.99, P<0.01), the MDA content obviously increased (3.72 +/- 0.30 vs 5.57 +/- 0.64, P<0.01). After intervened by astragaloside for 24 h, the proliferative and adhesive capacities of EPCs were significantly improved. The SOD contents of astragaloside intervention groups were obviously improved and the MDA content obviously lowered.

CONCLUSIONSAstragaloside showed significant protection on Ox-LDL damaged EPCs. Its mechanism might be correlated with antioxidative damage.

Cells, Cultured ; Endothelial Cells ; cytology ; drug effects ; metabolism ; Humans ; Leukocytes, Mononuclear ; metabolism ; Lipoproteins, LDL ; metabolism ; Malondialdehyde ; metabolism ; Oxidation-Reduction ; Oxidative Stress ; drug effects ; Saponins ; pharmacology ; Stem Cells ; cytology ; drug effects ; metabolism ; Triterpenes ; pharmacology

Vertically aligned, laterally spaced nanoscale titanium nanotubes were grown on a titanium surface by anodization, and the growth of chondroprogenitors on the resulting surfaces was investigated. Surfaces bearing nanotubes of 70 to 100 nm in diameter were found to trigger the morphological transition to a cortical actin pattern and rounded cell shape (both indicative of chondrocytic differentiation), as well as the up-regulation of type II collagen and integrin beta4 protein expression through the down-regulation of Erk activity. Inhibition of Erk signaling reduced stress fiber formation and induced the transition to the cortical actin pattern in cells cultured on 30-nm-diameter nanotubes, which maintained their fibroblastoid morphologies in the absence of Erk inhibition. Collectively, these results indicate that a titanium-based nanotube surface can support chondrocytic functions among chondroprogenitors, and may therefore be useful for future cartilaginous applications.
Animals ; Apoptosis ; Cell Differentiation/*drug effects ; Cells, Cultured ; Chick Embryo ; Chickens ; Chondrocytes/cytology/drug effects/metabolism ; Chondrogenesis/*drug effects ; Collagen Type II/metabolism ; Immunohistochemistry ; Integrin beta4/metabolism ; Mesenchymal Stem Cells/*cytology/*drug effects/metabolism ; Nanotubes/*chemistry ; Titanium/*chemistry/*pharmacology