Animals ; Cell Division ; Drosophila melanogaster ; cytology ; enzymology ; Female ; Stem Cells ; cytology ; enzymology ; Ubiquitin-Protein Ligases ; metabolism

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 investigate whether adipose-derived stem cells (ADSCs) induced by retinoic acid (RA) in vitro express primordial germ cell marker alkaline phosphatase (ALP) and vasa.

METHODSADSCs were isolated from adult female SD rats and cultured in vitro. The third passage of ADSCs was identified by adipogenic differentiation, osteogenic differentiation and cell surface marker detection. The ADSCs were treated with 1×10(-5), 1×10(-6), or 1×10(-7) mol/L RA for 7 or 14 days, and the cellular expression of ALP was detected. vasa mRNA expression in ADSCs treated with 1×10(-5) mol/L RA for 7 days was detected using RT-PCR.

RESULTSThe OD value of ADSCs treated with 1×10(-5), 1×10(-6), or 1×10(-7) mol/L RA was 0.59∓0.04, 0.27∓0.07, and 0.15∓0.03 after a 7-day treatment, and was 0.42∓0.02, 0.34∓0.01, and 0.19∓0.02 after a 14-day treatment, respectively, demonstrating significantly enhanced ALP expression in RA-treated ADSCs compared with that in the control cells (0.07∓0.01 and 0.07∓0.01 at 7 and 14 days, respectively, P<0.01). The ADSCs showed a negative vasa mRNA expression after 1×10(-5) mol/L RA treatment for 7 days.

CONCLUSIONRA-induced ADSCs express ALP, a marker of primordial germ cells, but does not express the primordial germ cell marker vasa.

Adipose Tissue ; cytology ; Adult Stem Cells ; cytology ; enzymology ; Alkaline Phosphatase ; metabolism ; Animals ; Cell Differentiation ; Cells, Cultured ; Female ; Germ Cells ; cytology ; metabolism ; Rats ; Rats, Sprague-Dawley ; Tretinoin ; pharmacology

Cyclooxygenase-2 (COX-2) is known to modulate bone metabolism, including bone formation and resorption. Because cartilage serves as a template for endochondral bone formation and because cartilage development is initiated by the differentiation of mesenchymal cells into chondrocytes (Ahrens et al., 1977; Sandell and Adler, 1999; Solursh, 1989), it is of interest to know whether COX-2 expression affect chondrocyte differentiation. Therefore, we investigated the effects of COX-2 protein on differentiation in rabbit articular chondrocyte and chick limb bud mesenchymal cells. Overexpression of COX-2 protein was induced by the COX-2 cDNA transfection. Ectopic expression of COX-2 was sufficient to causes dedifferentiation in articular chondrocytes as determined by the expression of type II collagen via Alcian blue staining and Western blot. Also, COX-2 overexpression caused suppression of SOX-9 expression, a major transcription factor that regulates type II collagen expression, as indicated by the Western blot and RT-PCR. We further examined ectopic expression of COX-2 in chondrifying mesenchymal cells. As expected, COX-2 cDNA transfection blocked cartilage nodule formation as determined by Alcian blue staining. Our results collectively suggest that COX-2 overexpression causes dedifferentiation in articular chondrocytes and inhibits chondrogenic differentiation of mesenchymal cells.
Animals ; Cartilage, Articular/cytology ; Cell Differentiation ; Cells, Cultured ; Chick Embryo ; Chondrocytes/*cytology/enzymology ; Chondrogenesis ; Collagen Type II/metabolism ; Cyclooxygenase 2/*biosynthesis/genetics ; Interleukin-1beta/pharmacology ; Mesenchymal Stem Cells/*cytology/enzymology ; Rabbits ; SOX9 Transcription Factor/metabolism

Many studies have reported that an electromagnetic field can promote osteogenic differentiation of mesenchymal stem cells. However, experimental results have differed depending on the experimental and environmental conditions. Optimization of electromagnetic field conditions in a single, identified system can compensate for these differences. Here we demonstrated that specific electromagnetic field conditions (that is, frequency and magnetic flux density) significantly regulate osteogenic differentiation of adipose-derived stem cells (ASCs) in vitro. Before inducing osteogenic differentiation, we determined ASC stemness and confirmed that the electromagnetic field was uniform at the solenoid coil center. Then, we selected positive (30/45 Hz, 1 mT) and negative (7.5 Hz, 1 mT) osteogenic differentiation conditions by quantifying alkaline phosphate (ALP) mRNA expression. Osteogenic marker (for example, runt-related transcription factor 2) expression was higher in the 30/45 Hz condition and lower in the 7.5 Hz condition as compared with the nonstimulated group. Both positive and negative regulation of ALP activity and mineralized nodule formation supported these responses. Our data indicate that the effects of the electromagnetic fields on osteogenic differentiation differ depending on the electromagnetic field conditions. This study provides a framework for future work on controlling stem cell differentiation.
Adipose Tissue/*cytology ; Alkaline Phosphatase/metabolism ; Biological Markers/metabolism ; Bone Matrix/metabolism ; Calcification, Physiologic/genetics ; *Cell Differentiation/genetics ; Core Binding Factor Alpha 1 Subunit/metabolism ; *Electromagnetic Fields ; Humans ; *Osteogenesis/genetics ; Reproducibility of Results ; Stem Cells/*cytology/enzymology/metabolism

OBJECTIVETo elucidate gene expressions of phase II enzymes in the mouse embryonic stem (ES) cell-derived hepatocytes.

METHODSEmbryoid body (EB) formation cultures were applied in directed differentiation of ES to hepatic-like cells. The expressions of hepatic-specific genes, including AFP, ALB, Cyp7a1, were detected by RT-PCR during the differentiation course. Albumin was detected by immunocyto- chemistry. The gene expressions of mGST1 and UGTs family, including UGT1a1, UGT1a6, UGT1a9 and UGT2b5, were investigated using RT-PCR.

RESULTSA notable gene expression of AFP and ALB was observed on d 8. On d 18, AFP gene failed to express, while ALB and Cyp7a1 genes were detected.Albumin-positive staining was detected in hepatic-like cells. Phase II enzyme genes expressed in variance during the differentiation course, UGT1a1 and UGT1a9 were expressed stably, UGT1a6 expression increased gradually, and UGT2b5 failed to express. Little mGST1 gene expression could been detected in the early course until d 18. In addition, all the enzymes gene expressions in the derived hepatocytes on d 18 were similar to those from mature mouse hepatocytes.

CONCLUSIONSMouse ES cell-derived mature hepatocytes express phase II enzyme UGTs and mGST1 genes similar to those in mature hepatocytes. The system may offer an alternative animal testing model related to phase enzymes in further research.

Animals ; Cell Differentiation ; Cells, Cultured ; Embryonic Stem Cells ; cytology ; enzymology ; Fluorescent Antibody Technique ; Glucuronosyltransferase ; genetics ; metabolism ; Glutathione S-Transferase pi ; genetics ; metabolism ; Hepatocytes ; cytology ; enzymology ; Isoenzymes ; genetics ; metabolism ; Male ; Mice ; Mice, Inbred BALB C ; Microsomes ; enzymology ; Reverse Transcriptase Polymerase Chain Reaction

Recently, reactive oxygen species (ROS) have been studied as a regulator of differentiation into specific cell types in embryonic stem cells (ESCs). However, ROS role in human ESCs (hESCs) is unknown because mouse ESCs have been used mainly for most studies. Herein we suggest that ROS generation may play a critical role in differentiation of hESCs; ROS enhances differentiation of hESCs into bi-potent mesendodermal cell lineage via ROS-involved signaling pathways. In ROS-inducing conditions, expression of pluripotency markers (Oct4, Tra 1-60, Nanog, and Sox2) of hESCs was decreased, while expression of mesodermal and endodermal markers was increased. Moreover, these differentiation events of hESCs in ROS-inducing conditions were decreased by free radical scavenger treatment. hESC-derived embryoid bodies (EBs) also showed similar differentiation patterns by ROS induction. In ROS-related signaling pathway, some of the MAPKs family members in hESCs were also affected by ROS induction. p38 MAPK and AKT (protein kinases B, PKB) were inactivated significantly by buthionine sulfoximine (BSO) treatment. JNK and ERK phosphorylation levels were increased at early time of BSO treatment but not at late time point. Moreover, MAPKs family-specific inhibitors could prevent the mesendodermal differentiation of hESCs by ROS induction. Our results demonstrate that stemness and differentiation of hESCs can be regulated by environmental factors such as ROS.
Biological Markers/metabolism ; Cell Differentiation/*drug effects ; Cell Line ; Cell Lineage/*drug effects ; Cells, Cultured ; Down-Regulation/drug effects ; Embryo, Mammalian/cytology/drug effects/metabolism ; Embryonic Stem Cells/*cytology/*drug effects/enzymology ; Endoderm/*cytology/drug effects ; Enzyme Activation/drug effects ; Free Radical Scavengers/pharmacology ; Humans ; Mesoderm/*cytology/drug effects ; Mitogen-Activated Protein Kinases/metabolism ; Pluripotent Stem Cells/cytology/metabolism ; Reactive Oxygen Species/metabolism/*pharmacology ; Up-Regulation/drug effects

Melanoma inhibiting activity/cartilage-derived retinoic acid-sensitive protein (MIA/CD-RAP) is a small soluble protein secreted from malignant melanoma cells and from chondrocytes. Recently, we revealed that MIA/CD-RAP can modulate bone morphogenetic protein (BMP)2-induced osteogenic differentiation into a chondrogenic direction. In the current study we aimed to find the molecular details of this MIA/CD-RAP function. Direct influence of MIA on BMP2 by protein-protein-interaction or modulating SMAD signaling was ruled out experimentally. Instead, we revealed inhibition of ERK signaling by MIA/CD-RAP. This inhibition is regulated via binding of MIA/CD-RAP to integrin alpha5 and abolishing its activity. Active ERK signaling is known to block chondrogenic differentiation and we revealed induction of aggrecan expression in chondrocytes by treatment with MIA/CD-RAP or PD098059, an ERK inhibitor. In in vivo models we could support the role of MIA/CD-RAP in influencing osteogenic differentiation negatively. Further, MIA/CD-RAP-deficient mice revealed an enhanced calcified cartilage layer of the articular cartilage of the knee joint and disordered arrangement of chondrocytes. Taken together, our data indicate that MIA/CD-RAP stabilizes cartilage differentiation and inhibits differentiation into bone potentially by regulating signaling processes during differentiation.
Animals ; Bone Morphogenetic Proteins/metabolism ; Cartilage/*cytology/metabolism ; *Cell Differentiation ; Chondrocytes/cytology/enzymology ; Extracellular Matrix Proteins/deficiency/*metabolism ; Extracellular Signal-Regulated MAP Kinases/metabolism ; Humans ; Integrin alpha5/metabolism ; Mesenchymal Stem Cells/cytology/metabolism ; Mice ; Neoplasm Proteins/deficiency/*metabolism ; Osteogenesis ; Protein Binding ; Signal Transduction ; Smad Proteins/metabolism

To investigate the expression of telomerase in cord blood hematopoietic stem/progenitor cells during their committed differentiation in vitro and provide an index of monitoring the proliferating potential of the hematopoietic stem/progenitor cells and security for clinical application. Human CD34 positive cells were isolated from umbilical cord blood by using magnetic cell sorting system (MACS), and were induced to differentiation with hematopoietic growth factors (SCF + IL3 + IL6 + GCSF and SCF + IL3 + IL6 + EPO) in a liquid culture system. The telomerase activity and the cytalytic subunit of telomerase (hTERT) of the cells were analysed during different periods of culture by using TRAP-PCR, TRAP-ELISA, Western blot and RT-PCR techniques, respectively. The results showed that a peak of cell growth was achieved on day 14 - 21 during induction of differentiation in vitro. Total cell number could increase 1006.4 +/- 103.2 times and could not increase there after. Telomerase activity and hTERT expression were low in freshly isolated cord blood CD34(+) cells and increased after about 7 days of culture in addition of cytokine combinations of SCF + IL3 + IL6 + GCSF and SCF + IL3 + IL6 + EPO, respectively. The telomerase activity and hTERT decreased after 14 days of culture and were not detected after 28 days of culture. It was concluded that the hematopoietic stem/progenitor cells can be expanded in large number in vitro and do not have the character of immortality and the telomerase activity could be a useful index in hematopoiesis regulation.
Antigens, CD34 ; blood ; Blotting, Western ; Cell Differentiation ; DNA-Binding Proteins ; Enzyme-Linked Immunosorbent Assay ; Fetal Blood ; cytology ; Hematopoietic Stem Cells ; cytology ; enzymology ; Humans ; Polymerase Chain Reaction ; RNA, Messenger ; analysis ; Telomerase ; genetics ; metabolism

Mesenchymal stem cells (MSCs) secrete a variety of neuroregulatory molecules, such as nerve growth factor, brain-derived neurotrophic factor, and glial cell-derived neurotrophic factor, which upregulate tyrosine hydroxylase (TH) gene expression in PC12 cells. Enhancing TH gene expression is a critical step for treatment of Parkinson's disease (PD). The objective of this study was to assess the effects of co-culturing PC12 cells with MSCs from feline bone marrow on TH protein expression. We divided the study into three groups: an MSC group, a PC12 cell group, and the combined MSC + PC12 cell group (the co-culture group). All cells were cultured in DMEM-HG medium supplemented with 10% fetal bovine serum for three days. Thereafter, the cells were examined using western blot analysis and immunocytochemistry. In western blots, the co-culture group demonstrated a stronger signal at 60 kDa than the PC12 cell group (p < 0.001). TH was not expressed in the MSC group, either in western blot or immunocytochemistry. Thus, the MSCs of feline bone marrow can up-regulate TH expression in PC12 cells. This implies a new role for MSCs in the neurodegenerative disease process.
Animals ; Antigens, Surface/metabolism ; Blotting, Western ; Cats/*physiology ; Cell Culture Techniques ; Cells, Cultured ; *Gene Expression Regulation, Enzymologic ; Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating)/metabolism ; Immunohistochemistry ; Mesenchymal Stem Cells/*cytology/metabolism ; Microscopy, Phase-Contrast ; PC12 Cells/cytology/*enzymology ; Rats ; Tyrosine 3-Monooxygenase/*metabolism