Axl encodes the tyrosine-protein kinase receptor, participating in the proliferation and migration of many cells. This study examined the role of Axl in functions of endothelial progenitor cells (EPCs). Axl was detected by RT-PCR and Western blotting in both placentas and EPCs from normal pregnancy and preeclampsia patients. The Axl inhibitor, BMS777-607, was used to inhibit the Axl signalling pathway in EPCs. Cell proliferation, differentiation, migration and adhesion were measured by CCK-8 assay, cell differentiation assay, Transwell assay, and cell adhesion assay, respectively. Results showed the expression levels of Axl mRNA and protein were significantly higher in both placentas and EPCs from preeclampsia patients than from normal pregnancy (P<0.05). After treatment with BMS777-607, proliferation, differentiation, migration and adhesion capability of EPCs were all significantly decreased. Our study suggests Axl may play a role in the function of EPCs, thereby involving in the pathogenesis of preeclampsia.
Adult ; Aminopyridines ; pharmacology ; Blood Pressure ; Case-Control Studies ; Cell Adhesion ; drug effects ; Cell Differentiation ; drug effects ; Cell Movement ; drug effects ; Cell Proliferation ; drug effects ; Female ; Fetal Blood ; cytology ; enzymology ; Gene Expression Regulation ; Gestational Age ; Human Umbilical Vein Endothelial Cells ; drug effects ; enzymology ; pathology ; Humans ; Placenta ; metabolism ; physiopathology ; Pre-Eclampsia ; blood ; genetics ; physiopathology ; Pregnancy ; Primary Cell Culture ; Protein Kinase Inhibitors ; pharmacology ; Proto-Oncogene Proteins ; antagonists & inhibitors ; genetics ; metabolism ; Pyridones ; pharmacology ; RNA, Messenger ; antagonists & inhibitors ; genetics ; metabolism ; Receptor Protein-Tyrosine Kinases ; antagonists & inhibitors ; genetics ; metabolism ; Stem Cells ; drug effects ; enzymology ; pathology

OBJECTIVETo evaluate the effects of ginsenoside Rg-1 on the proliferation and osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) and to explore the possible application on the alveolar bone regeneration.

METHODSTo determine the optimum concentration, the effects of ginsenoside Rg-1 ranging from 10 to 100 μmol/L were evaluated by 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide, alkaline phosphatase activity and calcium deposition. Expressions of runt-related transcription factor 2, collagen alpha-2(I) chain, osteopontin, osteocalcin protein were examined using real-time polymerase chain reaction.

RESULTSCompared with the control group, a certain concentration (10 μmol/L) of the Rg-1 solution significantly enhanced the proliferation and osteogenic differentiation of hPDLSCs (P<0.05). However, concentrations that exceeds 100 μmol/L led to cytotoxicity whereas concentrations below 10 nmol/L showed no significant effect as compared with the control.

CONCLUSIONGinsenoside Rg-1 can enhance the proliferation and osteogenic differentiation of hPDLSCs at an optimal concentration of 10 μmol/L.

Adolescent ; Alkaline Phosphatase ; metabolism ; Biomarkers ; metabolism ; Calcification, Physiologic ; drug effects ; Cell Differentiation ; drug effects ; Cell Proliferation ; drug effects ; Cell Separation ; Cell Shape ; drug effects ; Cells, Cultured ; Flow Cytometry ; Ginsenosides ; pharmacology ; Humans ; Osteoblasts ; drug effects ; metabolism ; Osteogenesis ; drug effects ; genetics ; Periodontal Ligament ; cytology ; Real-Time Polymerase Chain Reaction ; Stem Cells ; cytology ; drug effects ; enzymology ; Time Factors ; Young Adult

OBJECTIVETo investigate the effects and underlying molecular mechanisms of icariin (ICA) on self-renewal and differentiation of neural stem cells (NSCs).

METHODSNSCs were derived from forebrains of mice embryos by mechanical dissociation into single cell suspension. The self-renewal of NSCs was measured by neurosphere formation assay. The proliferation of NSCs was detected by water-soluble tetrazolium (WST) and 5-ethynyl-2'-deoxyuridine (EdU) incorporation assay. Protein expression of neuron-specific marker tubulin-βIII(TuJ1) and astrocyte-specific marker glial fibrillary acidic protein (GFAP) were measured by immunofluorescence and Western blotting. Using microarray, the differentially expressed genes (DEGs) were screened between NSCs with or without ICA treatment. The signaling pathways enriched by these DEGs and their role in mediating effects of ICA were analyzed.

RESULTSICA significantly promoted neurosphere formation of NSCs cultured in growth protocol in a dose-dependent manner and achieved the maximum effects at 100 nmol/L. ICA also increased optical absorbance value and EdU incorporation into nuclei of NSCs. ICA had no significant effects on the percentage of TuJ1 or GFAP-positive cells, and TuJ1 or GFAP protein expression in NSCs cultured in differentiation protocol. A total of 478 genes were found to be differentially regulated. Among signaling pathways significantly enriched by DEGs, mitogen activated protein kinase (MAPK) pathway was of interest. Blockade of extracellular signal-regulated kinase (ERK)/MAPK, other than p38/MAPK subfamily pathway partially abolished effects of ICA on neurosphere formation and EdU incorporation of NSCs.

CONCLUSIONICA can promote the selfrenewal of NSCs at least partially through ERK/MAPK signaling pathway.

Animals ; Cell Aggregation ; drug effects ; genetics ; Cell Differentiation ; drug effects ; genetics ; Cell Proliferation ; drug effects ; Cell Survival ; drug effects ; genetics ; Deoxyuridine ; analogs & derivatives ; metabolism ; Extracellular Signal-Regulated MAP Kinases ; metabolism ; Female ; Flavonoids ; pharmacology ; Gene Expression Regulation ; drug effects ; MAP Kinase Signaling System ; drug effects ; genetics ; Mice ; Neural Stem Cells ; cytology ; drug effects ; enzymology

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 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

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

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

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

BACKGROUNDMesenchymal stem cells (MSCs) transplantation is of therapeutic potential after ischemic injury in both experimental and clinical studies. Clinically, elderly patients are more vulnerable to acute myocardial infarction (AMI). But little is known about the characteristics of young donor-derived MSCs transplanted to old patients with AMI. The present study was designed to investigate the effect of transplanted MSCs from rats of different ages on the improvement of heart function after AMI.

METHODSMSCs from Sprague-Dawley (SD) rats were isolated and cultured in vitro. The apoptosis characteristics of MSCs were observed under conditions of ischemia and anoxia. SD rats underwent MI received intramyocardial injection of MSCs from young donor rats (n = 8), old donor rats (n = 8), respectively. AMI control group received equal volume physiological saline. Immunofluorescence was used to observe the differentiation of the grafted cells into cardiomyocytes. Four weeks after cell transplantation, reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry for vascular endothelial growth factor (VEGF), VIII-factor immunohistochemistry for vessel density, TUNEL, caspase-3 for cardiomyocyte apoptosis, echocardiography and hemodynamic detection for heart function were performed.

RESULTSThe apoptosis rate of the old donor-derived MSCs group was significantly higher than that of the young donor-derived MSCs group under conditions of ischemia and anoxia (P < 0.05). Engrafted MSCs survived, proliferated and differentiated into myocardium-like cells. VEGF gene expression and capillary density in the old donor-derived group were lower than those in the young donor-derived group but higher than those in the control group (P < 0.05). The transplantation of old donor-derived MSCs attenuated apoptosis of cardiomyocytes in the peri-infarct region compared with the control group and the effect was elevated in young donor-derived MSCs (P < 0.05). The heart functions (left ventricle ejection fraction (LVEF), left ventricle fractional shortening (LVFS)) were improved more significantly in the old donor-derived MSCs group than in the control group and the heart function in the young donor-derived MSCs group further improved (P < 0.05).

CONCLUSIONSYoung donor-derived MSCs can improve heart function significantly through angiogenesis and decreasing cardiomyocyte apoptosis when transplanted to the infarcted area.

Age Factors ; Animals ; Apoptosis ; Caspase 3 ; metabolism ; Cells, Cultured ; Flow Cytometry ; Immunohistochemistry ; In Situ Nick-End Labeling ; Male ; Mesenchymal Stem Cell Transplantation ; methods ; Mesenchymal Stromal Cells ; cytology ; Myocardial Infarction ; physiopathology ; surgery ; Myocytes, Cardiac ; cytology ; enzymology ; Rats ; Rats, Sprague-Dawley ; Reverse Transcriptase Polymerase Chain Reaction ; Vascular Endothelial Growth Factor A ; genetics ; metabolism