Drug resistance and relapse are the major challenge for current treatment of acute leukemia. It is critical for ultimately curing leukemia to overcome chemoresistance of leukemic stem cells (LSC) and to eradicate LSC. Recent studies have found that abnormal activated Hedgehog (HH) signaling pathway plays an important role in a wide variety of tumors and regulates multi-drug resistance of LSC. This review briefly summarizes the molecular mechanism of HH signal pathway inducing drug resistance of LSC and leading to novel strategies for eradicating LSC.
Drug Resistance, Neoplasm ; Hedgehog Proteins ; metabolism ; Humans ; Leukemia ; metabolism ; Neoplastic Stem Cells ; drug effects ; Signal Transduction

OBJECTIVETo investigate the roles of telomere and telomerase in the effect of ginsenoside Rg1 to delay hematopoietic stem cell senescence.

METHODSca-1(+) HSC was isolated by magnetic cell sorting(MACS) and divided into five groups: the control group, the aged model group, the Rg1 group, the Rg1 treated aged group and the Rg1 delayed aged group. The changes of cells were observed by senescence-associated beta-Galactosidase (SA-beta-Gal) staining. Cell cycle assay and culture of mixed hematopoietic progenitor cell were used to investigate the effect of ginsenoside Rg1 to delay Sca-1(+) HSC senescence. Telomere length and telomerase activity were detected by southern blotting and TRAP-PCR-SYBR Green staining.

RESULTCompared with aged model group, the percentage of positive cells expressed SA-beta-Gal and the number of cells entered G1 phase were decreased and the number of colony of mixed hematopoietic progenitor was increased. It showed markedly decreased in the shortening of telomere length and reinforcing in the telomerase activity to Rg1 treated aged group and Rg1 delayed aged group. The change of Rg1 delayed aged group was significantly higher than Rg1 treated aged group.

CONCLUSIONActivation of telomerase and prolonging of telomere length might be involved in the process of ginsenoside Rg1 to delay and treat the senescence of Sca-1(+) HSC.

Cellular Senescence ; drug effects ; Ginsenosides ; pharmacology ; Hematopoietic Stem Cells ; drug effects ; physiology ; Telomerase ; metabolism ; Telomere ; drug effects

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

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

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

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

OBJECTIVETo explore the effects of stem cell factor (SCF) on proliferation, transmigration, capillary tube formation of human umbilical vein endothelial cells (HUVEC) and on the chemotaxis of CD133(+) cells.

METHODSIn the presence of blank control, SCF, vascular endothelial growth factor (VEGF), anti-human SCF (anti-SCF) or human IgG, the difference in proliferation capacity of HUVEC was analyzed by MTT and CCK-8 methods, and wound scratch assay and three-dimensional in vitro Matrigel assay were used for transmigration and capillary tube formation of HUVEC, respectively. In addition, the chemotaxis of CD133(+) cells sorted from human umbilical cord blood by flow cytometry was investigated by Transwell migration assay.

RESULTSSCF didn't improve the proliferative capacity of HUVEC, but significantly enhanced the transmigration capacity, and increased capillary tube formation in a dose-dependent manner. The number of intact tubules [(30.0 ± 3.4)/10(5) HUVEC] formed by HUVECs in the presence of the optimal concentration of SCF (100 ng/ml) was remarkably higher than that in blank control group [(5.0 ± 2.6)/10(5) HUVEC, P < 0.01]. SCF also significantly induced a chemotactic response of CD133(+) cells, the transmembrane migration cell number into Transwell lower chamber was significantly higher in SCF group [(118.0 ± 6.5)/10(4) CD133(+) cells] than in blank control group [(47.0 ± 4.7)/10(4) CD133(+) cells, P < 0.01 ].

CONCLUSIONSSCF significantly promotes the transmigration and capillary tube formation of HUVEC, and induces a chemotactic response of CD133(+) cells. SCF/c-kit signaling possibly plays a critical role in regulating angiogenesis of vascular endothelial cells and vasculogenesis of endothelial progenitor cells.

Cell Movement ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Human Umbilical Vein Endothelial Cells ; cytology ; Humans ; Neovascularization, Physiologic ; drug effects ; Sincalide ; metabolism ; Stem Cell Factor ; pharmacology

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

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

OBJECTIVETo explore the effect of Chinese herbs for Shen invigorating and blood activating (CHSIBA) on the number of endothelial progenitor cells (EPCs) in the bone marrow and the peripheral blood and the signaling pathway of bone marrow matrix metalloproteinase 9 (MMP-9) of the myocardial infarction (MI) model rats.

METHODSThe MI rat model was established by ligation. Thirty successfully modeled rats were randomly divided into the high dose CHSIBA group, the low dose CHSIBA group, and the model group, 10 in each group. Besides, another 10 normal rats were recruited as the blank group. Rats in the high dose CHSIBA group and the low dose CHSIBA group were administered with CHSIBA at 3 g/kg and 1.5 g/kg body weight by gastrogavage (by adding them in 4 mL physiological saline), once daily. Rats in the model group and the blank group were administered with 4 mL physiological saline once daily. The EPCs were collected from the bone marrow and the peripheral blood 4 weeks later. Seven days later the CD34/CD133 phenotype was identified in collected sticking wall cells using flow cytometry. The MMP-9 and water soluble Kit ligand (sKitL) were detected using Western blot. The expressions of vascular endothelial growth factor (VEGF) and stromal cell-derived factor-1alpha (SDF-1alpha) were detected using ELISA.

RESULTSThe CD34/CD133 positive rate and the EPC quantity in the bone marrow and the peripheral blood were higher in the high dose CHSIBA group and the low dose CHSIBA group than in the model group (P < 0.05, P < 0.01). Besides, the expressions of VEGF, SDF-1alpha, MMP-9, and sKitL in the bone marrow and the peripheral blood were also higher in the high dose CHSIBA group and the low dose CHSIBA group than in the model group (P < 0.05, P < 0.01).

CONCLUSIONCHSIBA could activate MMP-9 signaling pathway, increase its upstream and downstream signal expression levels, and mobilize EPCs in the bone marrow to enter the blood circulation.

Animals ; Bone Marrow Cells ; drug effects ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; Endothelial Cells ; drug effects ; metabolism ; Male ; Matrix Metalloproteinase 9 ; metabolism ; Myocardial Infarction ; metabolism ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; Stem Cells ; drug effects ; metabolism