技术资料

Histone deacetylase inhibitors (HDI) have been shown to increase differentiation-related gene expression in several tumor-derived cell lines by hyperacetylating core histones. Effects of HDI on primary cultured cells,however,have hardly been investigated. In the present study,the ability of trichostatin A (TSA),a prototype hydroxamate HDI,to counteract the loss of liver-specific functions in primary rat hepatocyte cultures has been investigated. Upon exposure to TSA,it was found that the cell viability of the cultured hepatocytes and their albumin secretion as a function of culture time were increased. TSA-treated hepatocytes also better maintained cytochrome P450 (CYP)-mediated phase I biotransformation capacity,whereas the activity of phase II glutathione S-transferases (GST) was not affected. Western blot and qRT-PCR analysis of CYP1A1,CYP2B1 and CYP3A11 protein and mRNA levels,respectively,further revealed that TSA acts at the transcriptional level. In addition,protein expression levels of the liver-enriched transcription factors (LETFs) hepatic nuclear factor 4 alpha (HNF4alpha) and CCAAT/enhancer binding protein alpha (C/EBPalpha) were accordingly increased by TSA throughout culture time. In conclusion,these findings indicate that TSA plays a major role in the preservation of the differentiated hepatic phenotype in culture. It is suggested that the effects of TSA on CYP gene expression are mediated via controlling the expression of LETFs. View Publication

Numerous factors that can influence the proliferation and differentiation in vitro of cells at various stages of hematopoiesis have been identified,but the mechanisms used by stromal cells to regulate the cycling status of the most primitive human hematopoietic cells are still poorly understood. Previous studies of long-term cultures (LTC) of human marrow have suggested that cytokine-induced variations in stromal cell production of one or more stimulators and inhibitors of hematopoiesis may be important. To identify the specific regulators involved,we performed Northern analyses on RNA extracted from human marrow LTC adherent layers,or stromal cell types derived from or related to those present in the adherent layer. These analyses showed marked increases in interleukin-1 beta (IL-1 beta),IL-6,and granulocyte colony-stimulating factor (G-CSF) mRNA levels within 8 hours after treatments that lead to the activation within 2 days of primitive hematopoietic progenitors in such cultures. Increases in granulocyte-macrophage (GM)-CSF and M-CSF mRNA were also sometimes seen. Bioassays using cell lines responsive to G-CSF,GM-CSF,and IL-6 showed significant elevation in growth factor levels 24 hours after IL-1 beta stimulation. Neither IL-3 nor IL-4 mRNA was detectable at any time. In contrast,transforming growth factor-beta (TGF-beta) mRNA and nanogram levels of TGF-beta bioactivity in the medium were detected at all times in established LTC,and these levels were not consistently altered by any of the manipulations that stimulated hematopoietic growth factor production and primitive progenitor cycling. We also found that addition of anti-TGF-beta antibody could prolong or reactivate primitive progenitor proliferation when added to previously stimulated or quiescent cultures,respectively. Together,these results indicate a dominant negative regulatory role of endogenously produced TGF-beta in unperturbed LTC,with activation of primitive hematopoietic cells being achieved by mechanisms that stimulate stromal cells to produce G-CSF,GM-CSF,and IL-6. Given the similarities between the LTC system and the marrow microenvironment,it seems likely that the control of human stem cell activation in vivo may involve similar variations in the production of these factors by stromal cells. View Publication

The success rate is generally higher when cloning mice from embryonic stem (ES) cell nuclei than from somatic cell nuclei,suggesting that the embryonic nature or the undifferentiated state of the donor cell increases cloning efficiency. We assessed the developmental ability of cloned embryos derived from cultured neural stem cell (NSC) nuclei and compared the success rate with that of embryos cloned from other donor cells such as differentiated NSCs,cumulus cells,Sertoli cells and ES cells in the mouse. The transfer of two-cell cloned embryos derived from cultured NSC nuclei into surrogate mothers produced five live cloned mice. However,the success rate (0.5%) was higher in embryos cloned from cultured NSC nuclei than from differentiated NSCs (0%),but lower than that obtained by cloning mice from other cell nuclei (2.2-3.5%). Although the in vitro developmental potential to the two-cell stage of the cloned embryos derived from NSC nuclei (73%) was similar to that of the cloned embryos derived from other somatic cell nuclei (e.g.,85% in Sertoli cells and 75% in cumulus cells),the developmental rate to the morula-blastocyst stage was only 7%. This rate is remarkably lower than that produced from other somatic cells (e.g.,50% in Sertoli cells and 54% in cumulus cells). These results indicate that the undifferentiated state of neural cells does not enhance the cloning efficiency in mice and that the arrest point for in vitro development of cloned embryos depends on the donor cell type. View Publication

Various growth factors are known to stimulate both early and late stages of human hematopoietic cell development in semisolid assay systems,but their role as microenvironmental regulators is poorly understood. To address this problem,we developed a novel coculture system in which highly purified primitive human hematopoietic cells were seeded onto an irradiated feeder layer of cells from a murine marrow-derived stromal cell line (M2-10B4) previously engineered by retroviral-mediated gene transfer to produce specific human factors. Effects on cells at very early,intermediate,and late stages of hematopoiesis were then evaluated by assessing the number of clonogenic cell precursors (long-term culture initiating cells [LTC-IC]),clonogenic cells,and mature granulocyte and macrophage progeny present in the cultures after 5 weeks. In the absence of any feeders,cells at all stages of hematopoiesis decreased to very low levels. In contrast,maintenance of LTC-IC was found to be supported by control murine stromal cells as effectively as by standard human marrow adherent layers. The presence of granulocyte colony-stimulating factor (G-CSF) and interleukin-3-producing M2-10B4 cells in combination was able to further enhance the maintenance and early differentiation of these cells without a decline in their proliferative potential as measured by the clonogenic output per LTC-IC. However,this effect was lost if granulocyte-macrophage CSF (GM-CSF)-producing feeders were also present. On the other hand,in the presence of GM-CSF-producing feeders,the output of mature granulocytes and macrophages increased 20-fold. These findings show that it is possible to selectively improve the maintenance of very primitive human hematopoietic cells in vitro or their output of mature progeny by appropriate manipulation of the long-term marrow culture system. Further exploitation of this approach should facilitate investigation of the mechanisms operative within the human marrow microenvironment in vivo and the design of protocols for in vitro manipulation of human marrow for future therapeutic applications. View Publication

Hematopoietic stem cells (HSCs) have the capacity to self-renew and the potential to differentiate into all of the mature blood cell types. The ability to prospectively identify and isolate HSCs has been the subject of extensive investigation since the first transplantation studies implying their existence almost 50 years ago. Despite significant advances in enrichment protocols,the continuous in vitro propagation of human HSCs has not yet been achieved. This chapter describes current procedures used to phenotypically and functionally characterize candidate human HSCs and initial efforts to derive permanent human HSC lines. View Publication

Transformed,oncogenic precursors,possessing both defining neural-stem-cell properties and the ability to initiate intracerebral tumours,have been identified in human brain cancers. Here we report that bone morphogenetic proteins (BMPs),amongst which BMP4 elicits the strongest effect,trigger a significant reduction in the stem-like,tumour-initiating precursors of human glioblastomas (GBMs). Transient in vitro exposure to BMP4 abolishes the capacity of transplanted GBM cells to establish intracerebral GBMs. Most importantly,in vivo delivery of BMP4 effectively blocks the tumour growth and associated mortality that occur in 100% of mice after intracerebral grafting of human GBM cells. We demonstrate that BMPs activate their cognate receptors (BMPRs) and trigger the Smad signalling cascade in cells isolated from human glioblastomas (GBMs). This is followed by a reduction in proliferation,and increased expression of markers of neural differentiation,with no effect on cell viability. The concomitant reduction in clonogenic ability,in the size of the CD133+ population and in the growth kinetics of GBM cells indicates that BMP4 reduces the tumour-initiating cell pool of GBMs. These findings show that the BMP-BMPR signalling system--which controls the activity of normal brain stem cells--may also act as a key inhibitory regulator of tumour-initiating,stem-like cells from GBMs and the results also identify BMP4 as a novel,non-cytotoxic therapeutic effector,which may be used to prevent growth and recurrence of GBMs in humans. View Publication

Cancer cells exhibit increased glycolysis for ATP production due,in part,to respiration injury (the Warburg effect). Because ATP generation through glycolysis is less efficient than through mitochondrial respiration,how cancer cells with this metabolic disadvantage can survive the competition with other cells and eventually develop drug resistance is a long-standing paradox. We report that mitochondrial respiration defects lead to activation of the Akt survival pathway through a novel mechanism mediated by NADH. Respiration-deficient cells (rho(-)) harboring mitochondrial DNA deletion exhibit dependency on glycolysis,increased NADH,and activation of Akt,leading to drug resistance and survival advantage in hypoxia. Similarly,chemical inhibition of mitochondrial respiration and hypoxia also activates Akt. The increase in NADH caused by respiratory deficiency inactivates PTEN through a redox modification mechanism,leading to Akt activation. These findings provide a novel mechanistic insight into the Warburg effect and explain how metabolic alteration in cancer cells may gain a survival advantage and withstand therapeutic agents. View Publication

BACKGROUND: Endothelial progenitor cells (EPCs) are present in peripheral blood and can promote postnatal angiogenesis. The number and function of circulating EPCs are altered in diabetics. This study sought to investigate whether the number and functional properties of EPCs from patients with type II diabetes could be improved by pioglitazone. METHODS: For this randomized controlled study,we recruited 36 type II diabetic patients on metformin monotherapy with a glycohemoglobin A1c of textless7%. Patients were separated into pioglitazone (n = 24) and control (n = 12) groups. The number and functional activity of EPCs,and the brachial artery flow-mediated dilation were determined before and after pioglitazone treatment (8 weeks) as an add-on therapy to metformin. In addition,direct effects of pioglitazone on EPCs were also investigated. RESULTS: After pioglitazone treatment,the numbers of circulating EPCs significantly increased (from 0.44% +/- 0.14% to 0.89% +/- 0.29%,P = .01). The migratory response and the adhesive capacity to fibronectin and collagen were improved by 158%,34%,and 83%,respectively (all P textless .05). Treatment with pioglitazone significantly lowered triglyceride,very low density lipoprotein cholesterol,and high-sensitivity C-reactive protein (hsCRP) levels,and increased high-density lipoprotein levels and insulin sensitivity (all P textless .05). The increase in the number of circulating EPCs and the improvement in the migratory response after pioglitazone treatment were independently correlated to the decrease in hsCRP levels (P textless or = .01). The increase in the adhesive capacity was independently correlated to the decreases in very low density lipoprotein cholesterol (P = .01) and hsCRP levels (P = .03). In addition,pioglitazone was also demonstrated to have direct effects on increasing EPC proliferation and colony formation,and attenuating EPC apoptosis (all P textless .05,versus the controls). There were no significant changes in flow-mediated dilation in either group. CONCLUSIONS: Pioglitazone significantly increased the number and improved the functional properties of EPCs in type II diabetic patients through direct effects and/or anti-inflammation and lipid modification effects. View Publication

Human embryonic stem cells (hESCs) provide great opportunities for regenerative medicine,pharmacological and toxicological investigation,and the study of human embryonic development. These applications require proper derivation,maintenance,and extensive characterization of undifferentiated cells before being used for differentiation into cells of interest. Undifferentiated hESCs possess several unique features,including their extensive proliferation capacity in the undifferentiated state,ability to maintain a normal karyotype after long-term culture,expression of markers characteristic of stem cells,high constitutive telomerase activity,and capacity to differentiate into essentially all somatic cell types. This chapter will summarize the current development in culture conditions and provide technical details for the evaluation and characterization of hESCs. View Publication

The essentially infinite expansion potential and pluripotency of human embryonic stem cells (hESCs) makes them attractive for cell-based therapeutics. In contrast to mouse embryonic stem cells (mESCs),hESCs normally undergo high rates of spontaneous apoptosis and differentiation,making them difficult to maintain in culture. Here we demonstrate that p53 protein accumulates in apoptotic hESCs induced by agents that damage DNA. However,despite the accumulation of p53,it nevertheless fails to activate the transcription of its target genes. This inability of p53 to activate its target genes has not been observed in other cell types,including mESCs. We further demonstrate that p53 induces apoptosis of hESCs through a mitochondrial pathway. Reducing p53 expression in hESCs in turn reduces both DNA damage-induced apoptosis as well as spontaneous apoptosis. Reducing p53 expression also reduces spontaneous differentiation and slows the differentiation rate of hESCs. Our studies reveal the important roles of p53 as a critical mediator of human embryonic stem cells survival and differentiation. View Publication

The mTOR complex 2 (mTORC2) containing mTOR and rictor is thought to be rapamycin insensitive and was recently shown to regulate the prosurvival kinase AKT by phosphorylation on Ser473. We investigated the molecular effects of mTOR inhibition by the rapamycin derivatives (RDs) temsirolimus (CCI-779) and everolimus (RAD001) in acute myeloid leukemia (AML) cells. Unexpectedly,RDs not only inhibited the mTOR complex 1 (mTORC1) containing mTOR and raptor with decreased p70S6K,4EPB1 phosphorylation,and GLUT1 mRNA,but also blocked AKT activation via inhibition of mTORC2 formation. This resulted in suppression of phosphorylation of the direct AKT substrate FKHR and decreased transcription of D-cyclins in AML cells. Similar observations were made in samples from patients with hematologic malignancies who received RDs in clinical studies. Our study provides the first evidence that rapamycin derivatives inhibit AKT signaling in primary AML cells both in vitro and in vivo,and supports the therapeutic potential of mTOR inhibition strategies in leukemias. View Publication

BACKGROUND: The purpose of this study was to determine whether murine mesenchymal stem cells (MSC) are able to home to the viable myocardium when injected intravenously and attenuate cardiac dysfunction and ventricular remodeling associated with myocardial infarction. METHODS AND RESULTS: Murine bone marrow cells were negatively selected for lineage markers and adherent MSC differentiated into adipocytes and osteocytes following treatment in culture. Two weeks after coronary occlusion that resulted in a permanent transmural infarct we observed a significant drop in LV systolic pressure,dP/dt(max),dP/dt(min),ESPVR and E(max) and a significant increase in end-diastolic volume in vivo. Femoral vein injection of MSC 1 h after occlusion attenuated the cardiac dysfunction without altering infarct size,or end-diastolic volume. Injected MSC pre-labeled with fluorescent paramagnetic microspheres were observed scattered in noninfarcted regions of the myocardium. Flow cytometry of whole heart digests after intravenous injection of MSC labeled with either fluorescent microspheres or fluorescent PKH26 dye demonstrated that infarcted hearts from mice that received MSC injections contained significantly more cells that integrated into the heart (20x) than those from uninfarcted controls. CONCLUSION: We conclude that intravenously injected MSC were able to home to viable myocardium and preserve systolic function by 2 weeks following ligation. The preserved contractility is likely an MSC-mediated paracrine response since infarct morphology was unchanged and labeled cells observed at two weeks exhibited the same characteristics as the injected MSC. These data underscore the importance of using MSC as a potential therapeutic intervention in preserving cardiac function following infarction. View Publication