技术资料

Thrombocytopenia is a critical problem that occurs in many hematologic diseases,as well as after cancer therapy and radiation exposure. Platelet transfusion is the most commonly used therapy but has limitations of alloimmunization,availability,and expense. Thus,the development of safe,small,molecules to enhance platelet production would be advantageous for the treatment of thrombocytopenia. Herein,we report that an important lipid mediator and a peroxisome proliferator-activated receptor gamma (PPARgamma) ligand called 15-deoxy-Delta(12,14) prostaglandin J(2) (15d-PGJ(2)),increases Meg-01 maturation and platelet production. 15d-PGJ(2) also promotes platelet formation from culture-derived mouse and human megakaryocytes and accelerates platelet recovery after in vivo radiation-induced bone marrow injury. Interestingly,the platelet-enhancing effects of 15d-PGJ(2) in Meg-01 cells are independent of PPARgamma,but dependent on reactive oxygen species (ROS) accumulation; treatment with antioxidants such as glutathione ethyl ester (GSH-EE); or N-acetylcysteine (NAC) attenuate 15d-PGJ(2)-induced platelet production. Collectively,these data support the concept that megakaryocyte redox status plays an important role in platelet generation and that small electrophilic molecules may have clinical efficacy for improving platelet numbers in thrombocytopenic patients. View Publication

BACKGROUND: Human embryonic stem cells (hESCs) are a potentially inexhaustible source of cells for replacement therapy. However,successful preclinical and clinical progress requires efficient and controlled differentiation towards the specific differentiated cell fate. METHODS: We previously developed a strategy to generate blast cells (BCs) from hESCs that were capable of differentiating into vascular structures as well as into all hematopoietic cell lineages. Although the BCs were shown to repair damaged vasculature in multiple animal models,the large-scale generation of cells under these conditions was challenging. Here we report a simpler and more efficient method for robust generation of hemangioblastic progenitors. RESULTS: In addition to eliminating several expensive factors that are unnecessary,we demonstrate that bone morphogenetic protein (BMP)-4 and VEGF are necessary and sufficient to induce hemangioblastic commitment and development from hESCs during early stages of differentiation. BMP-4 and VEGF significantly upregulate T-brachyury,KDR,CD31 and Lmo2 gene expression,while dramatically downregulating Oct-4 expression. The addition of basic FGF during growth and expansion was found to further enhance BC development,consistently generating approximately 1 x 10(8) BCs from one six well plate of hESCs. CONCLUSION: This new method represents a significantly improved system for generating hemangioblasts from hESCs,and although simplified,results in an eightfold increase in cell yield. View Publication

Mesenchymal stem cells (MSC) can differentiate into osteoblasts,adipocytes,chondrocytes and myoblasts. It has been suggested that a reciprocal relationship exists between the differentiation of MSC into osteoblasts and adipocytes. Peroxisome proliferator-activated receptor gamma2 (PPARgamma2) is a key element for the differentiation into adipocytes. Activation of the nuclear protein deacetylase Sirt1 has recently been shown to decrease adipocyte development from preadipocytes via inhibition of PPARgamma2. In vitro,MSC differentiate to osteoblasts when exposed to bone-inducing medium. However,adipocytes are also developed. In the present study we have targeted Sirt1 to control adipocyte development during differentiation of MSC into osteoblasts. The finding that resveratrol and isonicotinamide markedly inhibited adipocyte and promoted osteoblast differentiation demonstrates an interesting alternative to PPARgamma antagonists. These results are important for the evolving field of cell-based tissue engineering,but may also be relevant in the search for new treatments of osteoporosis. View Publication

Given their self-renewing and pluripotent capabilities,human embryonic stem cells (hESCs) are well poised as a cellular source for tissue regeneration therapy. However,the host immune response against transplanted hESCs is not well characterized. In fact,controversy remains as to whether hESCs have immune-privileged properties. To address this issue,we used in vivo bioluminescent imaging to track the fate of transplanted hESCs stably transduced with a double-fusion reporter gene consisting of firefly luciferase and enhanced GFP. We show that survival after transplant is significantly limited in immunocompetent as opposed to immunodeficient mice. Repeated transplantation of hESCs into immunocompetent hosts results in accelerated hESC death,suggesting an adaptive donor-specific immune response. Our data demonstrate that transplanted hESCs trigger robust cellular and humoral immune responses,resulting in intragraft infiltration of inflammatory cells and subsequent hESC rejection. Moreover,we have found CD4(+) T cells to be an important modulator of hESC immune-mediated rejection. Finally,we show that immunosuppressive drug regimens can mitigate the anti-hESC immune response and that a regimen of combined tacrolimus and sirolimus therapies significantly prolongs survival of hESCs for up to 28 days. Taken together,these data suggest that hESCs are immunogenic,trigger both cellular and humoral-mediated pathways,and,as a result,are rapidly rejected in xenogeneic hosts. This process can be mitigated by a combined immunosuppressive regimen as assessed by molecular imaging approaches. View Publication

Some cases of pre-B cell acute lymphoblastic leukemia (pre-B-ALL) are caused by the Philadelphia (Ph) chromosome-encoded BCR-ABL oncogene,and these tend to have a poor prognosis. Inhibitors of the PI3K/AKT pathway reduce BCR-ABL-mediated transformation in vitro; however,the specific PI3K isoforms involved are poorly defined. Using a murine model of Ph+ pre-B-ALL,we found that deletion of both Pik3r1 and Pik3r2,genes encoding class IA PI3K regulatory isoforms,severely impaired transformation. BCR-ABL-dependent pre/pro-B cell lines could be established at low frequency from progenitors that lacked these genes,but the cells were smaller,proliferated more slowly,and failed to cause leukemia in vivo. These cell lines displayed nearly undetectable PI3K signaling function and were resistant to the PI3K inhibitor wortmannin. However,they maintained activation of mammalian target of rapamycin (mTOR) and were more sensitive to rapamycin. Treatment with rapamycin caused feedback activation of AKT in WT cell lines but not PI3K-deficient lines. A dual inhibitor of PI3K and mTOR,PI-103,was more effective than rapamycin at suppressing proliferation of mouse pre-B-ALL and human CD19+CD34+)Ph+ ALL leukemia cells treated with the ABL kinase inhibitor imatinib. Our findings provide mechanistic insights into PI3K dependency in oncogenic networks and provide a rationale for targeting class IA PI3K,alone or together with mTOR,in the treatment of Ph+ ALL. View Publication

Adult hippocampal neurogenesis is stimulated by chronic administration of antidepressants (ADs) and by voluntary exercise. Neural progenitor cells (NPCs) in the dentate gyrus (DG) that are capable of continuous proliferation and neuronal differentiation are the source of such structural plasticity. Here we report that mice lacking the receptor tyrosine kinase TrkB in hippocampal NPCs have impaired proliferation and neurogenesis. When exposed to chronic ADs or wheel-running,no increase in proliferation or neurogenesis is observed. Ablation of TrkB also renders these mice behaviorally insensitive to antidepressive treatment in depression- and anxiety-like paradigms. In contrast,mice lacking TrkB only in differentiated DG neurons display typical neurogenesis and respond normally to chronic ADs. Thus,our data establish an essential cell-autonomous role for TrkB in regulating hippocampal neurogenesis and behavioral sensitivity to antidepressive treatments,and support the notion that impairment of the neurogenic niche is an etiological factor for refractory responses to an antidepressive regimen. View Publication

This study was to evaluate the enhancement value of chloroquine (CQ) in cancer cell killing when used in combination with Akt inhibitors. The results showed that the combination of CQ and Akt inhibitors is much more effective than either one alone. Importantly,the CQ-mediated chemosensitization of cell killing effects by Akt inhibitors is cancer specific. In particular,when combined with 10 microM CQ,1,3-dihydro-1-(1-((4-(6-phenyl-1H-imidazo[4,5-g]quinoxalin-7-yl)phenyl)methyl)-4-piperidinyl)-2H-benzimidazol-2-one (an Akt1 and 2 inhibitor; compound 8) killed cancer cells 10-120 times more effectively than normal cells. Thus,CQ is a very effective and cancer-specific chemosensitizer when used in combination with Akt inhibitors. View Publication

Medulloblastoma is the most common malignant brain tumor in children,but the cells from which it arises remain unclear. Here we examine the origin of medulloblastoma resulting from mutations in the Sonic hedgehog (Shh) pathway. We show that activation of Shh signaling in neuronal progenitors causes medulloblastoma by 3 months of age. Shh pathway activation in stem cells promotes stem cell proliferation but only causes tumors after commitment to-and expansion of-the neuronal lineage. Notably,tumors initiated in stem cells develop more rapidly than those initiated in progenitors,with all animals succumbing by 3-4 weeks. These studies suggest that medulloblastoma can be initiated in progenitors or stem cells but that Shh-induced tumorigenesis is associated with neuronal lineage commitment. View Publication

The mutagenic enzyme activation-induced cytidine deaminase (AID) is required for immunoglobulin class switch recombination (CSR) and somatic hypermutation (SHM) in germinal center (GC) B cells. Deregulated expression of AID is associated with various B-cell malignancies and,currently,it remains unclear how AID activity is extinguished to avoid illegitimate mutations. AID has also been shown to be alternatively spliced in malignant B cells,and there is limited evidence that this also occurs in normal blood B cells. The functional significance of these splice variants remains unknown. Here we show that normal GC human B cells and blood memory B cells similarly express AID splice variants and show for the first time that AID splicing variants are singly expressed in individual normal B cells as well as malignant B cells from chronic lymphocytic leukemia patients. We further demonstrate that the alternative AID splice variants display different activities ranging from inactivation of CSR to inactivation or heightened SHM activity. Our data therefore suggest that CSR and SHM are differentially switched off by varying the expression of splicing products of AID at the individual cell level. Most importantly,our findings suggest a novel tumor suppression mechanism by which unnecessary AID mutagenic activities are promptly contained for GC B cells. View Publication

BACKGROUND Pluripotent embryonic stem (ES) cells,which have the capacity to give rise to all tissue types in the body,show great promise as a versatile source of cells for regenerative therapy. However,the basic mechanisms of lineage specification of pluripotent stem cells are largely unknown,and generating sufficient quantities of desired cell types remains a formidable challenge. Small molecules,particularly those that modulate key developmental pathways like the bone morphogenetic protein (BMP) signaling cascade,hold promise as tools to study in vitro lineage specification and to direct differentiation of stem cells toward particular cell types. METHODOLOGY/ PRINCIPAL FINDINGS We describe the use of dorsomorphin,a selective small molecule inhibitor of BMP signaling,to induce myocardial differentiation in mouse ES cells. Cardiac induction is very robust,increasing the yield of spontaneously beating cardiomyocytes by at least 20 fold. Dorsomorphin,unlike the endogenous BMP antagonist Noggin,robustly induces cardiomyogenesis when treatment is limited to the initial 24-hours of ES cell differentiation. Quantitative-PCR analyses of differentiating ES cells indicate that pharmacological inhibition of BMP signaling during the early critical stage promotes the development of the cardiomyocyte lineage,but reduces the differentiation of endothelial,smooth muscle,and hematopoietic cells. CONCLUSIONS/ SIGNIFICANCE Administration of a selective small molecule BMP inhibitor during the initial stages of ES cell differentiation substantially promotes the differentiation of primitive pluripotent cells toward the cardiomyocytic lineage,apparently at the expense of other mesodermal lineages. Small molecule modulators of developmental pathways like dorsomorphin could become versatile pharmacological tools for stem cell research and regenerative medicine. View Publication

Self-renewal of human embryonic stem cells (ESCs) is promoted by FGF and TGFbeta/Activin signaling,and differentiation is promoted by BMP signaling,but how these signals regulate genes critical to the maintenance of pluripotency has been unclear. Using a defined medium,we show here that both TGFbeta and FGF signals synergize to inhibit BMP signaling; sustain expression of pluripotency-associated genes such as NANOG,OCT4,and SOX2; and promote long-term undifferentiated proliferation of human ESCs. We also show that both TGFbeta- and BMP-responsive SMADs can bind with the NANOG proximal promoter. NANOG promoter activity is enhanced by TGFbeta/Activin and FGF signaling and is decreased by BMP signaling. Mutation of putative SMAD binding elements reduces NANOG promoter activity to basal levels and makes NANOG unresponsive to BMP and TGFbeta signaling. These results suggest that direct binding of TGFbeta/Activin-responsive SMADs to the NANOG promoter plays an essential role in sustaining human ESC self-renewal. View Publication

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