技术资料

Human induced pluripotent stem cells (iPSCs) derived cardiomyocytes (iCMCs) would provide an unlimited cell source for regenerative medicine and drug discoveries. The objective of our study is to generate functional cardiomyocytes from human iPSCs and to develop a novel method of measuring contractility of CMCs. In a series of experiments,adult human skin fibroblasts (HSF) and human umbilical vein endothelial cells (HUVECs) were treated with a combination of pluripotent gene DNA and mRNA under specific conditions. The iPSC colonies were identified and differentiated into various cell lineages,including CMCs. The contractile activity of CMCs was measured by a novel method of frame-by-frame cross correlation (particle image velocimetry-PIV) analysis. Our treatment regimen transformed 4% of HSFs into iPSC colonies at passage 0,a significantly improved efficiency compared with use of either DNA or mRNA alone. The iPSCs were capable of differentiating both in vitro and in vivo into endodermal,ectodermal and mesodermal cells,including CMCs with<88% of cells being positive for troponin T (CTT) and Gata4 by flow cytometry. We report a highly efficient combination of DNA and mRNA to generate iPSCs and functional iCMCs from adult human cells. We also report a novel approach to measure contractility of iCMCs. View Publication

Neural progenitor cell (NPC) migration is an essential process for brain development,adult neurogenesis,and neuroregeneration after brain injury. Stromal cell-derived factor-1 (SDF-1,CXCL12) and its traditional receptor CXCR4 are well known to regulate NPC migration. However,the discovery of CXCR7,a newly identified CXCL12 receptor,adds to the dynamics of the existing CXCL12/CXCR4 pair. Antagonists for either CXCR4 or CXCR7 blocked CXCL12-mediated NPC migration in a transwell chemotaxis assay,suggesting that both receptors are required for CXCL12 action. We derived NPC cultures from Cxcr4 knockout (KO) mice and used transwell and stripe assays to determine the cell migration. NPCs derived from Cxcr4 KO mice polarized and migrated in response to CXCL12 gradient,suggesting that CXCR7 could serve as an independent migration receptor. Furthermore,Cxcr4 KO NPCs transplanted into the adult mouse striatum migrated in response to the adjacent injection of CXCL12,an effect that was blocked by a CXCR7 antagonist,suggesting that CXCR7 also mediates NPC migration in vivo. Molecular mechanism studies revealed that CXCR7 interact with Rac1 in the leading edge of the polarized NPCs in the absence of CXCR4. Both CXCR7 and Rac1 are required for extracellular signal-regulated kinases (ERK) 1/2 activation and subsequent NPC migration,indicating that CXCR7 could serve as a functional receptor in CXCL12-mediated NPC migration independent of CXCR4. Together these results reveal an essential role of CXCR7 for CXCL12-mediated NPC migration that will be important to understand neurogenesis during development and in adulthood. View Publication

BACKGROUND Tumor cells present high levels of oxidative stress. Cancer therapeutics exploiting such biochemical changes by increasing reactive oxygen species (ROS) production or decreasing intracellular ROS scavengers could provide a powerful treatment strategy. METHODS To test the effect of our compound,obtusaquinone (OBT),we used several cell viability assays on seven different glioblastoma (GBM) cell lines and primary cells and on 12 different cell lines representing various cancer types in culture as well as on subcutaneous (n = 7 mice per group) and two intracranial GBM (n = 6-8 mice per group) and breast cancer (n = 6 mice per group) tumor models in vivo. Immunoblotting,immunostaining,flow cytometry,and biochemical assays were used to investigate the OBT mechanism of action. Histopathological analysis (n = 2 mice per group) and blood chemistry (n = 2 mice per group) were used to test for any compound-related toxicity. Statistical tests were two-sided. RESULTS OBT induced rapid increase in intracellular ROS levels,downregulation of cellular glutathione levels and increase in its oxidized form,and activation of cellular stress pathways and DNA damage,subsequently leading to apoptosis. Oxidative stress is believed to be the main mechanism through which this compounds targets cancer cells. OBT was well tolerated in mice,slowed tumor growth,and statistically prolonged survival in GBM tumor models. The ratio of median survival in U251 intracranial model in OBT vs control was 1.367 (95% confidence interval [CI] of ratio = 1.031 to 1.367,P = .008). Tumor growth inhibition was also observed in a mouse breast cancer model (average tumor volume per mouse,OBT vs control: 36.3 vs 200.4mm(3),difference = 164.1mm(3),95% CI =72.6 to 255.6mm(3),P = .005). CONCLUSIONS Given its properties and efficacy in cancer killing,our results suggest that OBT is a promising cancer therapeutic. View Publication

Gliomas represent approximately 30% of all central nervous system tumors and 80% of malignant brain tumors. To understand the molecular mechanisms underlying the malignant progression of low-grade gliomas with mutations in IDH1 (encoding isocitrate dehydrogenase 1),we studied paired tumor samples from 41 patients,comparing higher-grade,progressed samples to their lower-grade counterparts. Integrated genomic analyses,including whole-exome sequencing and copy number,gene expression and DNA methylation profiling,demonstrated nonlinear clonal expansion of the original tumors and identified oncogenic pathways driving progression. These include activation of the MYC and RTK-RAS-PI3K pathways and upregulation of the FOXM1- and E2F2-mediated cell cycle transitions,as well as epigenetic silencing of developmental transcription factor genes bound by Polycomb repressive complex 2 in human embryonic stem cells. Our results not only provide mechanistic insight into the genetic and epigenetic mechanisms driving glioma progression but also identify inhibition of the bromodomain and extraterminal (BET) family as a potential therapeutic approach. View Publication

The present studies examined the toxic interaction between the non-coxib celecoxib derivative OSU-03012 and phosphodiesterase 5 (PDE5) inhibitors,and also determined the roles of endoplasmic reticulum stress response regulators in cell survival. PDE5 inhibitors interacted in a greater than additive fashion with OSU-03012 to kill parental glioma and stem-like glioma cells. Knockdown of the endoplasmic reticulum stress response proteins IRE1 or XBP1 enhanced the lethality of OSU-03012,and of [OSU-03012 + PDE5 inhibitor] treatment. Pan-caspase and caspase-9 inhibition did not alter OSU-03012 lethality but did abolish enhanced killing in the absence of IRE1 or XBP1. Expression of the mitochondrial protective protein BCL-XL or the caspase-8 inhibitor c-FLIP-s,or knockdown of death receptor CD95 or the death receptor caspase-8 linker protein FADD,suppressed killing by [OSU-03012 + PDE5 inhibitor] treatment. CD95 activation was blocked by the nitric oxide synthase inhibitor L-NAME. Knockdown of the autophagy regulatory proteins Beclin1 or ATG5 protected the cells from OSU-03012 and from [OSU-03012 + PDE5 inhibitor] toxicity. Knockdown of IRE1 enhanced OSU-03012/[OSU-03012 + PDE5 inhibitor]-induced JNK activation,and inhibition of JNK suppressed the elevated killing caused by IRE1 knockdown. Knockdown of CD95 blunted JNK activation. Collectively,our data demonstrate that PDE5 inhibitors recruit death receptor signaling to enhance OSU-03012 toxicity in glioblastoma multiforme (GBM) cells. View Publication

Tumor-initiating cells are a subpopulation in aggressive cancers that exhibit traits shared with stem cells,including the ability to self-renew and differentiate,commonly referred to as stemness. In addition,such cells are resistant to chemo- and radiation therapy posing a therapeutic challenge. To uncover stemness-associated functions in glioma-initiating cells (GICs),transcriptome profiles were compared to neural stem cells (NSCs) and gene ontology analysis identified an enrichment of Ca2+ signaling genes in NSCs and the more stem-like (NSC-proximal) GICs. Functional analysis in a set of different GIC lines regarding sensitivity to disturbed homeostasis using A23187 and Thapsigargin,revealed that NSC-proximal GICs were more sensitive,corroborating the transcriptome data. Furthermore,Ca2+ drug sensitivity was reduced in GICs after differentiation,with most potent effect in the NSC-proximal GIC,supporting a stemness-associated Ca2+ sensitivity. NSCs and the NSC-proximal GIC line expressed a larger number of ion channels permeable to potassium,sodium and Ca2+. Conversely,a higher number of and higher expression levels of Ca2+ binding genes that may buffer Ca2+,were expressed in NSC-distal GICs. In particular,expression of the AMPA glutamate receptor subunit GRIA1,was found to associate with Ca2+ sensitive NSC-proximal GICs,and decreased as GICs differentiated along with reduced Ca2+ drug sensitivity. The correlation between high expression of Ca2+ channels (such as GRIA1) and sensitivity to Ca2+ drugs was confirmed in an additional nine novel GIC lines. Calcium drug sensitivity also correlated with expression of the NSC markers nestin (NES) and FABP7 (BLBP,brain lipid-binding protein) in this extended analysis. In summary,NSC-associated NES+/FABP7+/GRIA1+ GICs were selectively sensitive to disturbances in Ca2+ homeostasis,providing a potential target mechanism for eradication of an immature population of malignant cells. View Publication

Neurogenesis decreases during aging causing a progressive cognitive decline but it is still controversial whether proliferation defects in neurogenic niches result from a loss of neural stem cells or from an impairment of their progression through the cell cycle. Using an accurate fluorescence-activated cell sorting technique,we show that the pool of neural stem cells is maintained in the subventricular zone of middle-aged mice while they have a reduced proliferative potential eventually leading to the subsequent decrease of their progeny. In addition,we demonstrate that the G1 phase is lengthened during aging specifically in activated stem cells,but not in transit-amplifying cells,and directly impacts on neurogenesis. Finally,we report that inhibition of TGFβ signaling restores cell cycle progression defects in stem cells. Our data highlight the significance of cell cycle dysregulation in stem cells in the aged brain and provide an attractive foundation for the development of anti-TGFβ regenerative therapies based on stimulating endogenous neural stem cells. View Publication

Glioblastoma (GBM) is associated with poor prognosis despite aggressive surgical resection,chemotherapy,and radiation therapy. Unfortunately,this standard therapy does not target glioma cancer stem cells (GCSCs),a subpopulation of GBM cells that can give rise to recurrent tumors. GBMs express human cytomegalovirus (HCMV) proteins,and previously we found that the level of expression of HCMV immediate-early (IE) protein in GBMs is a prognostic factor for poor patient survival. In this study,we investigated the relation between HCMV infection of GBM cells and the presence of GCSCs. Primary GBMs were characterized by their expression of HCMV-IE and GCSCs marker CD133 and by patient survival. The extent to which HCMV infection of primary GBM cells induced a GCSC phenotype was evaluated in vitro. In primary GBMs,a large fraction of CD133-positive cells expressed HCMV-IE,and higher co-expression of these two proteins predicted poor patient survival. Infection of GBM cells with HCMV led to upregulation of CD133 and other GSCS markers (Notch1,Sox2,Oct4,Nestin). HCMV infection also promoted the growth of GBM cells as neurospheres,a behavior typically displayed by GCSCs,and this phenotype was prevented by either chemical inhibition of the Notch1 pathway or by treatment with the anti-viral drug ganciclovir. GBM cells that maintained expression of HCMV-IE failed to differentiate into neuronal or astrocytic phenotypes. Our findings imply that HCMV infection induces phenotypic plasticity of GBM cells to promote GCSC features and may thereby increase the aggressiveness of this tumor. View Publication

The role of tumor stem cells in benign tumors such as pituitary adenomas remains unclear. In this study,we investigated whether the cells within pituitary adenomas that spontaneously develop in Rb+/- mice are hierarchically distributed with a subset being responsible for tumor growth. Cells derived directly from such tumors grew as spheres in serum-free culture medium supplemented with epidermal growth factor and basic fibroblast growth factor. Some cells within growing pituitary tumor spheres (PTS) expressed common stem cell markers (Sca1,Sox2,Nestin,and CD133),but were devoid of hormone-positive differentiated cells. Under subsequent differentiating conditions (matrigel-coated growth surface),PTS expressed all six pituitary hormones. We next searched for specific markers of the stem cell population and isolated a Sca1(+) cell population that showed increased sphere formation potential,lower mRNA hormone expression,higher expression of stem cell markers (Notch1,Sox2,and Nestin),and increased proliferation rates. When transplanted into non-obese diabetic-severe combined immunodeficiency gamma mice brains,Sca1(+) pituitary tumor cells exhibited higher rates of tumor formation (brain tumors observed in 11/11 (100%) vs 7/12 (54%) of mice transplanted with Sca1(+) and Sca1(-) cells respectively). Magnetic resonance imaging and histological analysis of brain tumors showed that tumors derived from Sca1(+) pituitary tumor cells were also larger and plurihormonal. Our findings show that Sca1(+) cells derived from benign pituitary tumors exhibit an undifferentiated expression profile and tumor-proliferative advantages,and we propose that they could represent putative pituitary tumor stem/progenitor cells. View Publication

BACKGROUND AND PURPOSE HIV-1 glycoprotein Gp120 induces apoptosis in rodent and human neurons in vitro and in vivo.HIV-1/Gp120 is involved in the pathogenesis of HIV-associated dementia (HAD) and inhibits proliferation of adult neural progenitor cells (NPCs) in glial fibrillary acidic protein (GFAP)/Gp120 transgenic (Tg) mice. As cannabinoids exert neuroprotective effects in several model systems,we examined the protective effects of the CB receptor agonist AM1241 on Gp120-mediated insults on neurogenesis. EXPERIMENTAL APPROACH We assessed the effects of AM1241 on survival and apoptosis in cultures of human and murine NPCs with immunohistochemical and TUNEL techniques. Neurogenesis in the hippocampus of GFAP/Gp120 transgenic mice in vivo was also assessed by immunohistochemistry. KEY RESULTS AM1241 inhibited in vitroGp120-mediated neurotoxicity and apoptosis of primary human and murine NPCs and increased their survival. AM1241 also promoted differentiation of NPCs to neuronal cells. While GFAP/Gp120 Tg mice exhibited impaired neurogenesis,as indicated by reduction in BrdU cells and doublecortin (DCX) cells,and a decrease in cells with proliferating cell nuclear antigen (PCNA),administration of AM1241 to GFAP/Gp120 Tg mice resulted in enhanced in vivo neurogenesis in the hippocampus as indicated by increase in neuroblasts,neuronal cells,BrdU cells and PCNA cells. Astrogliosis and gliogenesis were decreased in GFAP/Gp120 Tg mice treated with AM1241,compared with those treated with vehicle. CONCLUSIONS AND IMPLICATIONS The CB receptor agonist rescued impaired neurogenesis caused by HIV-1/Gp120 insult. Thus,CB receptor agonists may act as neuroprotective agents,restoring impaired neurogenesis in patients with HAD. View Publication