技术资料

The prion protein (PrP) is highly conserved and ubiquitously expressed,suggesting that it plays an important physiological function. However,despite decades of investigation,this role remains elusive. Here,by using animal and cellular models,we unveil a key role of PrP in the DNA damage response. Exposure of neurons to a genotoxic stress activates PRNP transcription leading to an increased amount of PrP in the nucleus where it interacts with APE1,the major mammalian endonuclease essential for base excision repair,and stimulates its activity. Preventing the induction of PRNP results in accumulation of abasic sites in DNA and impairs cell survival after genotoxic treatment. Brains from Prnp(-/-) mice display a reduced APE1 activity and a defect in the repair of induced DNA damage in vivo. Thus,PrP is required to maintain genomic stability in response to genotoxic stresses. View Publication

The ability of high-risk neuroblastoma to survive unfavorable growth conditions and multimodal therapy has produced an elusive childhood cancer with remarkably poor prognosis. A novel phenomenon enabling neuroblastoma to survive selection pressure is its capacity for reversible adaptive plasticity. This plasticity allows cells to transition between highly proliferative anchorage dependent (AD) and slow growing,anoikis-resistant anchorage independent (AI) phenotypes. Both phenotypes are present in established mouse and human tumors. The differential gene expression profile of the two cellular phenotypes in the mouse Neuro2a cell line delineated pathways of proliferation in AD cells or tyrosine kinase activation/ apoptosis inhibition in AI cells. A 20 fold overexpression of inhibitor of differentiation 2 (Id2) was identified in AD cells while up-regulation of genes involved in anoikis resistance like PI3K/Akt,Erk,Bcl2 and integrins was observed in AI cells. Similarly,differential expression of Id2 and other genes of interest were also observed in the AD and AI phenotypes of human neuroblastoma cell lines,SK-N-SH and IMR-32; as well as in primary human tumor specimens. Forced down-regulation of Id2 in AD cells or overexpression in AI cells induced the cells to gain characteristics of the other phenotype. Id2 binds both TGFβ and Smad2/3 and appears critical for maintaining the proliferative phenotype at least partially through negative regulation of the TGFβ/Smad pathway. Simultaneously targeting the differential molecular pathways governing reversible adaptive plasticity resulted in 50% cure of microscopic disease and delayed tumor growth in established mouse neuroblastoma tumors. We present a mechanism that accounts for reversible adaptive plasticity and a molecular basis for combined targeted therapies in neuroblastoma. View Publication

Glioblastoma (GBM) is a common and malignant tumor with a poor prognosis. Glioblastoma stem cells (GSCs) have been reported to be involved in tumorigenesis,tumor maintenance and therapeutic resistance. Thus,to discover novel candidate therapeutic drugs for anti-GBM and anti-GSCs is an urgent need. We hypothesized that if treatment with a drug could reverse,at least in part,the gene expression signature of GBM and GSCs,this drug may have the potential to inhibit pathways essential in the formation of GBM and thereby treat GBM. Here,we collected 356 GBM gene signatures from public databases and queried the Connectivity Map. We systematically evaluated the in vitro antitumor effects of 79 drugs in GBM cell lines. Of the drugs screened,thioridazine was selected for further characterization because it has potent anti-GBM and anti-GSCs properties. When investigating the mechanisms underlying the cytocidal effects of thioridazine,we found that thioridazine induces autophagy in GBM cell lines,and upregulates AMPK activity. Moreover,LC3-II was upregulated in U87MG sphere cells treated with thioridazine. In addition,thioridazine suppressed GBM tumorigenesis and induced autophagy in vivo. We not only repurposed the antipsychotic drug thioridazine as a potent anti-GBM and anti-GSCs agent,but also provided a new strategy to search for drugs with anticancer and anticancer stem cell properties. View Publication

Stem cell-based therapies have been reported in protecting cerebral infarction-induced neuronal dysfunction and death. However,most studies used rat/mouse neuron as model cell when treated with stem cell or exosomes. Whether these findings can be translated from rodent to humans has been in doubt. Here,we used human embryonic stem cell-derived neurons to detect the protective potential of exosomes against ischemia. Neurons were treated with in vitro oxygen-glucose deprivation (OGD) for 1 h. For treatment group,different exosomes were derived from neuron,embryonic stem cell,neural progenitor cell and astrocyte differentiated from H9 human embryonic stem cell and added to culture medium 30 min after OGD (100 μg/mL). Western blotting was performed 12 h after OGD,while cell counting and electrophysiological recording were performed 48 h after OGD. We found that these exosomes attenuated OGD-induced neuronal death,Mammalian target of rapamycin (mTOR),pro-inflammatory and apoptotic signaling pathway changes,as well as basal spontaneous synaptic transmission inhibition in varying degrees. The results implicate the protective effect of exosomes on OGD-induced neuronal death and dysfunction in human embryonic stem cell-derived neurons,potentially through their modulation on mTOR,pro-inflammatory and apoptotic signaling pathways. View Publication

BACKGROUND The molecular mechanisms that determine social behavior are poorly understood. Pheromones play a critical role in social recognition in most animals,including mice,but how these are converted into behavioral responses is largely unknown. Here,we report that the absence of the small GTPase M-Ras affects social behavior in mice. RESULTS In their interactions with other males,Mras(-/-) males exhibited high levels of territorial aggression and social investigations,and increased fear-related behavior. They also showed increased mating behavior with females. Curiously,increased aggression and mating behaviors were only observed when Mras(-/-) males were paired with Mras(-/-) partners,but were significantly reduced when paired with wild-type (WT) mice. Since mice use pheromonal cues to identify other individuals,we explored the possibility that pheromone detection may be altered in Mras(-/-) mice. Unlike WT mice,Mras(-/-) did not show a preference for exploring unfamiliar urinary pheromones or unfamiliar isogenic mice. Although this could indicate that vomeronasal function and/or olfactory learning may be compromised in Mras(-/-) mice,these observations were not fully consistent with the differential behavioral responses to WT and Mras(-/-) interaction partners by Mras(-/-) males. In addition,induction of c-fos upon pheromone exposure or in response to mating was similar in WT and Mras (-/-) mice,as was the ex vivo expansion of neural progenitors with EGF. This indicated that acute pheromone detection and processing was likely intact. However,urinary metabolite profiles differed between Mras(-/-) and WT males. CONCLUSIONS The changes in behaviors displayed by Mras(-/-) mice are likely due to a complex combination of factors that may include an inherent predisposition to increased aggression and sexual behavior,and the production of distinct pheromones that could override the preference for unfamiliar social odors. Olfactory and/or social learning processes may thus be compromised in Mras(-/-) mice. View Publication

Glioblastomas (GBMs) are highly aggressive,invasive brain tumors with bad prognosis and unmet medical need. These tumors are heterogeneous being constituted by a variety of cells in different states of differentiation. Among these,cells endowed with stem properties,tumor initiating/propagating properties and particularly resistant to chemo- and radiotherapies are designed as the real culprits for tumor maintenance and relapse after treatment. These cells,termed cancer stem-like cells,have been designed as prominent targets for new and more efficient cancer therapies. G-protein coupled receptors (GPCRs),a family of membrane receptors,play a prominent role in cell signaling,cell communication and crosstalk with the microenvironment. Their role in cancer has been highlighted but remains largely unexplored. Here,we report a descriptive study of the differential expression of the endo-GPCR repertoire in human glioblastoma cancer stem-like cells (GSCs),U-87 MG cells,human astrocytes and fetal neural stem cells (f-NSCs). The endo-GPCR transcriptome has been studied using Taqman Low Density Arrays. Of the 356 GPCRs investigated,138 were retained for comparative studies between the different cell types. At the transcriptomic level,eight GPCRs were specifically expressed/overexpressed in GSCs. Seventeen GPCRs appeared specifically expressed in cells with stem properties (GSCs and f-NSCs). Results of GPCR expression at the protein level using mass spectrometry and proteomic analysis are also presented. The comparative GPCR expression study presented here gives clues for new pathways specifically used by GSCs and unveils novel potential therapeutic targets. View Publication

Fetal-onset hydrocephalus affects 1 to 3 per 1,000 live births. It is not only a disorder of cerebrospinal fluid dynamics but also a brain disorder that corrective surgery does not ameliorate. We hypothesized that cell junction abnormalities of neural stem cells (NSCs) lead to the inseparable phenomena of fetal-onset hydrocephalus and abnormal neurogenesis. We used bromodeoxyuridine labeling,immunocytochemistry,electron microscopy,and cell culture to study the telencephalon of hydrocephalic HTx rats and correlated our findings with those in human hydrocephalic and nonhydrocephalic human fetal brains (n = 12 each). Our results suggest that abnormal expression of the intercellular junction proteins N-cadherin and connexin-43 in NSC leads to 1) disruption of the ventricular and subventricular zones,loss of NSCs and neural progenitor cells; and 2) abnormalities in neurogenesis such as periventricular heterotopias and abnormal neuroblast migration. In HTx rats,the disrupted NSC and progenitor cells are shed into the cerebrospinal fluid and can be grown into neurospheres that display intercellular junction abnormalities similar to those of NSC of the disrupted ventricular zone; nevertheless,they maintain their potential for differentiating into neurons and glia. These NSCs can be used to investigate cellular and molecular mechanisms underlying this condition,thereby opening the avenue for stem cell therapy. View Publication

BACKGROUND Transplanting autologous patient-derived enteric neuronal stem/progenitor cells (ENSCs) is an innovative approach to replacing missing enteric neurons in patients with Hirschsprung disease (HSCR). Using autologous cells eliminates immunologic and ethical concerns raised by other cell sources. However,whether postnatal aganglionic bowel is permissive for transplanted ENSCs and whether ENSCs from HSCR patients can be successfully isolated,cultured,and transplanted in vivo remains unknown. METHODS ENSCs isolated from the ganglionic intestine of Ednrb(-/-) mice (HSCR-ENSCs) were characterized immunohistochemically and evaluated for their capacity to proliferate and differentiate in vitro. Fluorescently labeled ENSCs were co-cultured ex vivo with aganglionic Ednrb(-/-) colon. For in vivo transplantation,HSCR-ENSCs were labeled with lentivirus expressing green fluorescent protein (GFP) and implanted into aganglionic embryonic chick gut in ovo and postnatal aganglionic Ednrb(-/-) rectum in vivo. KEY RESULTS HSCR-ENSCs maintain normal capacity self-renewal and neuronal differentiation. Moreover,the Ednrb(-/-) aganglionic environment is permissive to engraftment by wild-type ENSCs ex vivo and supports migratrion and neuroglial differentiation of these cells following transplantation in vivo. Lentiviral GFP-labeled HSCR-ENSCs populated embryonic chick hindgut and postnatal colon of Ednrb(-/-) HSCR,with cells populating the intermuscular layer and forming enteric neurons and glia. CONCLUSIONS & INFERENCES ENSCs can be isolated and cultured from mice with HSCR,and transplanted into the aganglionic bowel of HSCR littermates to generate enteric neuronal networks. These results in an isogenic model establish the potential of using autologous-derived stem cells to treat HSCR and other intestinal neuropathies. View Publication

The embryonic neuroepithelium gives rise to the entire central nervous system in vivo,making it an important tissue for developmental studies and a prospective cell source for regenerative applications. Current protocols for deriving homogenous neuroepithelial cultures from human pluripotent stem cells (hPSCs) consist of either embryoid body-mediated neuralization followed by a manual isolation step or adherent differentiation using small molecule inhibitors. Here,we report that hPSCs maintained under chemically defined,feeder-independent,and xeno-free conditions can be directly differentiated into pure neuroepithelial cultures ([mt]90% Pax6(+)/N-cadherin(+) with widespread rosette formation) within 6 days under adherent conditions,without small molecule inhibitors,and using only minimalistic medium consisting of Dulbecco's modified Eagle's medium/F-12,sodium bicarbonate,selenium,ascorbic acid,transferrin,and insulin (i.e.,E6 medium). Furthermore,we provide evidence that the defined culture conditions enable this high level of neural conversion in contrast to hPSCs maintained on mouse embryonic fibroblasts (MEFs). In addition,hPSCs previously maintained on MEFs could be rapidly converted to a neural compliant state upon transfer to these defined conditions while still maintaining their ability to generate all three germ layers. Overall,this fully defined and scalable protocol should be broadly useful for generating therapeutic neural cells for regenerative applications. View Publication

Blood-brain barrier (BBB) models are often used to investigate BBB function and screen brain-penetrating therapeutics,but it has been difficult to construct a human model that possesses an optimal BBB phenotype and is readily scalable. To address this challenge,we developed a human in vitro BBB model comprising brain microvascular endothelial cells (BMECs),pericytes,astrocytes and neurons derived from renewable cell sources. First,retinoic acid (RA) was used to substantially enhance BBB phenotypes in human pluripotent stem cell (hPSC)-derived BMECs,particularly through adherens junction,tight junction,and multidrug resistance protein regulation. RA-treated hPSC-derived BMECs were subsequently co-cultured with primary human brain pericytes and human astrocytes and neurons derived from human neural progenitor cells (NPCs) to yield a fully human BBB model that possessed significant tightness as measured by transendothelial electrical resistance (˜5,000 $\$(2)). Overall,this scalable human BBB model may enable a wide range of neuroscience studies. View Publication

Schizophrenia has been considered a devastating clinical syndrome rather than a single disease. Nevertheless,the mechanisms behind the onset of schizophrenia have been only partially elucidated. Several studies propose that levels of trace elements are abnormal in schizophrenia; however,conflicting data generated from different biological sources prevent conclusions being drawn. In this work,we used synchrotron radiation X-ray microfluorescence spectroscopy to compare trace element levels in neural progenitor cells (NPCs) derived from two clones of induced pluripotent stem cell lines of a clozapine-resistant schizophrenic patient and two controls. Our data reveal the presence of elevated levels of potassium and zinc in schizophrenic NPCs. Neural cells treated with valproate,an adjunctive medication for schizophrenia,brought potassium and zinc content back to control levels. These results expand the understanding of atomic element imbalance related to schizophrenia and may provide novel insights for the screening of drugs to treat mental disorders. ?? 2014 Elsevier B.V. View Publication

Efficient derivation of large-scale motor neurons (MNs) from human pluripotent stem cells is central to the understanding of MN development,modelling of MN disorders in vitro and development of cell-replacement therapies. Here we develop a method for rapid (20 days) and highly efficient (˜70%) differentiation of mature and functional MNs from human pluripotent stem cells by tightly modulating neural patterning temporally at a previously undefined primitive neural progenitor stage. This method also allows high-yield (textgreater250%) MN production in chemically defined adherent cultures. Furthermore,we show that Islet-1 is essential for formation of mature and functional human MNs,but,unlike its mouse counterpart,does not regulate cell survival or suppress the V2a interneuron fate. Together,our discoveries improve the strategy for MN derivation,advance our understanding of human neural specification and MN development,and provide invaluable tools for human developmental studies,drug discovery and regenerative medicine. View Publication