参考文献

Gonadotropin-releasing hormone (GnRH) is a hypothalamic decapeptide essential for fertility in vertebrates. Human male patients lacking GnRH and treated with hormone therapy can remain fertile after cessation of treatment suggesting that new GnRH neurons can be generated during adult life. We used zebrafish to investigate the neurogenic potential of the adult hypothalamus. Previously we have characterized the development of GnRH cells in the zebrafish linking genetic pathways to the differentiation of neuromodulatory and endocrine GnRH cells in specific regions of the brain. Here,we developed a new method to obtain neural progenitors from the adult hypothalamus in vitro. Using this system,we show that neurospheres derived from the adult hypothalamus can be maintained in culture and subsequently differentiate glia and neurons. Importantly,the adult derived progenitors differentiate into neurons containing GnRH and the number of cells is increased through exposure to either testosterone or GnRH,hormones used in therapeutic treatment in humans. Finally,we show in vivo that a neurogenic niche in the hypothalamus contains GnRH positive neurons. Thus,we demonstrated for the first time that neurospheres can be derived from the hypothalamus of the adult zebrafish and that these neural progenitors are capable of producing GnRH containing neurons. View Publication

BACKGROUND In glioblastoma (GBM),Id1 serves as a functional marker for self-renewing cancer stem-like cells. We investigated the mechanism by which cyclooxygenase-2 (Cox-2)-derived prostaglandin E2 (PGE2) induces Id1 and increases GBM self-renewal and radiation resistance. METHODS Mouse and human GBM cells were stimulated with dimethyl-PGE2 (dmPGE2),a stabilized form of PGE2,to test for Id1 induction. To elucidate the signal transduction pathway governing the increase in Id1,a combination of short interfering RNA knockdown and small molecule inhibitors and activators of PGE2 signaling were used. Western blotting,quantitative real-time (qRT)-PCR,and chromatin immunoprecipitation assays were employed. Sphere formation and radiation resistance were measured in cultured primary cells. Immunohistochemical analyses were carried out to evaluate the Cox-2-Id1 axis in experimental GBM. RESULTS In GBM cells,dmPGE2 stimulates the EP4 receptor leading to activation of ERK1/2 MAPK. This leads,in turn,to upregulation of the early growth response1 (Egr1) transcription factor and enhanced Id1 expression. Activation of this pathway increases self-renewal capacity and resistance to radiation-induced DNA damage,which are dependent on Id1. CONCLUSIONS In GBM,Cox-2-derived PGE2 induces Id1 via EP4-dependent activation of MAPK signaling and the Egr1 transcription factor. PGE2-mediated induction of Id1 is required for optimal tumor cell self-renewal and radiation resistance. Collectively,these findings identify Id1 as a key mediator of PGE2-dependent modulation of radiation response and lend insight into the mechanisms underlying radiation resistance in GBM patients. View Publication

Adipose tissue-derived mesenchymal stromal cells (AT-MSCs) are good candidates for cell therapy due to the accessibility of fat tissue and the abundance of AT-MSCs therein. Neurospheres are free-floating spherical condensations of cells with neural stem/progenitor cell (NSPC) characteristics that can be derived from AT-MSCs. The aims of this study were to examine the influence of oxygen (O2) tension on generation of neurospheres from canine AT-MSCs (AT-cMSCs) and to develop a hypoxic cell culture system to enhance the survival and therapeutic benefit of generated neurospheres. AT-cMSCs were cultured under varying oxygen tensions (1%,5% and 21%) in a neurosphere culture system. Neurosphere number and area were evaluated and NSPC markers were quantified using real-time quantitative PCR (qPCR). Effects of oxygen on neurosphere expression of hypoxia inducible factor 1,α subunit (HIF1A) and its target genes,erythropoietin receptor (EPOR),chemokine (C-X-C motif) receptor 4 (CXCR4) and vascular endothelial growth factor (VEGF),were quantified by qPCR. Neural differentiation potential was evaluated in 21% O2 by cell morphology and qPCR. Neurospheres were successfully generated from AT-cMSCs at all O2 tensions. Expression of nestin mRNA (NES) was significantly increased after neurosphere culture and was significantly higher in 1% O2 compared to 5% and 21% O2. Neurospheres cultured in 1% O2 had significantly increased levels of VEGF and EPOR. There was a significant increase in CXCR4 expression in neurospheres generated at all O2 tensions. Neurosphere culture under hypoxia had no negative effect on subsequent neural differentiation. This study suggests that generation of neurospheres under hypoxia could be beneficial when considering these cells for neurological cell therapies. View Publication

BACKGROUND Fabry disease (FD) is a lysosomal storage disease in which glycosphingolipids (GB3) accumulate in organs of the human body,leading to idiopathic hypertrophic cardiomyopathy and target organ damage. Its pathophysiology is still poorly understood. OBJECTIVES We aimed to generate patient-specific induced pluripotent stem cells (iPSC) from FD patients presenting cardiomyopathy to determine whether the model could recapitulate key features of the disease phenotype and to investigate the energy metabolism in Fabry disease. METHODS Peripheral blood mononuclear cells from a 30-year-old Chinese man with a diagnosis of Fabry disease,GLA gene (IVS4+919G>A) mutation were reprogrammed into iPSCs and differentiated into iPSC-CMs and energy metabolism was analyzed in iPSC-CMs. RESULTS The FD-iPSC-CMs recapitulated numerous aspects of the FD phenotype including reduced GLA activity,cellular hypertrophy,GB3 accumulation and impaired contractility. Decreased energy metabolism with energy utilization shift to glycolysis was observed,but the decreased energy metabolism was not modified by enzyme rescue replacement (ERT) in FD-iPSCs-CMs. CONCLUSION This model provided a promising in vitro model for the investigation of the underlying disease mechanism and development of novel therapeutic strategies for FD. This potential remedy for enhancing the energetic network and utility efficiency warrants further study to identify novel therapies for the disease. View Publication

The cell surface marker CD133 has been proposed as a brain tumor stem cell marker. However,there have been substantial controversies regarding the necessity and role of CD133 in tumorigenesis. This study aimed to characterize CD133(+) cells in brain tumors. Human brain tumor specimens and whole blood were collected from the same patients (N=12). We carried out dual FACS staining for CD133/CD34 and functional tumorigenesis and angiogenesis analyses of CD133(+) cells from different origins. We also investigated the in vivo tumorigenic potential and histological characteristics of four distinct groups on the basis of expression of CD133/CD34 markers (CD133(+),CD133(+)/CD34(+),CD133(+)/CD34(-),and CD133(-)). CD133(+) brain tumor cells coexpressed significantly higher positivity for CD34 (70.7±5.2% in CD133(+) vs. 12.3±4.2% in CD133(-) cells,P<0.001). CD133(+) brain tumor cells formed neurosphere-like spheroids and differentiated into multiple nervous system lineages unlike CD133(+) blood cells. They showed biological characteristics of endothelial cells,including vWF expression,LDL uptake and tube formation in vitro,unlike CD133(-) brain tumors cells. Pathologic analysis of brains implanted with CD133(+) cells showed large,markedly hypervascular tumors with well-demarcated boundary. CD133(+)/CD34(-) cells produced smaller but highly infiltrative tumors. Notably,pure angiogenic cell fractions (CD133(+)/CD34(+)) and CD133(-) tumor cells did not generate tumors in vivo. Our data suggest the presence of a distinct subpopulation of CD133(+) cells isolated from human brain tumors,with characteristics of endothelial progenitor cells (EPCs). View Publication

Neurogenesis in the adult brain is important for memory and learning,and the alterations in neural stem cells (NSCs) may be an important part of Alzheimer's disease pathogenesis. The phosphatidylinositol 3-kinase (PI3K) pathway has been suggested to play an important role in neuronal cell survival and is highly involved in adult neurogenesis. Recently,coenzyme Q10 (CoQ10) was found to affect the PI3K pathway. We investigated whether CoQ10 could restore amyloid β (Aβ)25-35 oligomer-inhibited proliferation of NSCs by focusing on the PI3K pathway. To evaluate the effects of CoQ10 on Aβ25-35 oligomer-inhibited proliferation of NSCs,NSCs were treated with several concentrations of CoQ10 and/or Aβ25-35 oligomers. BrdU labeling,Colony Formation Assays,and immunoreactivity of Ki-67,a marker of proliferative activity,showed that NSC proliferation decreased with Aβ25-35 oligomer treatment,but combined treatment with CoQ10 restored it. Western blotting showed that CoQ10 treatment increased the expression levels of p85α PI3K,phosphorylated Akt (Ser473),phosphorylated glycogen synthase kinase-3β (Ser9),and heat shock transcription factor,which are proteins related to the PI3K pathway in Aβ25-35 oligomers-treated NSCs. To confirm a direct role for the PI3K pathway in CoQ10-induced restoration of proliferation of NSCs inhibited by Aβ25-35 oligomers,NSCs were pretreated with a PI3K inhibitor,LY294002; the effects of CoQ10 on the proliferation of NSCs inhibited by Aβ25-35 oligomers were almost completely blocked. Together,these results suggest that CoQ10 restores Aβ25-35 oligomer-inhibited proliferation of NSCs by activating the PI3K pathway. View Publication

BACKGROUND Transplantation of enteric neural stem cells (ENSC) holds promise as a potential therapy for enteric neuropathies,including Hirschsprung disease. Delivery of transplantable cells via laparotomy has been described,but we propose a novel,minimally invasive endoscopic method of cell delivery. METHODS Enteric neural stem cells for transplantation were cultured from dissociated gut of postnatal donor mice. Twelve recipient mice,including Ednrb(-/-) mice with distal colonic aganglionosis,underwent colonoscopic injection of ENSC under direct vision using a 30-gauge Hamilton needle passed through a rigid cystoureteroscope. Cell engraftment,survival,and neuroglial differentiation were studied 1-4 weeks after the procedure. KEY RESULTS All recipient mice tolerated the procedure without complications and survived to sacrifice. Transplanted cells were found within the colonic wall in 9 of 12 recipient mice with differentiation into enteric neurons and glia. CONCLUSIONS & INFERENCES Endoscopic injection of ENSC is a safe and reliable method for cell delivery,and can be used to deliver a large number of cells to a specific area of disease. This minimally invasive endoscopic approach may prove beneficial to future human applications of cell therapy for neurointestinal disease. 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

MicroRNAs play an important role in T cell responses. However,how microRNAs regulate CD8 T cell memory remains poorly defined. Here,we found that miR-150 negatively regulates CD8 T cell memory in vivo. Genetic deletion of miR-150 disrupted the balance between memory precursor and terminal effector CD8 T cells following acute viral infection. Moreover,miR-150-deficient memory CD8 T cells were more protective upon rechallenge. A key circuit whereby miR-150 repressed memory CD8 T cell development through the transcription factor c-Myb was identified. Without miR-150,c-Myb was upregulated and anti-apoptotic targets of c-Myb,such as Bcl-2 and Bcl-xL,were also increased,suggesting a miR-150-c-Myb survival circuit during memory CD8 T cell development. Indeed,overexpression of non-repressible c-Myb rescued the memory CD8 T cell defects caused by overexpression of miR-150. Overall,these results identify a key role for miR-150 in memory CD8 T cells through a c-Myb-controlled enhanced survival circuit. View Publication

A capability for analyzing complex cellular communication among tissues is important in drug discovery and development,and in vitro technologies for doing so are required for human applications. A prominent instance is communication between the gut and the liver,whereby perturbations of one tissue can influence behavior of the other. Here,we present a study on human gut-liver tissue interactions under normal and inflammatory contexts,via an integrative multi-organ platform comprising human liver (hepatocytes and Kupffer cells),and intestinal (enterocytes,goblet cells,and dendritic cells) models. Our results demonstrated long-term (>2 weeks) maintenance of intestinal (e.g.,barrier integrity) and hepatic (e.g.,albumin) functions in baseline interaction. Gene expression data comparing liver in interaction with gut,versus isolation,revealed modulation of bile acid metabolism. Intestinal FGF19 secretion and associated inhibition of hepatic CYP7A1 expression provided evidence of physiologically relevant gut-liver crosstalk. Moreover,significant non-linear modulation of cytokine responses was observed under inflammatory gut-liver interaction; for example,production of CXCR3 ligands (CXCL9,10,11) was synergistically enhanced. RNA-seq analysis revealed significant upregulation of IFNα/β/γ signaling during inflammatory gut-liver crosstalk,with these pathways implicated in the synergistic CXCR3 chemokine production. Exacerbated inflammatory response in gut-liver interaction also negatively affected tissue-specific functions (e.g.,liver metabolism). These findings illustrate how an integrated multi-tissue platform can generate insights useful for understanding complex pathophysiological processes such as inflammatory organ crosstalk. Biotechnol. Bioeng. 2017;114: 2648-2659. textcopyright 2017 Wiley Periodicals,Inc. 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

Metabolic imaging of brain tumors using (13)C Magnetic Resonance Spectroscopy (MRS) of hyperpolarized [1-(13)C] pyruvate is a promising neuroimaging strategy which,after a decade of preclinical success in glioblastoma (GBM) models,is now entering clinical trials in multiple centers. Typically,the presence of GBM has been associated with elevated hyperpolarized [1-(13)C] lactate produced from [1-(13)C] pyruvate,and response to therapy has been associated with a drop in hyperpolarized [1-(13)C] lactate. However,to date,lower grade gliomas had not been investigated using this approach. The most prevalent mutation in lower grade gliomas is the isocitrate dehydrogenase 1 (IDH1) mutation,which,in addition to initiating tumor development,also induces metabolic reprogramming. In particular,mutant IDH1 gliomas are associated with low levels of lactate dehydrogenase A (LDHA) and monocarboxylate transporters 1 and 4 (MCT1,MCT4),three proteins involved in pyruvate metabolism to lactate. We therefore investigated the potential of (13)C MRS of hyperpolarized [1-(13)C] pyruvate for detection of mutant IDH1 gliomas and for monitoring of their therapeutic response. We studied patient-derived mutant IDH1 glioma cells that underexpress LDHA,MCT1 and MCT4,and wild-type IDH1 GBM cells that express high levels of these proteins. Mutant IDH1 cells and tumors produced significantly less hyperpolarized [1-(13)C] lactate compared to GBM,consistent with their metabolic reprogramming. Furthermore,hyperpolarized [1-(13)C] lactate production was not affected by chemotherapeutic treatment with temozolomide (TMZ) in mutant IDH1 tumors,in contrast to previous reports in GBM. Our results demonstrate the unusual metabolic imaging profile of mutant IDH1 gliomas,which,when combined with other clinically available imaging methods,could be used to detect the presence of the IDH1 mutation in vivo. View Publication