技术资料

Several progenitor cell populations have been reported to exist in hearts that play a role in cardiac turnover and/or repair. Despite the presence of cardiac stem and progenitor cells within the myocardium,functional repair of the heart after injury is inadequate. Identification of the signaling pathways involved in the expansion and differentiation of cardiac progenitor cells (CPCs) will broaden insight into the fundamental mechanisms playing a role in cardiac homeostasis and disease and might provide strategies for in vivo regenerative therapies. To understand and exploit cardiac ontogeny for drug discovery efforts,we developed an in vitro human induced pluripotent stem cell-derived CPC model system using a highly enriched population of KDR(pos)/CKIT(neg)/NKX2.5(pos) CPCs. Using this model system,these CPCs were capable of generating highly enriched cultures of cardiomyocytes under directed differentiation conditions. In order to facilitate the identification of pathways and targets involved in proliferation and differentiation of resident CPCs,we developed phenotypic screening assays. Screening paradigms for therapeutic applications require a robust,scalable,and consistent methodology. In the present study,we have demonstrated the suitability of these cells for medium to high-throughput screens to assess both proliferation and multilineage differentiation. Using this CPC model system and a small directed compound set,we identified activin-like kinase 5 (transforming growth factor-β type 1 receptor kinase) inhibitors as novel and potent inducers of human CPC differentiation to cardiomyocytes. Significance: Cardiac disease is a leading cause of morbidity and mortality,with no treatment available that can result in functional repair. This study demonstrates how differentiation of induced pluripotent stem cells can be used to identify and isolate cell populations of interest that can translate to the adult human heart. Two separate examples of phenotypic screens are discussed,demonstrating the value of this biologically relevant and reproducible technology. In addition,this assay system was able to identify novel and potent inducers of differentiation and proliferation of induced pluripotent stem cell-derived cardiac progenitor cells. View Publication

Transient global cerebral ischemia induces profound changes in the transcriptome of brain cells,which is partially associated with the induction or repression of genes that influence the ischemic response. However,the mechanisms responsible for the selective vulnerability of hippocampal neurons to global ischemia remain to be clarified. To identify molecular changes elicited by ischemic insults,we subjected hippocampal primary cultures to oxygen-glucose deprivation (OGD),an in vitro model for global ischemia that resulted in delayed neuronal death with an excitotoxic component. To investigate changes in the transcriptome of hippocampal neurons submitted to OGD,total RNA was extracted at early (7 h) and delayed (24 h) time points after OGD and used in a whole-genome RNA microarray. We observed that at 7 h after OGD there was a general repression of genes,whereas at 24 h there was a general induction of gene expression. Genes related with functions such as transcription and RNA biosynthesis were highly regulated at both periods of incubation after OGD,confirming that the response to ischemia is a dynamic and coordinated process. Our analysis showed that genes for synaptic proteins,such as those encoding for PICK1,GRIP1,TARPγ3,calsyntenin-2/3,SAPAP2 and SNAP-25,were down-regulated after OGD. Additionally,OGD decreased the mRNA and protein expression levels of the GluA1 AMPA receptor subunit as well as the GluN2A and GluN2B subunits of NMDA receptors,but increased the mRNA expression of the GluN3A subunit,thus altering the composition of ionotropic glutamate receptors in hippocampal neurons. Together,our results present the expression profile elicited by in vitro ischemia in hippocampal neurons,and indicate that OGD activates a transcriptional program leading to down-regulation in the expression of genes coding for synaptic proteins,suggesting that the synaptic proteome may change after ischemia. View Publication

NMDA receptors play a central role in shaping the strength of synaptic connections throughout development and in mediating synaptic plasticity mechanisms that underlie some forms of learning and memory formation in the CNS. In the hippocampus and the neocortex,GluN1 is combined primarily with GluN2A and GluN2B,which are differentially expressed during development and confer distinct molecular and physiological properties to NMDA receptors. The contribution of each subunit to the synaptic traffic of NMDA receptors and therefore to their role during development and in synaptic plasticity is still controversial. We report a critical role for the GluN2B subunit in regulating NMDA receptor synaptic targeting. In the absence of GluN2B,the synaptic levels of AMPA receptors are increased and accompanied by decreased constitutive endocytosis of GluA1-AMPA receptor. We used quantitative proteomic analysis to identify changes in the composition of postsynaptic densities from GluN2B(-/-) mouse primary neuronal cultures and found altered levels of several ubiquitin proteasome system components,in particular decreased levels of proteasome subunits. Enhancing the proteasome activity with a novel proteasome activator restored the synaptic levels of AMPA receptors in GluN2B(-/-) neurons and their endocytosis,revealing that GluN2B-mediated anchoring of the synaptic proteasome is responsible for fine tuning AMPA receptor synaptic levels under basal conditions. 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

Transplantation of Defined Populations of Differentiated Human Neural Stem Cell Progeny View Publication

Glioblastoma multiforme (GBM) is the most common and lethal form of intracranial tumor. We have established a lentivirus-induced mouse model of malignant gliomas,which faithfully captures the pathophysiology and molecular signature of mesenchymal human GBM. RNA-Seq analysis of these tumors revealed high nuclear factor κB (NF-κB) activation showing enrichment of known NF-κB target genes. Inhibition of NF-κB by either depletion of IκB kinase 2 (IKK2),expression of a IκBαM super repressor,or using a NEMO (NF-κB essential modifier)-binding domain (NBD) peptide in tumor-derived cell lines attenuated tumor proliferation and prolonged mouse survival. Timp1,one of the NF-κB target genes significantly up-regulated in GBM,was identified to play a role in tumor proliferation and growth. Inhibition of NF-κB activity or silencing of Timp1 resulted in slower tumor growth in both mouse and human GBM models. Our results suggest that inhibition of NF-κB activity or targeting of inducible NF-κB genes is an attractive therapeutic approach for GBM. View Publication

A mutation in the centrosomal-P4.1-associated protein (CPAP) causes Seckel syndrome with microcephaly,which is suggested to arise from a decline in neural progenitor cells (NPCs) during development. However,mechanisms ofNPCs maintenance remain unclear. Here,we report an unexpected role for the cilium inNPCs maintenance and identifyCPAPas a negative regulator of ciliary length independent of its role in centrosome biogenesis. At the onset of cilium disassembly,CPAPprovides a scaffold for the cilium disassembly complex (CDC),which includes Nde1,Aurora A,andOFD1,recruited to the ciliary base for timely cilium disassembly. In contrast,mutatedCPAPfails to localize at the ciliary base associated with inefficientCDCrecruitment,long cilia,retarded cilium disassembly,and delayed cell cycle re-entry leading to premature differentiation of patientiPS-derivedNPCs. AberrantCDCfunction also promotes premature differentiation ofNPCs in SeckeliPS-derived organoids. Thus,our results suggest a role for cilia in microcephaly and its involvement during neurogenesis and brain size control. View Publication

Hypoxic stressors contribute to neuronal death in many brain diseases. Astrocyte processes surround most neurons and are therefore anatomically well-positioned to shield them from hypoxic injury. Excitatory amino acid transporters (EAATs),represent the sole mechanism of active reuptake of glutamate into the astrocytes and neurons and are essential to dampen neuronal excitation following glutamate release at synapses. Glutamate clearance impairment from any factors is bound to result in an increase in hypoxic neuronal injury. The brain energy metabolism under hypoxic conditions depends on monocarboxylate transporters (MCTs) that are expressed by neurons and glia. Previous co-immunoprecipitation experiments revealed that MCT4 directly modulate EAAT1 in astrocytes. The reduction in both surface proteins may act synergistically to induce neuronal hyperexcitability and excitotoxicity. Therefore we hypothesized that astrocytes would respond to hypoxic conditions by enhancing their expression of MCT4 and EAAT1,which,in turn,would enable them to better support neurons to survive lethal hypoxia injury. An oxygen deprivation (OD) protocol was used in primary cultures of neurons,astrocytes,and astrocytes-neurons derived from rat hippocampus,with or without MCT4-targeted short hairpin RNA (shRNA) transfection. Cell survival,expression of MCT4,EAAT1,glial fibrillary acidic protein and neuronal nuclear antigen were evaluated. OD resulted in significant cell death in neuronal cultures and up-regulation of MCT4,EAAT1 expression respectively in primary cell cultures,but no injury in neuron-astrocyte co-cultures and astrocyte cultures. However,neuronal cell death in co-cultures was increased exposure to shRNA-MCT4 prior to OD. These findings demonstrate that the MCT4-mediated expression of EAAT1 is involved in the resistance to hypoxia injury in astrocyte-neuron co-cultures. View Publication

Little is known about monocyte differentiation in the lung mucosal environment and about how the epithelium shapes monocyte function. We studied the role of the soluble component of bronchial epithelial cells (BECs) obtained under basal culture conditions in innate and adaptive monocyte responses. Monocytes cultured in bronchial epithelial cell-conditioned media (BEC-CM) specifically upregulate CD141,CD123,and DC-SIGN surface levels andFLT3expression,as well as the release of IL-1β,IL-6,and IL-10. BEC-conditioned monocytes stimulate naive T cells to produce IL-17 through IL-1β mechanism and also trigger IL-10 production by memory T cells. Furthermore,monocytes cultured in an inflammatory environment induced by the cytokines IL-6,IL-8,IL-1β,IL-15,TNF-α,and GM-CSF also upregulate CD123 and DC-SIGN expression. However,only inflammatory cytokines in the epithelial environment boost the expression of CD141. Interestingly,we identified a CD141/CD123/DC-SIGN triple positive population in the bronchoalveolar lavage fluid (BALF) from patients with different inflammatory conditions,demonstrating that this monocyte population existsin vivo. The frequency of this monocyte population was significantly increased in patients with sarcoidosis,suggesting a role in inflammatory mechanisms. Overall,these data highlight the specific role that the epithelium plays in shaping monocyte responses. Therefore,the unraveling of these mechanisms contributes to the understanding of the function that the epithelium may playin vivo. View Publication

The recent Zika virus (ZIKV) outbreak,which mainly affected Brazil and neighbouring states,demonstrated the paucity of information concerning the epidemiology of several flaviruses,but also highlighted the lack of available agents with which to treat such emerging diseases. Here,we show that heparin,a widely used anticoagulant,while exerting a modest inhibitory effect on Zika Virus replication,fully prevents virus-induced cell death of human neural progenitor cells (NPCs). View Publication