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The vacuolar H+ ATPase (V-ATPase) is a proton pump responsible for acidification of cellular microenvironments,an activity exploited by tumors to survive,proliferate and resist to therapy. Despite few observations,the role of V-ATPase in human tumorigenesis remains unclear.We investigated the expression of ATP6V0C,ATP6V0A2,encoding two subunits belonging to the V-ATPase V0 sector and ATP6V1C,ATP6V1G1,ATPT6V1G2,ATP6V1G3,which are part of the V1 sector,in series of adult gliomas and in cancer stem cell-enriched neurospheres isolated from glioblastoma (GBM) patients. ATP6V1G1 expression resulted significantly upregulated in tissues of patients with GBM and correlated with shorter patients' overall survival independent of clinical variables.ATP6V1G1 knockdown in GBM neurospheres hampered sphere-forming ability,induced cell death,and decreased matrix invasion,a phenotype not observed in GBM monolayer cultures. Treating GBM organotypic cultures or neurospheres with the selective V-ATPase inhibitor bafilomycin A1 reproduced the effects of ATP6V1G1 siRNA and strongly suppressed expression of the stem cell markers Nestin,CD133 and transcription factors SALL2 and POU3F2 in neurospheres.These data point to ATP6V1G1 as a novel marker of poor prognosis in GBM patients and identify V-ATPase inhibition as an innovative therapeutic strategy for GBM. View Publication

Lung macrophage subpopulations have been identified based on size. We investigated characteristics of small and large macrophages in the alveolar spaces and lung interstitium of COPD patients and controls. Alveolar and interstitial cells were isolated from lung resection tissue from 88 patients. Macrophage subpopulation cell-surface expression of immunological markers and phagocytic ability were assessed by flow cytometry. Inflammatory related gene expression was measured. Alveolar and interstitial macrophages had subpopulations of small and large macrophages based on size and granularity. Alveolar macrophages had similar numbers of small and large cells; interstitial macrophages were mainly small. Small macrophages expressed significantly higher cell surface HLA-DR,CD14,CD38 and CD36 and lower CD206 compared to large macrophages. Large alveolar macrophages showed lower marker expression in COPD current compared to ex-smokers. Small interstitial macrophages had the highest pro-inflammatory gene expression levels,while large alveolar macrophages had the lowest. Small alveolar macrophages had the highest phagocytic ability. Small alveolar macrophage CD206 expression was lower in COPD patients compared to smokers. COPD lung macrophages include distinct subpopulations; Small interstitial and small alveolar macrophages with more pro-inflammatory and phagocytic function respectively,and large alveolar macrophages with low pro-inflammatory and phagocytic ability. View Publication

Realization of clinical potential of human pluripotent stem cells (hPSCs) in bone regenerative medicine requires development of simple and safe biomaterials for expansion of hPSCs followed by directing their lineage commitment to osteoblasts. In the present study,a chemically defined peptide-decorated polycaprolactone (PCL) nanofibrous microenvironment was prepared through electrospinning technology and subsequent conjugation with vitronectin peptide to promote the culture and osteogenic potential of hPSCs in vitro. The results indicated that hPSCs successfully proliferated and maintained their pluripotency on the biointerface of peptide-conjugated nanofibers without Matrigel under defined conditions. Moreover,the prepared niche exhibited an appealing ability in promoting directed differentiation of hPSCs to osteoblastic phenotype without embryoid body formation step,determined from the cell morphological alteration,alkaline phosphate activity,and osteogenesis-related gene expression,as well as protein production. Such well-defined,xeno-free,and safe nanofiber scaffolds that allow the survival and facilitate osteo-differentiation of hPSCs provide a novel platform for hPSCs differentiation via cell-nanofiber interplay,and possess great value in accelerating the translational perspectives of hPSCs in bone tissue engineering. View Publication

Human bocavirus 1 (HBoV1) is a human parvovirus that causes acute respiratory tract infections in young children. In this study,we confirmed that,when polarized/well-differentiated human airway epithelia are infected with HBoV1in vitro,they develop damage characterized by barrier function disruption and cell hypotrophy. Cell death mechanism analyses indicated that the infection induced pyroptotic cell death characterized by caspase-1 activation. Unlike infections with other parvoviruses,HBoV1 infection did not activate the apoptotic or necroptotic cell death pathway. When the NLRP3-ASC-caspase-1 inflammasome-induced pathway was inhibited by short hairpin RNA (shRNA),HBoV1-induced cell death dropped significantly; thus,NLRP3 mediated by ASC appears to be the pattern recognition receptor driving HBoV1 infection-induced pyroptosis. HBoV1 infection induced steady increases in the expression of interleukin 1α (IL-1α) and IL-18. HBoV1 infection was also associated with the marked expression of the antiapoptotic genesBIRC5andIFI6When the expression ofBIRC5and/orIFI6was inhibited by shRNA,the infected cells underwent apoptosis rather than pyroptosis,as indicated by increased cleaved caspase-3 levels and the absence of caspase-1.BIRC5and/orIFI6gene inhibition also significantly reduced HBoV1 replication. Thus,HBoV1 infection of human airway epithelial cells activates antiapoptotic proteins that suppress apoptosis and promote pyroptosis. This response may have evolved to confer a replicative advantage,thus allowing HBoV1 to establish a persistent airway epithelial infection. This is the first report of pyroptosis in airway epithelia infected by a respiratory virus.IMPORTANCEMicrobial infection of immune cells often induces pyroptosis,which is mediated by a cytosolic protein complex called the inflammasome that senses microbial pathogens and then activates the proinflammatory cytokines IL-1 and IL-18. While virus-infected airway epithelia often activate NLRP3 inflammasomes,studies to date suggest that these viruses kill the airway epithelial cells via the apoptotic or necrotic pathway; involvement of the pyroptosis pathway has not been reported previously. Here,we show for the first time that virus infection of human airway epithelia can also induce pyroptosis. Human bocavirus 1 (HBoV1),a human parvovirus,causes lower respiratory tract infections in young children. This study indicates that HBoV1 kills airway epithelial cells by activating genes that suppress apoptosis and thereby promote pyroptosis. This strategy appears to promote HBoV1 replication and may have evolved to allow HBoV1 to establish persistent infection of human airway epithelia. View Publication

Human bocavirus 1 (HBoV1) belongs to the genus Bocaparvovirus of the Parvoviridae family,and is an emerging human pathogenic respiratory virus. In vitro,HBoV1 infects well-differentiated/polarized primary human airway epithelium (HAE) cultured at an air-liquid interface (HAE-ALI). Although it is well known that autonomous parvovirus replication depends on the S phase of the host cells,we demonstrate here that the HBoV1 genome amplifies efficiently in mitotically quiescent airway epithelial cells of HAE-ALI cultures. Analysis of HBoV1 DNA in infected HAE-ALI revealed that HBoV1 amplifies its ssDNA genome following a typical parvovirus rolling-hairpin DNA replication mechanism. Notably,HBoV1 infection of HAE-ALI initiates a DNA damage response (DDR) with activation of all three phosphatidylinositol 3-kinase-related kinases (PI3KKs). We found that the activation of the three PI3KKs is required for HBoV1 genome amplification; and,more importantly,we identified that two Y-family DNA polymerases,Pol eta and Pol kappa,are involved in HBoV1 genome amplification. Overall,we have provided an example of de novo DNA synthesis (genome amplification) of an autonomous parvovirus in non-dividing cells,which is dependent on the cellular DNA damage and repair pathways. 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

Azidothymidine (AZT) is a synthetic,chain-terminating nucleoside analog used to treat HIV-1 infection. While AZT is not actively transported across the blood brain barrier,it does accumulate at high levels in cerebrospinal fluid,and subsequently diffuses into the overlying parenchyma. Due to the close anatomical proximity of the neurogenic niches to the ventricular system,we hypothesize that diffusion from CSF exposes neural stem/progenitor cells and their progeny to biologically relevant levels of AZT sufficient to perturb normal cell functions. We employed in vitro and in vivo models of mouse neurogenesis in order to assess the effects of AZT on developing and adult neurogenesis. Using in vitro assays we show that AZT reduces the population expansion potential of neural stem/progenitor cells by inducing senescence. Additionally,in a model of in vitro neurogenesis AZT severely attenuates neuroblast production. These effects are mirrored in vivo by clinically-relevant animal models. We show that in utero AZT exposure perturbs both population expansion and neurogenesis among neural stem/progenitor cells. Additionally,a short-term AZT regimen in adult mice suppresses subependymal zone neurogenesis. These data reveal novel negative effects of AZT on neural stem cell biology. Given that the sequelae of HIV infection often include neurologic deficits-subsumed under AIDS Dementia Complex (Brew,1999)-it is important to determine to what extent AZT negatively affects neurological function in ways that contribute to,or exacerbate,ADC in order to avoid attributing iatrogenic drug effects to the underlying disease process,and thereby skewing the risk/benefit analysis of AZT therapy. 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

Although neural stem cells (NSCs) sustain continuous neurogenesis throughout the adult lifespan of mammals,they progressively exhibit proliferation defects that contribute to a sharp reduction in subventricular neurogenesis during aging. However,little is known regarding the early age-related events in neurogenic niches. Using a fluorescence-activated cell sorting technique that allows for the prospective purification of the main neurogenic populations from the subventricular zone (SVZ),we demonstrated an early decline in adult neurogenesis with a dramatic loss of progenitor cells in 4 month-old young adult mice. Whereas the activated and quiescent NSC pools remained stable up to 12 months,the proliferative status of activated NSCs was already altered by 6 months,with an overall extension of the cell cycle resulting from a specific lengthening of G1. Whole genome analysis of activated NSCs from 2- and 6-month-old mice further revealed distinct transcriptomic and molecular signatures,as well as a modulation of the TGFβ signalling pathway. Our microarray study constitutes a cogent identification of new molecular players and signalling pathways regulating adult neurogenesis and its early modifications. View Publication

Quiescent neural stem cells (NSCs) are considered the reservoir for adult neurogenesis,generating new neurons throughout life. Until now,their isolation has not been reported,which has hampered studies of their regulatory mechanisms. We sorted by FACS quiescent NSCs and their progeny from the subventricular zone (SVZ) of adult mice according to the expression of the NSC marker LeX/CD15,the EGF receptor (EGFR) and the CD24 in combination with the vital DNA marker Hoechst 33342. Characterization of sorted cells showed that the LeX(bright)/EGFR-negative population was enriched in quiescent cells having an NSC phenotype. In contrast to proliferating NSCs and progenitors,the LeX(bright)/EGFR-negative cells,i.e. quiescent NSCs,resisted to a moderate dose of gamma-radiation (4Gy),entered the cell cycle two days after irradiation prior to EGFR acquisition and ultimately repopulated the SVZ. We further show that the GABAAR signaling regulates their cell cycle entry by using specific GABAAR agonists/antagonists and that the radiation-induced depletion of neuroblasts,the major GABA source,provoked their proliferation in the irradiated SVZ. Our study demonstrates that quiescent NSCs are specifically enriched in the LeX(bright)/EGFR-negative population,and identifies the GABAAR signaling as a regulator of the SVZ niche size by modulating the quiescence of NSCs. View Publication

Nervous system (NS) development relies on coherent upregulation of extensive sets of genes in a precise spatiotemporal manner. How such transcriptome-wide effects are orchestrated at the molecular level remains an open question. Here we show that 3'-untranslated regions (3' UTRs) of multiple neural transcripts contain AU-rich cis-elements (AREs) recognized by tristetraprolin (TTP/Zfp36),an RNA-binding protein previously implicated in regulation of mRNA stability. We further demonstrate that the efficiency of ARE-dependent mRNA degradation declines in the neural lineage because of a decrease in the TTP protein expression mediated by the NS-enriched microRNA miR-9. Importantly,TTP downregulation in this context is essential for proper neuronal differentiation. On the other hand,inactivation of TTP in non-neuronal cells leads to dramatic upregulation of multiple NS-specific genes. We conclude that the newly identified miR-9/TTP circuitry limits unscheduled accumulation of neuronal mRNAs in non-neuronal cells and ensures coordinated upregulation of these transcripts in neurons. View Publication

Human pluripotent stem cells derived cardiomyocytes (PSC-CMs) have been widely used for disease modeling,drug safety screening,and preclinical cell therapy to regenerate myocardium. Most studies have utilized PSC-CM grown in vitro for a relatively short period after differentiation. These PSC-CMs demonstrated structural,electrophysiological,and mechanical features of primitive cardiomyocytes. A few studies have extended in vitro PSC-CM culture time and reported improved maturation of structural and electromechanical properties. The degree of mitochondrial maturation,however,remains unclear. This study characterized the development of mitochondria during prolonged in vitro culture. PSC-CM demonstrated an improved mitochondrial maturation with prolonged culture,in terms of increased mitochondrial relative abundance,enhanced membrane potential,and increased activity of several mitochondrial respiratory complexes. These are in parallel with the maturation of other cellular components. However,the maturation of mitochondria in PSC-CMs grown for extended in vitro culture exhibits suboptimal maturation when compared with the maturation of mitochondria observed in the human fetal heart during similar time interval. View Publication