技术资料

Stem cell (SC) lines that capture the genetics of disease susceptibility provide new research tools. To assess the utility of mouse central nervous system (CNS) SC-containing neurosphere cultures for studying heritable neurodegenerative disease,we compared neurosphere cultures from transgenic mice that express human tau with the P301L familial frontotemporal dementia (FTD) mutation,rTg(tau(P301L))4510,with those expressing comparable levels of wild type human tau,rTg(tau(wt))21221. rTg(tau(P301L))4510 mice express the human tau(P301L) variant in their forebrains and display cellular,histological,biochemical and behavioral abnormalities similar to those in human FTD,including age-dependent differences in tau phosphorylation that distinguish them from rTg(tau(wt))21221 mice. We compared FTD-hallmark tau phosphorylation in neurospheres from rTg(tau(P301L))4510 mice and from rTg(tau(wt))21221 mice. The tau genotype-specific phosphorylation patterns in neurospheres mimicked those seen in mice,validating use of neurosphere cultures as models for studying tau phosphorylation. Genotype-specific tau phosphorylation was observed in 35 independent cell lines from individual fetuses; tau in rTg(tau(P301L))4510 cultures was hypophosphorylated in comparison with rTg(tau(wt))21221 as was seen in young adult mice. In addition,there were fewer human tau-expressing cells in rTg(tau(P301L))4510 than in rTg(tau(wt))21221 cultures. Following differentiation,neuronal filopodia-spine density was slightly greater in rTg(tau(P301L))4510 than rTg(tau(wt))21221 and control cultures. Together with the recapitulation of genotype-specific phosphorylation patterns,the observation that neurosphere lines maintained their cell line-specific-differences and retained SC characteristics over several passages supports the utility of SC cultures as surrogates for analysis of cellular disease mechanisms. View Publication

Considerable progress has been made in identifying signaling pathways that direct the differentiation of human pluripotent stem cells (hPSCs) into specialized cell types,including neurons. However,differentiation of hPSCs with extrinsic factors is a slow,step-wise process,mimicking the protracted timing of human development. Using a small-molecule screen,we identified a combination of five small-molecule pathway inhibitors that yield hPSC-derived neurons at textgreater75% efficiency within 10 d of differentiation. The resulting neurons express canonical markers and functional properties of human nociceptors,including tetrodotoxin (TTX)-resistant,SCN10A-dependent sodium currents and response to nociceptive stimuli such as ATP and capsaicin. Neuronal fate acquisition occurs about threefold faster than during in vivo development,suggesting that use of small-molecule pathway inhibitors could become a general strategy for accelerating developmental timing in vitro. The quick and high-efficiency derivation of nociceptors offers unprecedented access to this medically relevant cell type for studies of human pain. View Publication

Tumor necrosis factor related apoptosis-inducing ligand (TRAIL) induces apoptosis specifically in tumor cells and its efficacy has been tested in pre-clinical models by delivering it systemically as a purified ligand or via engineered stem cells (SC). However,about 50% of tumor lines are resistant to TRAIL and overcoming TRAIL resistance in aggressive tumors,such as glioblastoma-multiforme (GBM),and understanding the molecular dynamics of TRAIL-based combination therapies are critical to broadly use TRAIL as a therapeutic agent. In this study,we developed death receptor (DR)4/5-reporters that offer an imaging-based platform to identify agents that act in concert with a potent,secretable variant of TRAIL (S-TRAIL) by monitoring changes in DR4/5 expression. Utilizing these reporters,we show a differential regulation of DR4/5 when exposed to a panel of clinically relevant agents. A histone deacetylase inhibitor,MS-275,resulted in upregulation of DR4/5 in all GBM cell lines,and these changes could be followed in real time both in vitro and in vivo in mice bearing tumors and they correlated with increased TRAIL sensitivity. To further assess the dynamics of combinatorial strategies that overcome resistance of tumors to SC released S-TRAIL,we also engineered tumor cells to express live-cell caspase-reporters and SCs to express S-TRAIL. Utilizing DR4/5 and caspase reporters in parallel,we show that MS-275 sensitizes TRAIL-resistant GBM cells to stem cell (SC) delivered S-TRAIL by changing the time-to-death in vitro and in vivo. This study demonstrates the effectiveness of a combination of real-time reporters of TRAIL-induced apoptosis pathway in evaluating the efficacy of SC-TRAIL-based therapeutics and may have implications in targeting a broad range of cancers. View Publication

In the search for ways to combat degenerative neurological disorders,neurogenesis-stimulating factors are proving to be a promising area of research. In this study,we show that the hormonal factor prolactin (PRL) can activate a pool of latent precursor cells in the adult mouse hippocampus. Using an in vitro neurosphere assay,we found that the addition of exogenous PRL to primary adult hippocampal cells resulted in an approximate 50% increase in neurosphere number. In addition,direct infusion of PRL into the adult dentate gyrus also resulted in a significant increase in neurosphere number. Together these data indicate that exogenous PRL can increase hippocampal precursor numbers both in vitro and in vivo. Conversely,PRL null mice showed a significant reduction (approximately 80%) in the number of hippocampal-derived neurospheres. Interestingly,no deficit in precursor proliferation was observed in vivo,indicating that in this situation other niche factors can compensate for a loss in PRL. The PRL loss resulted in learning and memory deficits in the PRL null mice,as indicated by significant deficits in the standard behavioral tests requiring input from the hippocampus. This behavioral deficit was rescued by direct infusion of recombinant PRL into the hippocampus,indicating that a lack of PRL in the adult mouse hippocampus can be correlated with impaired learning and memory. View Publication

Glioblastoma multiforme is the most malignant type of primary brain tumor with a poor prognosis. These tumors consist of a heterogeneous population of malignant cells,including well-differentiated tumor cells and less differentiated cells with stem cell properties. These cancer stem cells,known as brain tumor initiating cells,likely contribute to glioma recurrence,as they are highly invasive,mobile,resistant to radiation and chemotherapy,and have the capacity to self-renew. Glioblastoma tumor cells release excitotoxic levels of glutamate,which may be a key process in the death of peritumoral neurons,formation of necrosis,local inflammation,and glioma-related seizures. Moreover,elevated glutamate levels in the tumor may act in paracrine and autocrine manner to activate glutamate receptors on glioblastoma tumor cells,resulting in proliferation and invasion. Using a previously described culturing condition that selectively promotes the growth of brain tumor initiating cells,which express the stem cell markers nestin and SOX-2,we characterize the expression of α-amino-3-hydroxy-5-methyl-4-isozolepropionic acid (AMPA)-type glutamate receptor subunits in brain tumor initiating cells derived from glioblastomas. Here we show for the first time that glioblastoma brain tumor initiating cells express high concentrations of functional calcium-permeable AMPA receptors,compared to the differentiated tumor cultures consisting of non-stem cells. Up-regulated calcium-permeable AMPA receptor expression was confirmed by immunoblotting,immunocytochemistry,and intracellular calcium imaging in response to specific agonists. Our findings raise the possibility that glutamate secretion in the GBM tumor microenvironment may stimulate brain tumor derived cancer stem cells. View Publication

In congenital mitochondrial DNA (mtDNA) disorders,a mixture of normal and mutated mtDNA (termed heteroplasmy) exists at varying levels in different tissues,which determines the severity and phenotypic expression of disease. Pearson marrow pancreas syndrome (PS) is a congenital bone marrow failure disorder caused by heteroplasmic deletions in mtDNA. The cause of the hematopoietic failure in PS is unknown,and adequate cellular and animal models are lacking. Induced pluripotent stem (iPS) cells are particularly amenable for studying mtDNA disorders,as cytoplasmic genetic material is retained during direct reprogramming. Here,we derive and characterize iPS cells from a patient with PS. Taking advantage of the tendency for heteroplasmy to change with cell passage,we isolated isogenic PS-iPS cells without detectable levels of deleted mtDNA. We found that PS-iPS cells carrying a high burden of deleted mtDNA displayed differences in growth,mitochondrial function,and hematopoietic phenotype when differentiated in vitro,compared to isogenic iPS cells without deleted mtDNA. Our results demonstrate that reprogramming somatic cells from patients with mtDNA disorders can yield pluripotent stem cells with varying burdens of heteroplasmy that might be useful in the study and treatment of mitochondrial diseases. STEM CELLS2013;31:1287–1297 View Publication

Within the two neurogenic niches of the adult mammalian brain,i.e.,the subventricular zone lining the lateral ventricle and the subgranular zone of the hippocampus,there exist distinct populations of proliferating neural precursor cells that differentiate to generate new neurons. Numerous studies have suggested that epigenetic regulation by histone-modifying proteins is important in guiding precursor differentiation during development; however,the role of these proteins in regulating neural precursor activity in the adult neurogenic niches remains poorly understood. Here we examine the role of an NAD(+) -dependent histone deacetylase,SIRT1,in modulating the neurogenic potential of neural precursors in the neurogenic niches of the adult mouse brain. We show that SIRT1 is expressed by proliferating adult subventricular zone and hippocampal neural precursors,although its transcript and protein levels are dramatically reduced during neural precursor differentiation. Utilizing a lentiviral-mediated delivery strategy,we demonstrate that abrogation of SIRT1 signaling by RNAi does not affect neural precursor numbers or their proliferation. However,SIRT1 knock down results in a significant increase in neuronal production in both the subventricular zone and the hippocampus. In contrast,enhancing SIRT1 signaling either through lentiviral-mediated SIRT1 overexpression or through use of the SIRT1 chemical activator Resveratrol prevents adult neural precursors from differentiating into neurons. Importantly,knock down of SIRT1 in hippocampal precursors in vivo,either through RNAi or through genetic ablation,promotes their neurogenic potential. These findings highlight SIRT1 signaling as a negative regulator of neuronal differentiation of adult subventricular zone and hippocampal neural precursors. textcopyright 2013 Wiley Periodicals,Inc. View Publication

A fundamental impediment to functional recovery from spinal cord injury (SCI) and traumatic brain injury is the lack of sufficient axonal regeneration in the adult central nervous system. There is thus a need to develop agents that can stimulate axon growth to re-establish severed connections. Given the critical role played by protein kinases in regulating axon growth and the potential for pharmacological intervention,small molecule protein kinase inhibitors present a promising therapeutic strategy. Here,we report a robust cell-based phenotypic assay,utilizing primary rat hippocampal neurons,for identifying small molecule kinase inhibitors that promote neurite growth. The assay is highly reliable and suitable for medium-throughput screening,as indicated by its Z'-factor of 0.73. A focused structurally diverse library of protein kinase inhibitors was screened,revealing several compound groups with the ability to strongly and consistently promote neurite growth. The best performing bioassay hit robustly and consistently promoted axon growth in a postnatal cortical slice culture assay. This study can serve as a jumping-off point for structure activity relationship (SAR) and other drug discovery approaches toward the development of drugs for treating SCI and related neurological pathologies. View Publication

UNLABELLED Herpes simplex virus 1 (HSV-1) establishes lifelong latent infections in the sensory neurons of the trigeminal ganglia (TG),wherein it retains the capacity to reactivate. The interferon (IFN)-driven antiviral response is critical for the control of HSV-1 acute replication. We therefore sought to further investigate this response in TG neurons cultured from adult mice deficient in a variety of IFN signaling components. Parallel experiments were also performed in fibroblasts isolated concurrently. We showed that HSV-1 replication was comparable in wild-type (WT) and IFN signaling-deficient neurons and fibroblasts. Unexpectedly,a similar pattern was observed for the IFN-sensitive vesicular stomatitis virus (VSV). Despite these findings,TG neurons responded to IFN-β pretreatment with STAT1 nuclear localization and restricted replication of both VSV and an HSV-1 strain deficient in γ34.5,while wild-type HSV-1 replication was unaffected. This was in contrast to fibroblasts in which all viruses were restricted by the addition of IFN-β. Taken together,these data show that adult TG neurons can mount an effective antiviral response only if provided with an exogenous source of IFN-β,and HSV-1 combats this response through γ34.5. These results further our understanding of the antiviral response of neurons and highlight the importance of paracrine IFN-β signaling in establishing an antiviral state. IMPORTANCE Herpes simplex virus 1 (HSV-1) is a ubiquitous virus that establishes a lifelong latent infection in neurons. Reactivation from latency can cause cold sores,blindness,and death from encephalitis. Humans with deficiencies in innate immunity have significant problems controlling HSV infections. In this study,we therefore sought to elucidate the role of neuronal innate immunity in the control of viral infection. Using neurons isolated from mice,we found that the intrinsic capacity of neurons to restrict virus replication was unaffected by the presence or absence of innate immunity. In contrast,neurons were able to mount a robust antiviral response when provided with beta interferon,a molecule that strongly stimulates innate immunity,and that HSV-1 can combat this response through the γ34.5 viral gene. Our results have important implications for understanding how the nervous system defends itself against virus infections. View Publication

Recombinant adeno-associated viruses (rAAV) have been widely used in gene therapy applications for central nervous system diseases. Though rAAV can efficiently target neurons and astrocytes in mouse brains,microglia,the immune cells of the brain,are refractile to rAAV. To identify AAV capsids with microglia-specific transduction properties,we initially screened the most commonly used serotypes,AAV1-9 and rh10,on primary mouse microglia cultures. While these capsids were not permissive,we then tested the microglial targeting properties of a newly characterized set of modified rAAV6 capsid variants with high tropism for monocytes. Indeed,these newly characterized rAAV6 capsid variants,specially a triply mutated Y731F/Y705F/T492V form,carrying a self-complementary genome and microglia-specific promoters (F4/80 or CD68) could efficiently and selectively transduce microglia in vitro. Delivery of these constructs in mice brains resulted in microglia-specific expression of green fluorescent protein,albeit at modest levels. We further show that CD68 promoter-driven expression of the inflammatory cytokine,interleukin-6,using this capsid variant leads to increased astrogliosis in the brains of wild-type mice. Our study describes the first instance of AAV-targeted microglial gene expression leading to functional modulation of the innate immune system in mice brains. This provides the rationale for utilizing these unique capsid/promoter combinations for microglia-specific gene targeting for modeling or functional studies. View Publication

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

Oxidative stress as a contributor to neuronal death during prion infection is supported by the fact that various oxidative damage markers accumulate in the brain during the course of this disease. The normal cellular substrate of the causative agent,the prion protein,is also linked with protective functions against oxidative stress. Our previous work has found that,in chronic prion infection,an apoptotic subpopulation of cells exhibit oxidative stress and the accumulation of oxidised lipid and protein aggregates with caspase recruitment. Given the likely failure of antioxidant defence mechanisms within apoptotic prion-infected cells,we aimed to investigate the role of the crucial antioxidant pathway components,superoxide dismutases (SOD) 1 and 2,in an in vitro model of chronic prion infection. Increased total SOD activity,attributable to SOD1,was found in the overall population coincident with a decrease in SOD2 protein levels. When apoptotic cells were separated from the total population,the induction of SOD activity in the infected apoptotic cells was lost,with activity reduced back to levels seen in mock-infected control cells. In addition,mitochondrial superoxide production was increased and mitochondrial numbers decreased in the infected apoptotic subpopulation. Furthermore,a pan-caspase probe colocalised with SOD2 outside of mitochondria within cytosolic aggregates in infected cells and inhibition of caspase activity was able to restore cellular levels of SOD2 in the whole unseparated infected population to those of mock-infected control cells. Our results suggest that prion propagation exacerbates an apoptotic pathway whereby mitochondrial dysfunction follows mislocalisation of SOD2 to cytosolic caspases,permitting its degradation. Eventually,cellular capacity to maintain oxidative homeostasis is overwhelmed,thus resulting in cell death. View Publication