技术资料

In vitro exposure of neural progenitor cell (NPC) populations to reduced O(2) (e.g. 3% versus 20%) can increase their proliferation,survival and neuronal differentiation. Our objective was to determine if an acute (textless1hr),in vivo exposure to intermittent hypoxia (AIH) alters expansion and/or differentiation of subsequent in vitro cultures of NPC from the subventricular zone (SVZ). Neonatal C57BL/6 mice (postnatal day 4) were exposed to an AIH paradigm (20×1 minute; alternating 21% and 10% O(2)). Immediately after AIH,SVZ tissue was isolated and NPC populations were cultured and assayed either as neurospheres (NS) or as adherent monolayer cells (MASC). AIH markedly increased the capacity for expansion of cultured NS and MASC,and this was accompanied by increases in a proliferation maker (Ki67),MTT activity and hypoxia-inducible factor-1α (HIF-1α) signaling in NS cultures. Peptide blockade experiments confirmed that proteins downstream of HIF-1α are important for both proliferation and morphological changes associated with terminal differentiation in NS cultures. Finally,immunocytochemistry and Western blotting experiments demonstrated that AIH increased expression of the neuronal fate determination transcription factor Pax6 in SVZ tissue,and this was associated with increased neuronal differentiation in cultured NS and MASC. We conclude that in vivo AIH exposure can enhance the viability of subsequent in vitro SVZ-derived NPC cultures. AIH protocols may therefore provide a means to prime" NPC prior to transplantation into the injured central nervous system." View Publication

Insight into the normal function of PrP(C),and how it can be subverted to produce neurotoxic effects,is provided by PrP molecules carrying deletions encompassing the conserved central region. The most neurotoxic of these mutants,Δ105-125 (called ΔCR),produces a spontaneous neurodegenerative illness when expressed in transgenic mice,and this phenotype can be dose-dependently suppressed by co-expression of wild-type PrP. Whether the toxic activity of ΔCR PrP and the protective activity or wild-type PrP are cell-autonomous,or can be exerted on neighboring cells,is unknown. To investigate this question,we have utilized co-cultures of differentiated neural stem cells derived from mice expressing ΔCR or wild-type PrP. Cells from the two kinds of mice,which are marked by the presence or absence of GFP,are differentiated together to yield neurons,astrocytes,and oligodendrocytes. As a surrogate read-out of ΔCR PrP toxicity,we assayed sensitivity of the cells to the cationic antibiotic,Zeocin. In a previous study,we reported that cells expressing ΔCR PrP are hypersensitive to the toxic effects of several cationic antibiotics,an effect that is suppressed by co-expression of wild type PrP,similar to the rescue of the neurodegenerative phenotype observed in transgenic mice. Using this system,we find that while ΔCR-dependent toxicity is cell-autonomous,the rescuing activity of wild-type PrP can be exerted in trans from nearby cells. These results provide important insights into how ΔCR PrP subverts a normal physiological function of PrP(C),and the cellular mechanisms underlying the rescuing process. View Publication

Exercise has been shown to positively augment adult hippocampal neurogenesis; however,the cellular and molecular pathways mediating this effect remain largely unknown. Previous studies have suggested that microglia may have the ability to differentially instruct neurogenesis in the adult brain. Here,we used transgenic Csf1r-GFP mice to investigate whether hippocampal microglia directly influence the activation of neural precursor cells. Our results revealed that an exercise-induced increase in neural precursor cell activity was mediated via endogenous microglia and abolished when these cells were selectively removed from hippocampal cultures. Conversely,microglia from the hippocampi of animals that had exercised were able to activate latent neural precursor cells when added to neurosphere preparations from sedentary mice. We also investigated the role of CX(3)CL1,a chemokine that is known to provide a more neuroprotective microglial phenotype. Intraparenchymal infusion of a blocking antibody against the CX(3)CL1 receptor,CX(3)CR1,but not control IgG,dramatically reduced the neurosphere formation frequency in mice that had exercised. While an increase in soluble CX(3)CL1 was observed following running,reduced levels of this chemokine were found in the aged brain. Lower levels of CX(3)CL1 with advancing age correlated with the natural decline in neural precursor cell activity,a state that could be partially alleviated through removal of microglia. These findings provide the first direct evidence that endogenous microglia can exert a dual and opposing influence on neural precursor cell activity within the hippocampus,and that signaling through the CX(3)CL1-CX(3)CR1 axis critically contributes toward this process. View Publication

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

Neurogenesis decreases during aging and following cranial radiotherapy,causing a progressive cognitive decline that is currently untreatable. However,functional neural stem cells remained present in the subventricular zone of high dose-irradiated and aged mouse brains. We therefore investigated whether alterations in the neurogenic niches are perhaps responsible for the neurogenesis decline. This hypothesis was supported by the absence of proliferation of neural stem cells that were engrafted into the vascular niches of irradiated host brains. Moreover,we observed a marked increase in TGF-β1 production by endothelial cells in the stem cell niche in both middle-aged and irradiated mice. In co-cultures,irradiated brain endothelial cells induced the apoptosis of neural stem/progenitor cells via TGF-β/Smad3 signalling. Strikingly,the blockade of TGF-β signalling in vivo using a neutralizing antibody or the selective inhibitor SB-505124 significantly improved neurogenesis in aged and irradiated mice,prevented apoptosis and increased the proliferation of neural stem/progenitor cells. These findings suggest that anti-TGF-β-based therapy may be used for future interventions to prevent neurogenic collapse following radiotherapy or during aging. View Publication