技术资料

BACKGROUND Neuronal stem cells (NSCs) are promising for neurointestinal disease therapy. Although NSCs have been isolated from intestinal musclularis,their presence in mucosa has not been well described. Mucosa-derived NSCs are accessible endoscopically and could be used autologously. Brain-derived Nestin-positive NSCs are important in endogenous repair and plasticity. The aim was to isolate and characterize mucosa-derived NSCs,determine their relationship to Nestin-expressing cells and to demonstrate their capacity to produce neuroglial networks in vitro and in vivo. METHODS Neurospheres were generated from periventricular brain,colonic muscularis (Musc),and mucosa-submucosa (MSM) of mice expressing green fluorescent protein (GFP) controlled by the Nestin promoter (Nestin-GFP). Neuronal stem cells were also grown as adherent colonies from intestinal mucosal organoids. Their differentiation potential was assessed using immunohistochemistry using glial and neuronal markers. Brain and gut-derived neurospheres were transplanted into explants of chick embryonic aneural hindgut to determine their fate. KEY RESULTS Musc- and MSM-derived neurospheres expressed Nestin and gave rise to cells of neuronal,glial,and mesenchymal lineage. Although Nestin expression in tissue was mostly limited to glia co-labelled with glial fibrillary acid protein (GFAP),neurosphere-derived neurons and glia both expressed Nestin in vitro,suggesting that Nestin+/GFAP+ glial cells may give rise to new neurons. Moreover,following transplantation into aneural colon,brain- and gut-derived NSCs were able to differentiate into neurons. CONCLUSIONS & INFERENCES Nestin-expressing intestinal NSCs cells give rise to neurospheres,differentiate into neuronal,glial,and mesenchymal lineages in vitro,generate neurons in vivo and can be isolated from mucosa. Further studies are needed for exploring their potential for treating neuropathies. 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

Differentiation of pluripotent and lineage restricted stem cells such as neural stem cells (NSCs) was studied on conducting substrates of various nature without perturbation of the genome with exogenous genetic material or chemical stimuli. Primary mouse adult neural stem cells (NSCs) and P19 pluripotent embryonal (P19 EC) carcinoma cells were used. Expression levels of neuronal markers β-III-tubulin and neurofilament were evaluated by immunochemistry and flow cytometry. It was shown that the ability of the substrate to induce differentiation directly correlated with its conductivity. Conducting substrates (conducting oxides or doped pi-conjugated organic polymers) with different morphology,structure,and conductivity mechanisms all promoted differentiation of NSC and P19 cells into neuronal lineage to a similar degree without use of additional factors such as poly-L-ornithine coating or retinoic acid,as verified by their morphology and upregulation of the neuronal markers but not astrocyte marker GFAP. However,substrates with low conductance below ca. 10(-4) S cm(-2) did not show this ability. Morphology of differentiating cells was visualized by atomic force microscopy. NSCs cells increased β-III-tubulin expression by 95% and P19 cells by over 30%. Our results suggest that the substrate conductivity is a key factor governing the cell fate. Differentiation of P19 cells into neuronal lineage on conducting substrates was attributed to downregualtion of Akt signaling pathway and increase in expression of dual oxidase 1 (DUOX 1). View Publication

The integration of microscale engineering,microfluidics,and AC electrokinetics such as dielectrophoresis has generated novel microsystems that enable quantitative analysis of cellular phenotype,function,and physiology. These systems are increasingly being used to assess diverse cell types,such as stem cells,so it becomes critical to thoroughly evaluate whether the systems themselves impact cell function. For example,engineered microsystems have been utilized to investigate neural stem/progenitor cells (NSPCs),which are of interest due to their potential to treat CNS disease and injury. Analysis by dielectrophoresis (DEP) microsystems determined that unlabeled NSPCs with distinct fate potential have previously unrecognized distinguishing electrophysiological characteristics,suggesting that NSPCs could be isolated by DEP microsystems without the use of cell type specific labels. To gauge the potential impact of DEP sorting on NSPCs,we investigated whether electric field exposure of varying times affected survival,proliferation,or fate potential of NSPCs in suspension. We found short-term DEP exposure (1 min or less) had no effect on NSPC survival,proliferation,or fate potential revealed by differentiation. Moreover,NSPC proliferation (measured by DNA synthesis and cell cycle kinetics) and fate potential were not altered by any length of DEP exposure (up to 30 min). However,lengthy exposure (textgreater5 min) to frequencies near the crossover frequency (50-100 kHz) led to decreased survival of NSPCs (maximum ∼30% cell loss after 30 min). Based on experimental observations and mathematical simulations of cells in suspension,we find that frequencies near the crossover frequency generate an induced transmembrane potential that results in cell swelling and rupture. This is in contrast to the case for adherent cells since negative DEP frequencies lower than the crossover frequency generate the highest induced transmembrane potential and damage for these cells. We clarify contrasting effects of DEP on adherent and suspended cells,which are related to the cell position within the electric field and the strength of the electric field at specific distances from the electrodes. Modeling of electrode configurations predicts optimal designs to induce cell movement by DEP while limiting the induced transmembrane potential. We find DEP electric fields are not harmful to stem cells in suspension at short exposure times,thus providing a basis for developing DEP-based applications for stem cells. View Publication

We describe a novel form of tumor cell plasticity characterized by reversible adaptive plasticity in murine and human neuroblastoma. Two cellular phenotypes were defined by their ability to exhibit adhered,anchorage dependent (AD) or sphere forming,anchorage independent (AI) growth. The tumor cells could transition back and forth between the two phenotypes and the transition was dependent on the culture conditions. Both cell phenotypes exhibited stem-like features such as expression of nestin,self-renewal capacity,and mesenchymal differentiation potential. The AI tumorspheres were found to be more resistant to chemotherapy and proliferated slower in vitro compared to the AD cells. Identification of specific molecular markers like MAP2,β-catenin,and PDGFRβ enabled us to characterize and observe both phenotypes in established mouse tumors. Irrespective of the phenotype originally implanted in mice,tumors grown in vivo show phenotypic heterogeneity in molecular marker signatures and are indistinguishable in growth or histologic appearance. Similar molecular marker heterogeneity was demonstrated in primary human tumor specimens. Chemotherapy or growth factor receptor inhibition slowed tumor growth in mice and promoted initial loss of AD or AI heterogeneity,respectively. Simultaneous targeting of both phenotypes led to further tumor growth delay with emergence of new unique phenotypes. Our results demonstrate that neuroblastoma cells are plastic,dynamic,and may optimize their ability to survive by changing their phenotype. Phenotypic switching appears to be an adaptive mechanism to unfavorable selection pressure and could explain the phenotypic and functional heterogeneity of neuroblastoma. 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

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

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

Ampakines are chemical compounds known to modulate the properties of ionotropic α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA)-subtype glutamate receptors. The functional effects attributed to ampakines involve plasticity and the increase in synaptic efficiency of neuronal circuits,a process that may be intimately associated with differentiation of newborn neurons. The subventricular zone (SVZ) is the main neurogenic niche of the brain,containing neural stem cells with brain repair potential. Accordingly,the identification of new pharmaceutical compounds with neurogenesis-enhancing properties is important as a tool to promote neuronal replacement based on the use of SVZ cells. The purpose of the present paper is to examine the possible proneurogenic effects of ampakine CX546 in cell cultures derived from the SVZ of early postnatal mice. We observed that CX546 (50 μm) treatment triggered an increase in proliferation,evaluated by BrdU incorporation assay,in the neuroblast lineage. Moreover,by using a cell viability assay (TUNEL) we found that,in contrast to AMPA,CX546 did not cause cell death. Also,both AMPA and CX546 stimulated neuronal differentiation as evaluated morphologically through neuronal nuclear protein (NeuN) immunocytochemistry and functionally by single-cell calcium imaging. Accordingly,short exposure to CX546 increased axonogenesis,as determined by the number and length of tau-positive axons co-labelled for the phosphorylated form of SAPK/JNK (P-JNK),and dendritogenesis (MAP2-positive neurites). Altogether,this study shows that ampakine CX546 promotes neurogenesis in SVZ cell cultures and thereby may have potential for future stem cell-based therapies. View Publication

The WNT pathway plays multiple roles in neural development and is crucial for establishment of the embryonic cerebellum. In addition,WNT pathway mutations are associated with medulloblastoma,the most common malignant brain tumor in children. However,the cell types within the cerebellum that are responsive to WNT signaling remain unknown. Here we investigate the effects of canonical WNT signaling on two important classes of progenitors in the developing cerebellum: multipotent neural stem cells (NSCs) and granule neuron precursors (GNPs). We show that WNT pathway activation in vitro promotes proliferation of NSCs but not GNPs. Moreover,mice that express activated β-catenin in the cerebellar ventricular zone exhibit increased proliferation of NSCs in that region,whereas expression of the same protein in GNPs impairs proliferation. Although β-catenin-expressing NSCs proliferate they do not undergo prolonged expansion or neoplastic growth; rather,WNT signaling markedly interferes with their capacity for self-renewal and differentiation. At a molecular level,mutant NSCs exhibit increased expression of c-Myc,which might account for their transient proliferation,but also express high levels of bone morphogenetic proteins and the cyclin-dependent kinase inhibitor p21,which might contribute to their altered self-renewal and differentiation. These studies suggest that the WNT pathway is a potent regulator of cerebellar stem cell growth and differentiation. 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

Neurogenesis persists in the rodent dentate gyrus (DG) throughout adulthood but declines with age and stress. Neural progenitor cells (NPCs) residing in the subgranular zone of the DG are regulated by an array of growth factors and respond to the microenvironment,adjusting their proliferation level to determine the rate of neurogenesis. Here we report that genetic deletion of neurofibromin (Nf1),a tumor suppressor with RAS-GAP activity,in adult NPCs enhanced DG proliferation and increased generation of new neurons in mice. Nf1 loss-associated neurogenesis had the functional effect of enhancing behavioral responses to subchronic antidepressants and,over time,led to spontaneous antidepressive-like behaviors. Thus,our findings establish an important role for the Nf1-Ras pathway in regulating adult hippocampal neurogenesis,and demonstrate that activation of adult NPCs is sufficient to modulate depression- and anxiety-like behaviors. View Publication