A. Gritti

A. Vescovi

D. Bottai

E. Bongarzone

F. Galbiati

M. I. Givogri

English

AIR Universita degli studi di Milano

Poster Session BP8: Stem Cell BiologyBP8-01A novel method to enrich for neural stem cellsC. Y. Brazel, R. J. Felling and S. W. LevisonDepartment of Neural and Behavioral Science, Penn State Collegeof Medicine, Hershey, PA, USAIsolating a pure population of neural stem cells (NSCs) has been difficultsince no exclusive surface markers have been identified in vivo or in vitro touse for panning or FACS purification. Current FACS protocols rely onnegative sorting or transgenic mice that express GFP from either the nestin orthe SOX2 promoter. While these methods produce highly enriched prepa-rations of nestin+ or SOX2+ cells, only a fraction of the sorted cells arecompetent to form neurospheres, suggesting that a subset of the positive cellsare stem cells. Here, we demonstrate that postnatal rat NSCs express lowlevels of pro-apoptotic molecules and resist PI3K inhibition, ERK1/2inhibition, and glutamate receptor stimulation as compared with lateoligodendrocyte progenitors. Therefore, we exploited the relative resistanceof NSCs to these apoptotic stimuli to develop a novel method for stem cellenrichment. We find that maintaining SVZ cultures in the presence ofLY294002 and PD98059, inhibitors of PI3K and ERK1/2 signaling, for 72 heliminates lineage-restricted precursors, whereas stem cells survive. Not onlydo the surviving cells form neurospheres, but 89% of these neurospheresgenerate neurons, astrocytes, and oligodendrocytes. Without this enrichmentstep, only 42% of the neurospheres are multipotent. Additionally, theneurospheres enriched using this procedure produce 3-times more secondaryneurospheres, further suggesting that this procedure increases the ratio ofstem cells to lineage-restricted progenitors. This relatively simple andinexpensive means of enriching for NSCs will facilitate gene profilingexperiments as well as other studies to reveal the fundamental properties ofneural stem cells.Acknowledgment: This study was supported in part by MH59950 awardedto SWL.BP8-02Lysophosphatidic acid and its receptors as novelregulators of neural stem/progenitor cell growth anddifferentiationS. I. Svetlov, T. N. Ignatova, K. W. Wang, R. L. Hayesand V. G. KukekovUniversity of Florida, Department of Neuroscience, Gainesville, FL,USANeural stem/progenitor cells are able to self-renew as well as differentiateinto neuronal cells and glia in vitro. These cells produce neurospheres inserum-free medium containing epidermal growth factor (EGF) and fibroblastgrowth factor (FGF2). We demonstrate that lysophosphatidic acid (LPA), alysophospholipid growth factor, initiated the formation of neurospheres fromdissociated mouse postnatal forebrain even in the absence of EGF and FGF2.The neurosphere growth induced by LPAwas slower than by EGF and FGF2,sustained for up to 3 months, and associated with proliferation of the cellsharboring Sca-1 antigen and AC133, markers of primitive stem cells ofhematopoietic and neural origin. Sca-1 and AC133 positive cells alsoexpressed LPA1, LPA2 and LPA3 subtypes of LPA receptor. The formationand growth of neurospheres by LPA was inhibited by diacylglycerol-pyrophosphate, an antagonist of LPA1/LPA3 receptors. When neurosphereswere attached to the matrix and then induced to spread and differentiate in thepresence of LPA, a significant delay of cell migration was observed ascompared with EGF/FGF2. LPA receptors were expressed in the developingattached neurosphere, and co-localized with bIII-tubulin and nestin, andCNPase, but no with GFAP, a marker of astrocyte lineage. Sca-1 antigen andAC133 were still detected in the residual core of neurospheres grownattached in the presence of LPA, but not EGF/FGF, and co-localized withLPA receptors. Collectively, these data reveal novel properties of LPA towardinitiation and regulation of neural stem/progenitor cell growth, maintainingundifferentiated phenotype as well as differentiation in vitro.BP8-03Dopamine specifically inhibits neural stem cellproliferation suggesting a novel mechanism ofantipsychotic drug actionT. K. Kippin,*, S. Kapur*, and D. van der Kooy**University of Toronto, Toronto, Ont., Canada; Medical Universityof South Carolina, Charleston, SC, USA; Center for Addiction andMental Health, Toronto, Ont., CanadaAntipsychotic drugs increase brain tissue volume during treatment ofschizophrenic patients. We examined the contribution of cell proliferation tothis phenomenon. In adult rats chronic haloperidol (2 mg/kg/day via s.c.osmotic pump) significantly increases the numbers of neural stem cells andconstitutively proliferating progenitors in the subependyma of the lateralventricle, and results in more new neurons and glia in the olfactory bulbs andstriatum, respectively. The increase in neural stem cells by haloperidol doesnot occur in dopamine D2 receptor null mice and peripheral D2 receptorantagonism does not alter neural stem cell number demonstrating that theeffect of haloperidol on neural stem cells is dependent on central D2receptors. D2 receptors are present in both adult lateral ventricle subepend-yma and in adult-derived neurospheres. Neurospheres are generated fromcells FACS for D2 receptors demonstrating that neural stem cells express D2receptors. Treatment of neurosphere cultures with dopamine or quinpirolesignificantly decreased the proliferation of neural stem cell from wildtype,but not D2 receptor null, mice and this effect is reversible by haloperidol.These data demonstrate that haloperidol increases cell proliferation in theadult mammalian brain by antagonizing dopamine at D2 receptors on neuralstem cells. These findings implicate a role for cell genesis in the therapeuticeffects of antipsychotic drugs.BP8-04Integration of transplanted neural progenitors in thebrain of arylsulfatase a deficient miceE. Bongarzone, F. Galbiati, D. Bottai, A. Gritti, A. Vescoviand M. I. GivogriSan Raffaele Scientific Institute, Milano, ItalyMetachromatic leukodystrophy (MLD) is a dysmyelinating disease due tomutations in the arylsulfatase A (ARSA) gene. Central dysmyelination isconsequent to oligodendrocyte cell death, likely due to the lysosomalaccumulation of sulfatides. We tested the potential of oligodendrocyteprogenitors (OPCs) and of multipotential neural progenitors (NSCs) tosurvive and integrate in the brain of mice affected of MLD. Two-day-oldMLD pups received a single injection (105 cells/brain) of eitherLacZ + OPCs or GFP + NSCs in the lateral ventricle. One day afterinjection (1 dpi), LacZ + OPCs were detected within the delivery area in theMLD and control brains. However, LacZ+ cells were also detected inthe contralateral MLD hemisphere, indicative of a robust migration in themutant. At later time points, grafted cells migrated preferentially to thesubventricular zone (SVZ), rostral migratory stream (RMS), olfactory bulb(OB), corpus callosum and cortex and to a lesser extent, to the thalamus,hypothalamus and cerebellum of the MLD brain. In wild type mice,LacZ + OPCs remained within the injection area in the ventricle but were notfound in white matter areas or the SVZ-RMS-OB pathway. Most ofLacZ + OPCs that migrated in the MLD SVZ-RMS-OB pathway remainedundifferentiated while most of the LacZ+ cells that moved in the cortex andcorpus callosum differentiated into PLP+/MBP+ myelin associated cells.GFP + NSCs showed similar patterns of migration but additionally, theyrobustly migrated to cortical areas where they adopted neuronal morphol-ogies. Treatment with these cells led to a mild amelioration of central nerveconduction. These findings demonstrate that transplanted neural progenitorscan survive, migrate and differentiate in the brain of MLD mice. 2004 International Society for Neurochemistry, Journal of Neurochemistry, 90 (Suppl. 1) 47

Integration of transplanted neural progenitors in the brain of arylsulfatase : a deficient mice

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Integration of transplanted neural progenitors in the brain of arylsulfatase : a deficient mice

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