Temporal profile of stem cell division, migration, and differentiation from subventricular zone to olfactory bulb after transient forebrain ischemia in gerbils.

The stage of neurogenesis can be divided into three steps: proliferation, migration, and differentiation. To elucidate their detailed relations after ischemia, the three steps were comprehensively evaluated, in the subventricular zone (SVZ) through the rostral migratory stream (RMS) to the olfactory bulb (OB), in adult gerbil brain after 5 minutes of transient forebrain ischemia. Bromodeoxyuridine (BrdU), highly polysialylated neural cell adhesion molecule (PSA-NCAM), neuronal nuclear antigen (NeuN), and glial fibrillary acidic protein (GFAP) were used as markers for proliferation, migration, and differentiation, respectively. The number of BrdU-labeled cells that coexpressed PSA-NCAM and the size of PSA-NCAM-positive cell colony increased in the SVZ with a peak at 10 d after transient ischemia. In the RMS, the number of BrdU-labeled cells that coexpressed PSA-NCAM increased, with a delayed peak at 30 d, when the size of RMS itself became larger and the number of surrounding GFAP-positive cells increased. In the OB, BrdU + NeuN double positive cells were detected at 30 and 60 d. NeuN staining and terminal deoxynucleotidyl dUTP nick-end labeling staining showed no neuronal cell loss around the SVZ, and in the RMS and the OB after transient ischemia. These findings indicate that transient forebrain ischemia enhances neural stem cell proliferation in the SVZ without evident neuronal cell loss, and has potential neuronal precursor migration with activation of GFAP-positive cells through the RMS to the OB.     

Neuroectodermal and microglial differentiation of bone marrow cells in the mouse spinal cord and sensory ganglia

There is now evidence that bone marrow (BM) can generate cells expressing neuronal antigens in adult mouse brain. In the present study, we examined the spinal cord and dorsal root ganglia (DRG) of adult mice 3 months after BM cell transplantation from transgenic donor mice expressing the enhanced green fluorescent protein (GFP). To determine whether GFP(+) cells acquire neuroectodermal phenotypes, we tested, by immunocytochemistry followed by confocal analysis, the coexpression of the astrocytic marker glial fibrillary acidic protein (GFAP) and the neuronal markers NeuN, neurofilament (NF), and class III beta-tubulin (TuJ1). Rare GFP(+) cells coexpressing TuJ1, NF, and NeuN were found both in spinal cord and in sensory ganglia. These cells have small dimensions and short cytoplasmic processes, probably reflecting an immature phenotype. Double GFP and GFAP positivity was found only in spinal cord. To determine whether cell fusion with endogenous cells occurred, we investigated the nuclear content of cells coexpressing GFP and neuronal or astrocytic markers, demonstrating that these cells have only one nucleus and a DNA ploidy that it is not different from that of surrounding neurons and astrocytes. Large numbers of GFP(+) cells are also positively stained for F4/80, a microglial-recognizing antibody, and present a characteristic microglial-like morphology both in spinal cord and, with a higher frequency, in sensory ganglia. These data support a potential role for BM-derived stem cells in spinal cord neuroneogenesis. They also confirm that the microglial compartment within the CNS and in DRG undergoes a relatively fast turnover, with the contribution of hematopoietic stem cells. Both these findings might prove useful for the development of treatments for spinal cord neurodegenerative and acquired disorders.     

Green fluorescent protein bone marrow cells express hematopoietic and neural antigens in culture and migrate within the neonatal rat brain

Finding a reliable source of alternative neural stem cells for treatment of various diseases and injuries affecting the central nervous system is a challenge. Numerous studies have shown that hematopoietic and nonhematopoietic progenitors derived from bone marrow (BM) under specific conditions are able to differentiate into cells of all three germ layers. Recently, it was reported that cultured, unfractionated (whole) adult BM cells form nestin-positive spheres that can later initiate neural differentiation (Kabos et al., 2002). The identity of the subpopulation of BM cells that contributes to neural differentiation remains unknown. We therefore analyzed the hematopoietic and neural features of cultured, unfractionated BM cells derived from a transgenic mouse that expresses green fluorescent protein (GFP) in all tissues. We also transplanted the BM cells into the subventricular zone (SVZ), a region known to support postnatal neurogenesis. After injection of BM cells into the neurogenic SVZ in neonatal rats, we found surviving GFP+ BM cells close to the injection site and in various brain regions, including corpus callosum and subcortical white matter. Many of the grafted cells were detected within the rostral migratory stream (RMS), moving toward the olfactory bulb (OB), and some cells reached the subependymal zone of the OB. Our in vitro experiments revealed that murine GFP+ BM cells retained their proliferation and differentiation potential and predominantly preserved their hematopoietic identity (CD45, CD90, CD133), although a few expressed neural antigens (nestin, glial fibrillary acdiic protein, TuJ1).     

Targeted in utero delivery of a retroviral vector for gene transfer in the rodent brain

In vivo application of viral vectors for gene transfer is a commonly used tool in anatomical and functional studies, as well as in development of neuroprotective and restorative strategies for therapy. Although the most common route of administration is via direct injection into the brain parenchyma in adult animals, a number of short-term studies have been performed in the developing central nervous system. Here we investigated the long-term transgene expression following in utero delivery of a retroviral vector encoding for the green fluorescent protein (GFP) marker gene at embryonic days 14.5-17.5 using an ultrasound-guided injection system. Intraparenchymal injections of the ganglionic eminence were compared with vector delivery to the intracerebroventricular space. Injections into the ganglionic eminences resulted in a predominantly unilateral transduction localized to the forebrain, giving rise to GFP-positive (GFP+) neurons and astrocytes in the striatum, olfactory bulb, cortex and hippocampus. When the vector was injected into the lateral ventricle, on the other hand, widespread expression of GFP was seen throughout the brain. The total number of GFP+ cells in the striatum was estimated to be between 20,000 and 50,000 cells using a computerized stereological quantification tool. Phenotypic characterization of these transduced cells using confocal microscopical analysis showed that 64% were NeuN+ neurons, 14% APC+ oligodendrocytes and 15% glial cells labelled with GFAP, S100beta and Iba1, when the vector injection was performed at E14.5. Delivery into later embryos resulted in a reduction in neuronal profiles with a reciprocal increase in glial cells.     

Generation of neural stem cell-like cells from bone marrow-derived human mesenchymal stem cells

Under appropriate culture conditions, bone marrow (BM)-derived mesenchymal stem cells are capable of differentiating into diverse cell types unrelated to their phenotypical embryonic origin, including neural cells. Here, we report the successful generation of neural stem cell (NSC)-like cells from BM-derived human mesenchymal stem cells (hMSCs). Initially, hMSCs were cultivated in a conditioned medium of human neural stem cells. In this culture system, hMSCs were induced to become NSC-like cells, which proliferate in neurosphere-like structures and express early NSC markers. Like central nervous system-derived NSCs, these BM-derived NSC-like cells were able to differentiate into cells expressing neural markers for neurons, astrocytes, and oligodendrocytes. Whole-cell patch clamp recording revealed that neuron-like cells, differentiated from NSC-like cells, exhibited electrophysiological properties of neurons, including action potentials. Transplantation of NSC-like cells into mouse brain confirmed that these NSC-like cells retained their capability to differentiate into neuronal and glial cells in vivo. Our data show that multipotent NSC-like cells can be efficiently produced from BM-derived hMSCs in culture and that these cells may serve as a useful alternative to human neural stem cells for potential clinical applications such as autologous neuroreplacement therapies.     

MARK2/Par-1 guides the directionality of neuroblasts migrating to the olfactory bulb

In rodents and most other mammals studied, neuronal precursors generated in the subventricular zone (SVZ) migrate to the adult olfactory bulb (OB) to differentiate into interneurons called granule and periglomerular cells. How the newborn cells navigate in the postnatal forebrain to reach precisely their target area is largely unknown. However, it is often thought that postnatal neurogenesis recapitulates the neuronal development occurring during embryogenesis.During brain development, intracellular kinases are key elements for controlling cell polarization as well as the coupling between polarization and cellular movement. We show here that the polarity kinase MARK2 maintains its expression in the postnatal SVZ-OB system. We therefore investigated the potential role of this kinase in adjusting postnatal neuroblast migration. We employed mouse brain slices maintained in culture, in combination with lentiviral vector injections designed to label neuronal precursors with GFP and to diminish the expression of MARK2. Time-lapse video microscopy was used to monitor neuroblast migration in the postnatal forebrain from SVZ precursors to cells populating the OB.We found that reduced MARK2 expression resulted in altered migratory patterns and stalled neuroblasts in the rostral migratory stream (RMS). In agreement with the observed migratory defects, we report a diminution of the proportion of cells reaching the OB layers. Our study reveals the involvement of MARK2 in the maintenance of the migratory direction in postnatally-generated neuroblasts and consequently on the control of the number of newly-generated neurons reaching and integrating the appropriate target circuits.     

Postnatal changes in the distribution and density of neuronal nuclei and doublecortin antigens in domestic chicks (Gallus domesticus)

To understand better the rate of neurogenesis and the distribution of new neurons in posthatch domestic chicks, we describe and compare the expression of the neuronal nuclei protein (NeuN, a.k.a. Fox-3) and doublecortin antigens in the whole brain of chicks 2 days, 8 days, and 14 weeks posthatch. In the forebrain ventricular and paraventricular zones, the density of bromodeoxyuridine-, NeuN-, and doublecortin-labeled cells was compared between chicks 24 hours and 7 days after an injection of bromodeoxyuridine (2 and 8 days posthatch, respectively). The distribution of NeuN-labeled neurons was similar to Nissl-stained tissue, with the exception of some areas where neurons did not express NeuN: cerebellar Purkinje cells and olfactory bulb mitral cells. The ventral tegmental area of 2-day-old chicks was also faintly labeled. The distribution of doublecortin was similar at all timepoints, with doublecortin-labeled profiles located throughout all forebrain areas as well as in the cerebellar granule cell layer. However, doublecortin labeling was not detectable in any midbrain or brainstem areas. Our data indicate that a significant number of new neurons is still formed in the telencephalon of posthatch domestic chicks, whereas subtelencephalic areas (except for the cerebellum) finish their neuronal expansion before hatching. Most newly formed cells in chicks leave the paraventricular zone after hatching, but a pool of neurons stays in the vicinity of the ventricular zone and matures in situ within 7 days. Proliferating cells often migrate laterally along forebrain laminae into still-developing brain areas.     

Neuronal replacement in the injured olfactory bulb

The adult forebrain subventricular zone contains neural stem cells that produce neurons destined for the olfactory bulb, where interneuron populations turnover throughout life. Forebrain injuries can stimulate production of these cells, and re-direct migrating precursors from the olfactory system to areas of damage, where their region-appropriate differentiation and long-term functional integration remain a matter for debate. Paradoxically, little is known about the ability of these progenitors to replace olfactory neurons lost to injury. Their innate capacity to generate bulb neurons may give them an advantage in this regard, and using injections of N-methyl-d-aspartate to kill mature olfactory bulb neurons, combined with bromodeoxyuridine labeling to monitor the fate of adult-born cells, we investigated the potential for injury-induced neurogenesis in this system. Widespread degeneration of bulb neurons did not affect the rate of cell proliferation in the subventricular zone, or cause neuroblasts to divert from their normal migratory route. However migration was slowed by the injury, leading to the accumulation and differentiation of neuroblasts as NeuN+ cells in the rostral migratory stream within 2 weeks of their birth. Despite this, a subset of new neurons successfully invaded the damaged bulb tissue, where they expressed neuronal markers including NeuN, calretinin, GABA, and tyrosine hydroxylase, with some surviving here for as long as 6 months. To test for functional integration of cells born post-injury, we also performed smaller NMDA lesions in restricted portions of the bulb granule cell layer and observed adult-born NeuN+ cells in these areas within 5 weeks, and BrdU+ cells that expressed the immediate-early gene c-fos following odor stimulation. These data suggest that the normal neurogenic capacity of the adult subventricular zone can be adapted to replace subsets of olfactory neurons lost to injury.     

Glial conversion of SVZ-derived committed neuronal precursors after ectopic grafting into the adult brain

In the adult mouse forebrain, large numbers of neuronal precursors, destined to become GABA- and dopamine-producing interneurons of the olfactory bulb (OB), are generated in the subventricular zone (SVZ). Although this neurogenic system represents a potential reservoir of stem and progenitor cells for brain repair approaches, information about the survival and differentiation of SVZ-derived cells in ectopic brain regions is still fragmentary. We show here that ectopic grafting of SVZ tissue gave rise to two morphologically distinguishable cell types displaying oligodendrocytic or astrocytic characteristics. Since SVZ tissue contains neuronal and glial progenitors, we used magnetic cell sorting to deplete A2B5+ glial progenitors from the dissociated SVZ and to positively select cells that express PSA-NCAM. This procedure allowed the purification of neuronal precursors expressing TUJ1, DCX and GAD65/67. Transplantation of these cells led again to the generation of the same two glial cell types, showing that committed interneuron precursors undergo glial differentiation outside their normal environment.     

Hematopoietic origin of microglial and perivascular cells in brain

BACKGROUND: Bone marrow (BM)-derived cells differentiate into a wide variety of cell types. BM contains a heterogeneous population of stem and progenitor cells including hematopoietic stem cells, marrow stromal cells, and perhaps other progenitor cells. To establish unequivocally the transdifferentiation capability of a hematopoietic cell to a nonhematopoietic cell (endothelial cells, neurons, and glial cells), it is imperative to demonstrate that a single cell or clone of that single cell (clonal analysis) differentiates into cells comprising vessels or other cells in the brain. METHODS: We generated mice that exhibited a high level of hematopoietic reconstitution from a single enhanced green fluorescent protein (EGFP) stem cell. To achieve this, we combined FACS sorting and cell culture to generate a population of cells derived from a single hematopoietic stem cell (Lin-, CD34-, c-kit+, and Sca-1+). Clonal populations of cells were then transplanted into lethally irradiated recipient mice. After 3-4 months of engraftment, some mice underwent middle cerebral artery (MCA) suture occlusion. EGFP immunocytochemistry and dual labeling was performed with cell-specific markers on tissue from various time points. RESULTS: In all transplanted mice, EGFP+ highly ramified cells were seen in the brain parenchyma. These cells stained with RCA120 lectin and had the characteristics of parenchymal microglial cells. In brains without infarction and in uninfarcted brain regions of mice that underwent MCA occlusion, there were many EGFP+ cells in a perivascular distribution, associated with both small and larger blood vessels. The cells were tightly apposed to the vessel wall and some had long processes that enveloped the endothelial cells. After MCA occlusion, there was an influx of EGFP expressing cells in the ischemic tissue that colocalized with the "neovascularization." These EGFP+ cells were wrapped around endothelial cells in an albuminal location and did not coexpress von Willebrand Factor or CD31. We detected rare dual-labeled EGFP and NeuN-expressing cells. We detected two staining patterns. The more frequent pattern was phagocytosis of NeuN cells by EGFP expressing cells. However, we also detected rarer cells where the EGFP and NeuN appeared to be colocalized by confocal microscopy. CONCLUSIONS: HSC differentiate into parenchymal microglial cells and perivascular cells in the brain. The numbers of these cells increase after cerebral ischemia. The HSC is therefore one source of parenchymal microglial cells and a source for perivascular cells. After a cerebral infarction, there are rare HSC-derived cells that stain with the neuronal marker, NeuN. However, the more common pattern appears to represent phagocytosis of damaged neurons by EGFP+ microglial cells.     

Hematopoietic growth factors pass through the blood-brain barrier in intact rats

We have previously demonstrated that receptors for hematopoietic growth factors, stem cell factor (SCF) and granulocyte-colony stimulating factor (G-CSF) are expressed in the neurons and the neural progenitor cells (NPCs) of the adult rat brain, and that systemic administration of SCF and G-CSF in the first week after induction of cortical brain ischemia (3 h–7 days post-ischemia) significantly improves functional outcome, augments NPC proliferation, and reduces infarct volume in rats. The purpose of the present study is to determine whether SCF and G-CSF pass through the blood–brain barrier (BBB) in intact rats. The growth factors were labeled with iodine (I¹²⁵), a radioactive compound. I¹²⁵-SCF and I¹²⁵-G-CSF were intravenously administered and the concentrations of I¹²⁵-SCF and I¹²⁵-G-CSF in the blood plasma and the brain were determined at 10, 30, 60, and 120 min after injection. We observed that both SCF and G-CSF were slowly and continuously transported from the blood stream to the brain in the same rate. In addition, both immunofluorescent staining and Western blots showed that receptors for SCF and G-CSF were expressed in the capillaries of the adult rat brain, suggesting that SCF and G-CSF entry to the brain may be mediated via receptor-mediated transport, one of the endogenous transports in the BBB. These data indicate that both SCF and G-CSF were able to pass through the BBB in intact animals. This observation will help in further exploring the physiological role of peripheral SCF and G-CSF in the brain and therapeutic possibility to chronic stroke.     

干细胞生长因子和粒细胞集落刺激因子与心肌梗死大鼠体内骨髓间充质干细胞的迁移*

背景：外周静脉移植间充质干细胞只有1%~5%的移植细胞能归巢到心肌梗死区域。 目的：观察干细胞生长因子、粒细胞集落刺激因子对骨髓间充质干细胞归巢的影响。 方法：采用贴壁培养法分离培养SD大鼠骨髓间充质干细胞,取传至3~5代细胞。建立SD大鼠急性心肌梗死模型,干细胞生长因子组、粒细胞集落刺激因子组、干细胞生长因子+粒细胞集落刺激因子组在骨髓间充质干细胞移植前3 d和移植后3 d单独或混合皮下注射干细胞生长因子、粒细胞集落刺激因子,骨髓间充质干细胞组不注射细胞因子。 结果与结论：荧光显微镜下观察,骨髓间充质干细胞迁移至心肌梗死组织,骨髓间充质干细胞组、干细胞生长因子组、粒细胞集落刺激因子组迁移至心肌梗死区的骨髓间充质干细胞数量没有明显的区别(P > 0.05),干细胞生长因子+粒细胞集落刺激因子组的骨髓间充质干细胞数量明显高于其他3组(P < 0.05)。免疫荧光组织化学显示,植入的部分骨髓间充质干细胞表达心肌特异蛋白cTnI。结果说明干细胞生长因子和粒细胞集落刺激因子两种细胞因子联合应用可以促进骨髓间充质干细胞归巢至心肌梗死区域,在体内微环境的诱导下,骨髓间充质干细胞能够转化为心肌样细胞。     

Origin and distribution of bone marrow-derived cells in the central nervous system in a mouse model of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is associated with increased numbers of microglia within the central nervous system (CNS). However, it is unknown whether the microgliosis results from proliferation of CNS resident microglia, or recruitment of bone marrow (BM)-derived microglial precursors. Here we assess the distribution and number of BM-derived cells in spinal cord using transplantation of green fluorescent protein (GFP)-labeled BM cells into myelo-ablated mice over-expressing human mutant superoxide dismutase 1 (mSOD), a murine model of ALS. Transplantation of GFP+ BM did not affect the rate of disease progression in mSOD mice. Mean numbers of microglia and GFP+ cells in spinal cords of control mice were not significantly different from those in asymptomatic mSOD mice and showed no change with animal age. The number of GFP+ cells and microglia (F4/80+ and CD11b+ cells) within the spinal cord of mSOD mice increased compared to age-matched controls at a time when mSOD mice exhibited disease symptoms, continuing up to disease end-stage. Although we observed an increase in the number of GFP+ cells in spinal cords of mSOD mice with disease symptoms, mean numbers of GFP+ F4/80+ cells comprised less than 20% of all F4/80+ cells and did not increase with disease progression. Furthermore, the relative rates of proliferation in CD45+GFP- and CD45+GFP+ cells were comparable. Thus, we demonstrate that the microgliosis present in spinal cord tissue of mSOD mice is primarily due to an expansion of resident microglia and not to the recruitment of microglial precursors from the circulation.     

Perisomatic‐targeting granule cells in the mouse olfactory bulb

Inhibitory interneurons in the hippocampus and neocortex are differentiated into several morphological and functional subtypes that innervate distinct subcellular domains of principal neurons. In the olfactory bulb (OB), odor information is processed by local neuronal circuits that include the major inhibitory interneuron, granule cells (GCs). All GCs reported to date target their inhibitory output synapses mainly to dendrites of mitral cells (MCs) and tufted cells (TCs) in the external plexiform layer (EPL). Here we identified a novel type of GC that targets output synapses selectively to the perisomatic region of MCs. In the OB of adult transgenic mice expressing green fluorescent protein (GFP) under the control of nestin gene regulatory regions, we observed cells in the granule cell layer (GCL) that have GC‐like morphology and strongly express GFP (referred to as type S cells). Type S cells expressed NeuN and GAD67, molecular markers for GCs. Intracellular labeling of type S cells revealed that their dendrites did not enter the EPL, but formed branches and spines within the GCL, internal plexiform layer, and mitral cell layer. Type S cells typically had huge spines at the ends of the apical dendrites. Some of the terminal spines attached to the perisomatic region of MCs and formed dendrosomatic reciprocal synapses with a presumed granule‐to‐mitral inhibitory synapse and a mitral‐to‐granule excitatory synapse. These findings indicate the morphological differentiation of GCs into dendritic‐targeting and perisomatic‐targeting subsets, and suggest the functional differentiation of the GC subsets in the processing of odor information in the OB. J. Comp. Neurol. 515:409–426, 2009. © 2009 Wiley‐Liss, Inc.     

Substantial migration of SVZ cells to the cortex results in the generation of new neurons in the excitotoxically damaged immature rat brain

Mammalian SVZ progenitors continuously generate new neurons in the olfactory bulb. After injury, changes in SVZ cell number suggest injury-induced migration. Studies that trace the migration of SVZ precursors into neurodegenerating areas are lacking. Previously, we showed a decrease in BrdU+SVZ cells following excitotoxic damage to the immature rat cortex. Here, we demonstrate that NMDA-induced injury forces endogenous Cell Tracker Green (CTG) labeled VZ/SVZ precursors out of the SVZ into the neurodegenerating cortex. CTG+/Nestin+/Filamin A+ precursors are closely associated with vimentin+/GFAP+/GLAST+ filaments and express both chemokine receptor CXCR4 and Robo1. In the cortex, SVZ-derived progenitors show a progressive expression of developing, migrating and mature neurons and glial markers. CTG+/GFAP+ astrocytes greatly outnumber CTG+/MAP2+/NeuN+ neurons. SVZ-derived progenitors differentiate into both tbr1+ cortical glutamatergic neurons and calretinin+ interneurons. But, there is little integration of these neurons into the existing circuitry, as seen by Fluorogold retrograde tracing from the internal capsule.     

Visualization of neurogenesis in the central nervous system using nestin promoter-GFP transgenic mice

Neurons are generated from neural progenitor cells not only during development but also in the mature brain. To develop an in vivo system for analyzing neurogenesis, we generated transgenic mice expressing green fluorescent protein (GFP) under the control of regulatory regions of the nestin gene. GFP fluorescence was observed in areas and during periods connected with neurogenesis, including embryonic neuroepithelium, neonatal cerebellum, and hippocampal dentate gyrus and rostral migratory pathway from the subventricular zone to the olfactory bulb in the adult. GFP-positive cells in the adult brain included immature neuronal cells expressing polysialylated NCAM. BrdU labeling experiments revealed that newly generated interneurons which migrated rostrally from the subventricular zone expressed GFP until they reached the olfactory bulb. These results indicate that nestin promoter-GFP transgenic mice can be utilized to visualize the regions of neurogenesis throughout the life of the animals and to follow the migration and differentiation of newly generated neurons.     

Neural stem and progenitor cells in nestin-GFP transgenic mice

Neural stem cells generate a wide spectrum of cell types in developing and adult nervous systems. These cells are marked by expression of the intermediate filament nestin. We used the regulatory elements of the nestin gene to generate transgenic mice in which neural stem cells of the embryonic and adult brain are marked by the expression of green fluorescent protein (GFP). We used these animals as a reporter line for studying neural stem and progenitor cells in the developing and adult nervous systems. In these nestin-GFP animals, we found that GFP-positive cells reflect the distribution of nestin-positive cells and accurately mark the neurogenic areas of the adult brain. Nestin-GFP cells can be isolated with high purity by using fluorescent-activated cell sorting and can generate multipotential neurospheres. In the adult brain, nestin-GFP cells are approximately 1,400-fold more efficient in generating neurospheres than are GFP-negative cells and, despite their small number, give rise to 70 times more neurospheres than does the GFP-negative population. We characterized the expression of a panel of differentiation markers in GFP-positive cells in the nestin-GFP transgenics and found that these cells can be divided into two groups based on the strength of their GFP signal: GFP-bright cells express glial fibrillary acidic protein (GFAP) but not betaIII-tubulin, whereas GFP-dim cells express betaIII-tubulin but not GFAP. These two classes of cells represent distinct classes of neuronal precursors in the adult mammalian brain, and may reflect different stages of neuronal differentiation. We also found unusual features of nestin-GFP-positive cells in the subgranular cell layer of the dentate gyrus. Together, our results indicate that GFP-positive cells in our transgenic animals accurately represent neural stem and progenitor cells and suggest that these nestin-GFP-expressing cells encompass the majority of the neural stem cells in the adult brain.     

GDNF is a chemoattractant factor for neuronal precursor cells in the rostral migratory stream

Olfactory bulb (OB) interneurons are generated from neuroblast cells derived from the anterior subventricular zone (SVZa) of the forebrain. The mechanisms guiding the rostral migration of these neuronal precursors are not well understood. Here, we show that glial cell line-derived neurotrophic factor (GDNF) is produced in the olfactory bulb but distributed along the rostral migratory stream (RMS) in a pattern concordant with the expression of its GPI-anchored receptor GFRalpha1. We demonstrate that GDNF is a chemoattractant factor for RMS-derived neuronal precursors, but not for SVZa neuroblast cells. In agreement with this, GDNF increased Cyclin-dependent kinase 5 (Cdk5) activity in RMS cells, a kinase critically involved in neuronal migration and guidance. GDNF-mediated cell chemoattraction was abrogated in RMS explants treated with the Cdk5 inhibitor Roscovitine as well as in RMS explants isolated from Ncam mutant mice. Chemical cross-linking assays showed that 125I-GDNF is able to interact directly with NCAM in RMS-derived cells. Taken together, these data demonstrate that GDNF is a direct chemoattractant factor for neuroblast cells migrating along the RMS and support the participation of NCAM during this guidance process.     

The transcription factor Foxg1 regulates telencephalic progenitor proliferation cell autonomously, in part by controlling Pax6 expression levels

Background

Postnatal olfactory bulb (OB) neurogenesis involves the generation of granule and periglomerular cells by neural stem cells (NSCs) located in the walls of the lateral ventricle (LV). Recent studies show that NSCs located in different regions of the LV give rise to different types of OB neurons. However, the molecular mechanisms governing neuronal specification remain largely unknown and new methods to approach these questions are needed.

Results

In this study, we refine electroporation of the postnatal forebrain as a technique to perform precise and accurate delivery of transgenes to NSCs located in distinct walls of the LV in the mouse. Using this method, we confirm and expand previous studies showing that NSCs in distinct walls of the LV produce neurons that invade different layers of the OB. Fate mapping of the progeny of radial glial cells located in these distinct LV walls reveals their specification into defined subtypes of granule and periglomerular neurons.

Conclusions

Our results provide a baseline with which future studies aiming at investigating the role of factors in postnatal forebrain neuronal specification can be compared. Targeted electroporation of defined LV NSC populations will prove valuable to study the genetic factors involved in forebrain neuronal specification.     

Human neural stem/progenitor cells, expanded in long-term neurosphere culture, promote functional recovery after focal ischemia in Mongolian gerbils

Transplantation of human neural stem cells (NSCs) is a promising potential therapy for neurologic dysfunctions after the hyperacute stage of stroke in humans, but large amounts of human NSCs must be expanded in long-term culture for such therapy. To determine their possible therapeutic potential for human stroke, human fetal neural stem/progenitor cells (NSPCs) (i.e., neurosphere-forming cells) were isolated originally from forebrain tissues of one human fetus, and expanded in long-term neurosphere culture (exceeding 24 weeks), then xenografted into the lesioned areas in the brains of Mongolian gerbils 4 days after focal ischemia. Sensorimotor and cognitive functions were evaluated during the 4 weeks after transplantation. The total infarction volume in the NSPC-grafted animals was significantly lower than that in controls. Approximately 8% of the grafted NSPCs survived, mainly in areas of selective neuronal death, and were costained with antibodies against neuronal nuclei antibody (NeuN), microtubule associated protein (MAP-2), glial fibrillary acidic protein (GFAP), and anti-2'3' cyclic nucleotide 3'-phosphodiesterase (CNPase). Synaptic structures between NSPCs-derived neurons and host neurons were observed. Furthermore, gradual improvement of neurologic functions was observed clearly in the NSPC-grafted animals, compared to that in controls. Human NSPCs, even from long-term culture, remarkably improved neurologic functions after focal ischemia in the Mongolian gerbil, and maintained their abilities to migrate around the infarction, differentiate into mature neurons, and form synapses with host neuronal circuits. These results indicate that in vitro-expanded human neurosphere cells are a potential source for transplantable material for treatment of stroke.