Osteopontin increases the proliferation of neural progenitor cells

We examined the role of osteopontin in the proliferation of neural progenitor cells in vitro. Osteopontin increased the proliferation of neural progenitor cells in the presence of FGF2 as measured by cell proliferation assay and bromodeoxy uridine incorporation studies. In addition, immunoblot analysis demonstrated an increase in the phosphorylation of retinoblastoma protein with a concurrent increase in the content of phospho-Akt and cyclin D1. These results indicate that osteopontin can upregulate the content of phospho-Akt, cyclin D1 and phospho-Rb to subsequently enhance the proliferation of neural progenitor cells in the presence of FGF2.     

Spatiotemporal expression of GM1 in murine medial pallial neural progenitor cells

The expression of gangliosides is developmentally regulated in the central nervous system. The expression of GM1 in the neural progenitor cells of the telencephalonic ventricular zone (VZ) has been reported in several studies. However, information on the spatial and temporal regulation of GM1 expression in the VZ is still lacking. In this study, we characterized the expression of GM1 in the developing mouse telencephalon. At E13, GM1 is expressed in neuronal cells as well as in the VZ. The initial expression of GM1 in the VZ is restricted to regions close to the medial pallium. Fluorescence-activated cell sorting (FACS) analysis and characterization of E14 GM1-positive cells showed that they contain progenitor cells that proliferate in response to epidermal growth factor (EGF) and/or basic fibroblast growth factor (bFGF) stimulation. The results obtained from quantitative gene expression analysis of region-specific genes (Emx1, Lhx2, Ngn1, Ngn2, Pax6, Dlx2, Gsh2, Mash1, and Nkx2.1), using real-time polymerase chain reaction indicate that FACS of GM1-expressing cells in the fetal forebrain enriches for the medial pallial neural progenitor cells.     

Focal ischemia induces transient expression of IL-6 in the substantia nigra pars reticulata

We examined the expression of IL-6 in the substantia nigra pars reticulata (SNr) at various time points after transient (3 h) middle cerebral artery occlusion (MCAO) in rats. The animals were killed at 1, 3, 7 or 14 days following operation. Coronal brain sections were processed for immunohistochemistry with antibodies against GFAP, OX-42 and IL-6 and for Nissl staining. Microglial activation was detected 3 and 7 days after ischemia. Reactive astrocytes have been found 7 and 14 days after ischemia. IL-6 expression was detected 3 and 7 days after ischemia. IL-6-positive cells beared the typical morphology of neurons. Distribution of IL-6-positive cells within the SNr was not homogenous. The lateral area of the SNr bears the highest number of IL-6-positive neurons while the central core bears the lowest. Quantification of intact neurons in the SNr 14 days after reperfusion shows that the highest amount of cell loss was found in the central core of the SNr and less neuronal cell loss was observed in the lateral area of the SNr. Thus, the SNr area with the highest IL-6 expression 3 and 7 days after ischemia bears the highest number of intact neurons 14 days after ischemia. This finding could be a clue for the neuroprotective role of IL-6 in the remote region SNr after focal cerebral ischemia.     

Sulfated glycoprotein-2 expression increases in rodent brain after transient global ischemia.

Sulfated glycoprotein-2 (SGP-2) is emerging as a prominent marker of neurodegeneration in mammalian brain. Regulation of brain SGP-2 was studied in adult male Wistar rats subjected to 30 min of forebrain ischemia by four vessel occlusion. By 3 days after the ischemic insult, SGP-2 RNA levels were increased two fold in caudate nucleus and hippocampus. SGP-2 protein levels assessed by immunoblots were markedly increased in both brain regions following ischemia. GFAP RNA levels also increased over 5 fold in caudate nucleus and hippocampus following the ischemic insult. Despite significant elevations in GFAP RNA, protein levels of GFAP assessed by immunoblot were only marginally affected. The elevated expression of SGP-2 in rodent brain following this and other experimental lesion paradigms (e.g., excitotoxic lesions, deafferentation) suggest some general involvement of SGP-2 in neurodegeneration and remodelling following neuronal injury.     

Fas activation increases neural progenitor cell survival

Although there is a sizable amount of research focusing on adult neural progenitor cells (NPCs) as a therapeutic approach for many neurodegenerative diseases, including multiple sclerosis, little is known about the pathways that govern NPC survival and apoptosis. Fas, a member of the death receptor superfamily, plays a well‐characterized role in the immune system, but its function in neural stem cells remains uncertain. Our study focuses on the effects of Fas on NPC survival in vitro. Activation of Fas by recombinant Fas ligand (FasL) did not induce apoptosis in murine NPCs in culture. In fact, both an increase in the amount of viable cells and a decrease in apoptotic and dying cells were observed with FasL treatment. Our data indicate that FasL‐mediated adult NPC neuroprotection is characterized by a reduction in apoptosis, but not increased proliferation. Further investigation of this effect revealed that the antiapoptotic effects of FasL are mediated by the up‐regulation of Birc3, an inhibitor of apoptosis protein (IAP). Conversely, the observed effect is not the result of altered caspase activation or FLIP (Fas‐associated death domain‐like interleukin‐1beta‐converting enzyme inhibitory protein) up‐regulation, which is known to inhibit caspase‐8‐mediated cell death in T cells. Our data indicate that murine adult NPCs are resistant to FasL‐induced cell death. Activation of Fas increased cell survival by decreasing apoptosis through Birc3 up‐regulation. These results describe a novel pathway involved in NPC survival. © 2009 Wiley‐Liss, Inc.     

Intravenous injection of neural progenitor cells improved depression-like behavior after cerebral ischemia

Poststroke depression (PSD) occurs in approximately one-third of stroke survivors and is one of the serious sequelae of stroke. The onset of PSD causes delayed functional recovery by rehabilitation and also increases cognitive impairment. However, appropriate strategies for the therapy against ischemia-induced depression-like behaviors still remain to be developed. Such behaviors have been associated with a reduced level of brain-derived neurotrophic factor (BDNF). In addition, accumulating evidence indicates the ability of stem cells to improve cerebral ischemia-induced brain injuries. However, it remains to be clarified as to the effect of neural progenitor cells (NPCs) on PSD and the association between BDNF level and PSD. Using NPCs, we investigated the effect of intravenous injection of NPCs on PSD. We showed that injection of NPCs improved ischemia-induced depression-like behaviors in the forced-swimming test and sucrose preference test without having any effect on the viable area between vehicle- and NPC-injected ischemic rats. The injection of NPCs prevented the decrease in the level of BDNF in the ipsilateral hemisphere. The levels of phosphorylated CREB, ERK and Akt, which have been implicated in events downstream of BDNF signaling, were also decreased after cerebral ischemia. NPC injection inhibited these decreases in the phosphorylation of CREB and ERK, but not that of Akt. Our findings provide evidence that injection of NPCs may have therapeutic potential for the improvement of depression-like behaviors after cerebral ischemia and that these effects might be associated with restoring BDNF-ERK-CREB signaling.     

The prostacyclin analogue iloprost increases circulating endothelial progenitor cells in patients with critical limb ischemia

Patients with critical limb ischemia (CLI) have low levels of endothelial progenitor cells (EPC). Iloprost has been demonstrated to stimulate vascular endothelial growth factor (VEGF) and promote angiogenesis. We investigated the effects of iloprost on EPC levels in vivo in CLI patients. Twenty-three patients with stage III and IV CLI were treated with iloprost for four weeks, improving clinical and instrumental parameters. Mononuclear cells isolated from peripheral blood were cultured to obtain "early" EPC, evaluated counting adherent cells with double positivity for acetylated low-density lipoprotein uptake and Ulex Europaeus lectin at flow cytometry. These cells also co-expressed the monocyte markers CD14 and CD45. Iloprost increased EPC number in the whole patient population: pre-treatment median: 13,812/ml; range: 1,263-83,648/ml; post-treatment median: 23,739/ml; range: 3,385-99,251/ml; p = 0.035, irrespective of age, sex, disease stage or atherosclerosis risk factors. In conclusion, iloprost increases EPC number in peripheral blood in vivo. Such an effect may have therapeutic relevance.     

Injection of neural progenitor cells improved learning and memory dysfunction after cerebral ischemia

Accumulating evidence indicates that stem cells have the ability to improve neurological deficits seen after cerebral ischemia. However, the effects of neural progenitor cells (NPCs) on cerebral ischemia-induced learning and memory dysfunction remain to be clarified. The purpose of the present study was to determine whether the injection of exogenous NPCs could prevent learning and memory dysfunction after cerebral ischemia. Sustained cerebral ischemia was produced by the injection of 700 microspheres into the right hemisphere of each rat. We demonstrated that injection of NPCs into the hippocampus at 10 min after the induction of cerebral ischemia reduced prolongation of the escape latency seen in acquisition and retention tests of the water maze task on Days 12-28 after cerebral ischemia. Injection of NPCs partially attenuated the decrease in viable areas of the ipsilateral hemisphere on Day 28 after the cerebral ischemia. We also demonstrated that injection of NPCs prevented the decrease in the level of BDNF seen at the early period after cerebral ischemia. These results suggest that the injection of exogenous NPCs into the hippocampus can prevent cerebral ischemia-induced learning and memory dysfunction, possibly through maintenance of the BDNF level.     

Isolation and characterization of murine neural stem/progenitor cells based on Prominin-1 expression

The identification of strategies for the isolation of neural stem cells (NSCs) has important implications for the understanding of their biology and the development of therapeutic applications. It has been previously described that human neural stem and progenitor cells (NSPCs) can be isolated from the central nervous system (CNS) using antibodies to prominin (CD133) and fluorescence-activated cell sorting (FACS). Although this antigen displayed an identical membrane topology in several human and murine tissues there was uncertainty as to the relationship between human and mouse prominin because of the low level of amino acid identity. Here we show that prominin expression can be used to identify and isolate also murine NSPCs from the developing or adult brain. Prominin is co-expressed with known neural stem markers like SOX 1-2, Musashi and Nestin. Moreover, neurosphere-forming cells with multipotency and self-renewal capacity reside within the prominin-positive fraction. Transplantation experiments show that CD133-positive cells give rise to neurons and glial cells in vivo, and that many neurons display appropriate phenotypic characteristics of the recipient tissues. The demonstration that CD133 is a stem cell antigen for murine NSPCs as it is for human NSPCs is useful for the investigation of mammal neurogenesis and development of preclinical tests of NSPCs transplantation in mouse analogues of human diseases.     

Pax6 directly modulate Sox2 expression in the neural progenitor cells

Pax6 is a key regulator in the neuronal fate determination as well as the proliferation of neural stem cells, but the mechanisms are still unknown. Our study shows that Pax6 regulate the proliferation of neural progenitor cells of cortical subventricular zone, through direct modulation of the Sox2 expression during the late developmental stage in mice. We found a dramatic decrease in the number of Sox2+ neural progenitor cells in the subventricular zone of E18.5 Pax6(-/-) mice. We confirmed that Pax6 could bind to the Sox2 promoter by chromatin immunoprecipitation assay and activate Sox2 expression by a luciferase reporter gene assay. Moreover, neural progenitors isolated from the Pax6(-/-) embryos showed a decreased neurosphere formation as well as proliferation.     

Focal epilepsy as a possible sign of transient subclinical ischemia

Late-onset partial epileptic seizures occurred in 10 patients with symptoms and/or signs of ischemic cerebrovascular disease (ICVD) and were associated with transient ischemic attacks in 7 of them. History, somatic and neurological examinations, laboratory and ancillary investigations and follow-up revealed no other disease which might be responsible for the seizures. The anatomical and temporal proximity of signs of ICVD indicate the latter as the most likely cause of seizures. Although the available neuroimaging did not allow us to rule out the presence of silent cerebral infarctions in all patients, it is conceivable that jacksonian seizures, and more rarely complex partial seizures, might occasionally represent a clinical sign due to transient cerebral ischemia and thus herald major cerebrovascular events.     

Glia as neural progenitor cells

Recent studies have substantially expanded our conception of the roles for glia in function and maintenance of the adult nervous system. Of these reports, several have re-examined the lineage relationships among neural stem cells, their early radial glial derivatives and their mitotically competent neurogenic daughters. These studies have highlighted the role of radial cells in development, and of their glial progeny postnatally, as both progenitors and regulators of neuronal production and phenotype. In the adult mammalian brain, radial cell populations are scant, but their glial derivatives participate in a gliovascular network that organizes not only the structural and functional architecture of the brain but also its generative niches for resident progenitors - glial as well as neuronal. As in other organs, these progenitors can reside as transit-amplifying pools, by which lineage-biased progenitors expand to replenish discrete mature phenotypes. This review will consider the types of transit-amplifying progenitor cells persistent in the adult mammalian CNS, and the extent to which these derive from glial phenotypes. It will also discuss the interactions of progenitor cells with their brethren that could specify their phenotype and fate, while defining the permissive niches for cell genesis in the adult CNS.     

Adult neural stem and progenitor cells modified to secrete GDNF can protect, migrate and integrate after intracerebral transplantation in rats with transient forebrain ischemia

Adult neural stem and progenitor cells (NSPCs) are important autologous transplantation tools in regenerative medicine, as they can secrete factors that protect the ischemic brain. We investigated whether adult NSPCs genetically modified to secrete more glial cell line-derived neurotrophic factor (GDNF) could protect against transient ischemia in rats. NSPCs were harvested from the subventricular zone of adult Wistar rats and cultured for 3 weeks in the presence of epidermal growth factor. The NSPCs were treated with fibre-mutant Arg-Gly-Asp adenovirus containing the GDNF gene (NSPC-GDNF) or enhanced green fluorescent protein (EGFP) gene (NSPC-EGFP; control group). In one experiment, cultured cells were transplanted into the right ischemic boundary zone of Wistar rat brains. One week later, animals underwent 90 min of intraluminal right middle cerebral artery occlusion followed by magnetic resonance imaging and behavioural tests. The NSPC-GDNF group had higher behavioural scores and lesser infarct volume than did controls at 1, 7 and 28 days postocclusion. In the second experiment, we transplanted NSPCs 3 h after ischemic insult. Compared to controls, rats receiving NSPC-GDNF had decreased infarct volume and better behavioural assessments at 7 days post-transplant. Animals were killed on day 7 and brains were collected for GDNF ELISA and morphological assessment. Compared to controls, more GDNF was secreted, more NSPC-GDNF cells migrated toward the ischemic core and more NSPC-GDNF cells expressed immature neuronal marker. Moreover, the NSPC-GDNF group showed more effective inhibition of microglial invasion and apoptosis. These findings suggest that NSPC-GDNF may be useful in treatment of cerebral ischemia.     

Chemokine receptor expression by neural progenitor cells in neurogenic regions of mouse brain

We previously demonstrated that chemokine receptors are expressed by neural progenitors grown as cultured neurospheres. To examine the significance of these findings for neural progenitor function in vivo, we investigated whether chemokine receptors were expressed by cells having the characteristics of neural progenitors in neurogenic regions of the postnatal brain. Using in situ hybridization we demonstrated the expression of CCR1, CCR2, CCR5, CXCR3, and CXCR4 chemokine receptors by cells in the dentate gyrus (DG), subventricular zone of the lateral ventricle, and olfactory bulb. The pattern of expression for all of these receptors was similar, including regions where neural progenitors normally reside. In addition, we attempted to colocalize chemokine receptors with markers for neural progenitors. In order to do this we used nestin-EGFP and TLX-LacZ transgenic mice, as well as labeling for Ki67, a marker for dividing cells. In all three areas of the brain we demonstrated colocalization of chemokine receptors with these three markers in populations of cells. Expression of chemokine receptors by neural progenitors was further confirmed using CXCR4-EGFP BAC transgenic mice. Expression of CXCR4 in the DG included cells that expressed nestin and GFAP as well as cells that appeared to be immature granule neurons expressing PSA-NCAM, calretinin, and Prox-1. CXCR4-expressing cells in the DG were found in close proximity to immature granule neurons that expressed the chemokine SDF-1/CXCL12. Cells expressing CXCR4 frequently coexpressed CCR2 receptors. These data support the hypothesis that chemokine receptors are important in regulating the migration of progenitor cells in postnatal brain.     

Long-term transgene expression in mouse neural progenitor cells modified with phiC31 integrase

Stem cells can potentially be utilized in combined gene/cell therapies for neural diseases. We examined the ability of the non-viral phiC31 integrase system to promote stable transgene expression in mouse neural progenitor cells (mNPCs). phiC31 integrase catalyzes the sequence-specific integration of attB-containing plasmids into pseudo attP sites in mammalian genomes, to produce long-term transgene expression. We achieved gene transfer by co-nucleofection of a plasmid carrying the luciferase marker gene and an attB site and a plasmid expressing integrase in mNPCs that had been generated in a neurosphere preparation. Luciferase expression was quantified in live cells for 8 weeks, revealing persistence of gene expression. Sequence-specific integration at a preferred pseudo attP site in the mouse genome was detected by using PCR. Furthermore, sustained transgene expression was demonstrated in genetically modified NPCs that were cultured in conditions that promoted either growth or differentiation into neurons and astrocytes. Our results demonstrate that the phiC31 integrase system produces stable transgene expression in adult mNPCs and their progeny and may be useful in strategies for combating neurodegenerative disorders.     

Three steps of neural stem cells development in gerbil dentate gyrus after transient ischemia.

The stage of neurogenesis can be divided into three steps: proliferation, migration, and differentiation. To elucidate detailed relations between these three steps after ischemia, the authors evaluated the three steps in the adult gerbil dentate gyrus (DG) after 5 minutes of transient global ischemia using bromodeoxyuridine (BrdU), highly polysialylated neural cell adhesion molecule (PSA-NCAM), and neuronal nuclear antigen (NeuN) and glial fibrillary acidic protein (GFAP) as markers for proliferation, migration, and differentiation, respectively. Bromodeoxyuridine-labeled cells increased approximately sevenfold, and PSA-NCAM-positive cells increased approximately threefold in the subgranular zone (SGZ) with a peak 10 days after ischemia. Bromodeoxyuridine-labeled cells with PSA-NCAM expression were first detected both in the SGZ and the granule cell layer (GCL) 20 days after ischemia and gradually decreased after that, whereas BrdU-labeled cells with NeuN gradually increased in the GCL until 60 days after ischemia. A few BrdU-labeled cells with GFAP expression were detected in DG after ischemia; no PSA-NCAM-positive cells with GFAP expression were detected, but the radial processes of glial cells were partly in contact with PSA-NCAM-positive cell bodies and dendrites. These results suggest that neural stem cell proliferation begins at the SGZ, and that the cells then migrate into the GCL and differentiate mainly into neuronal cells. The majority of these three steps finished in 2 months after transient global ischemia.     

Adult neural progenitor cells reactivate superbursting in mature neural networks

Behavioral recovery in animal models of human CNS syndromes suggests that transplanted stem cell derivatives can augment damaged neural networks but the mechanisms behind potentiated recovery remain elusive. Here we use microelectrode array (MEA) technology to document neural activity and network integration as rat primary neurons and rat hippocampal neural progenitor cells (NPCs) differentiate and mature. The natural transition from neuroblast to functional excitatory neuron consists of intermediate phases of differentiation characterized by coupled activity. High-frequency network-wide bursting or "superbursting" is a hallmark of early plasticity that is ultimately refined into mature stable neural network activity. Microelectrode array (MEA)-plated neurons transition through this stage of coupled superbursting before establishing mature neuronal phenotypes in vitro. When plated alone, adult rat hippocampal NPC-derived neurons fail to establish the synchronized bursting activity that neurons in primary and embryonic stem cell-derived cultures readily form. However, adult rat hippocampal NPCs evoke re-emergent superbursting in electrophysiologically mature rat primary neural cultures. Developmental superbursting is thought to accompany transient states of heightened plasticity both in culture preparations and across brain regions. Future work exploring whether NPCs can re-stimulate developmental states in injury models would be an interesting test of their regenerative potential.