Dose‐dependent effect of EGF on migration and differentiation of adult subventricular zone astrocytes

Adult neural stem cells (NSCs) are located in the subventricular zone (SVZ), a specialized brain niche located on the walls of the lateral ventricle. Under physiological conditions, NSCs generate a large number of young neurons and some oligodendrocytes, however the mechanisms controlling cell proliferation and migration are unclear. In vitro, epidermal growth factor (EGF) signaling has been shown to be an important mediator of cell proliferation and migration in the adult brain; however, the primary SVZ progenitors that respond to EGF are not well known. In this study, we isolated SVZ type‐B astrocytes and cultured them under different EGF concentrations. We found a dose‐dependent effect of EGF on proliferation rates and migration of SVZ type‐B astrocytes. We found that GFAP+ type‐B astrocytes gave rise to highly migratory and proliferating cells that expressed Olig2 and NG2. After EGF withdrawal, a significant number of EGF‐stimulated cells differentiated into S100β+/O4+ oligodendrocytes. This study provides new insights about the production of oligodendrocytes derived from the astrocyte NSCs residing in the adult SVZ. To be able to manipulate the endogenous adult progenitors, it is crucial to identify and isolate the responding primary precursors and determine the extracellular signals that regulate their cell division, migration, and fate. © 2010 Wiley‐Liss, Inc.     

Migrating and myelinating potential of subventricular zone neural progenitor cells in white matter tracts of the adult rodent brain

Adult neural stem cells in the subventricular zone (SVZ) produce neuronal progenitors that migrate along the rostral migratory stream (RMS) and generate olfactory interneurons. Here, we evaluate the migratory potential of SVZ cells outside the RMS and their capacity to generate oligodendrocytes in the adult brain. We show that SVZ cells migrate long distances when grafted into white matter tracts such as the cingulum (Ci) and corpus callosum (CC). Furthermore, 22 days postinjection, most present morphologic and phenotypic characteristics of cells committed to the oligodendrocyte lineage. Cells grafted in shiverer CC and Ci become MBP-positive oligodendrocytes, abundantly myelinating these white matter tracts. Type A progenitors are involved in this myelinating process. Altogether, this study reveals the migrating and myelinating potential of SVZ cells in a new environmental context. Therefore, SVZ cells stand as interesting candidates for the development of novel therapeutic strategies for demyelinating diseases.     

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.     

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.     

Oligodendrogenesis in the subventricular zone and the role of epidermal growth factor

Demyelinating diseases are characterized by an extensive loss of oligodendrocytes and myelin sheaths from axolemma. These neurological disorders are a common cause of disability in young adults, but so far, there is no effective treatment against them. It has been suggested that neural stem cells (NSCs) may play an important role in brain repair therapies. NSCs in the adult subventricular zone (SVZ), also known as Type-B cells, are multipotential cells that can self-renew and give rise to neurons and glia. Recent findings have shown that cells derived from SVZ Type-B cells actively respond to epidermal-growth-factor (EGF) stimulation becoming highly migratory and proliferative. Interestingly, a subpopulation of these EGF-activated cells expresses markers of oligodendrocyte precursor cells (OPCs). When EGF administration is removed, SVZ-derived OPCs differentiate into myelinating and pre-myelinating oligodendrocytes in the white matter tracts of corpus callosum, fimbria fornix and striatum. In the presence of a demyelinating lesion, OPCs derived from EGF-stimulated SVZ progenitors contribute to myelin repair. Given their high migratory potential and their ability to differentiate into myelin-forming cells, SVZ NSCs represent an important endogenous source of OPCs for preserving the oligodendrocyte population in the white matter and for the repair of demyelinating injuries.     

Disruption of postnatal progenitor migration and consequent abnormal pattern of glial distribution in the cerebrum following administration of methylmercury

Transplacental administration of methylmercury (MeHg) induces disruption of neuronal migration in the developing cerebral cortex. However, the effects of MeHg on glial progenitor migration remain unclear. To understand this, we performed double administration of MeHg and 5-bromo-2-deoxyuridine (BrdU) to neonatal rat pups on postnatal day 2 (P2), when glial cells are generated from progenitors in the subventricular zone (SVZ). Histopathological examination of a proportion of the MeHg-treated rats on P28 revealed no apparent abnormalities of cytoarchitecture or neuron count in either the primary motor or primary somatosensory cortex of the cerebrum. BrdU immunohistochemistry revealed abnormal accumulation of the labeled cells in the deeper layers of the cortices and underlying white matter of both areas, where an excessive number of astrocytes (glial fibrillary acidic protein- or S-100beta-immunolabeled cells) and oligodendrocytes (2',3'-cyclic-nucleotide 3'-phosphohydrolase-labeled cells) were located. Next, to investigate the migration of individual progenitors from the forebrain SVZ of P2 neonates, we labeled them in vivo with a retrovirus encoding green fluorescent protein (GFP), following administration of MeHg, and then examined the distribution pattern of the GFP-labeled cells in the P28 cerebrum. We found that the labeled cells developed into astrocytes and oligodendrocytes and were accumulated abnormally in the lateral white matter as well as in the adjacent deeper layer of the lateral cortex and lateral side of the striatum. Thus, exposure to MeHg in the gliogenic period induced irregular distribution of glia as a consequence of abnormal migration of the postnatal progenitors.     

Fibroblast growth factor-2 (FGF-2) and platelet-derived growth factor AB (PDGF AB) promote adult SVZ-derived oligodendrogenesis in vivo

The capacity of multipotential progenitor cells of the adult mammalian forebrain to generate myelin-forming oligodendrocytes was tested by grafting fragments of different regions of the subventricular zone (SVZ) of the lateral ventricle and the striatum of 6-month-old wild-type mice into the brain of neonate shiverer and wild-type mice. Without growth factor treatment, only few cells of the rostral SVZ survived and formed myelin after engraftment. Treating donors prior to transplantation with a single intraperitoneal injection of epidermal growth factor, basic fibroblast growth factor 2 (FGF-2), and platelet-derived growth factor AB (PDGF(AB)) vigorously promoted the survival, migration, and differentiation of the grafted SVZ cells into myelin-forming oligodendrocytes. In situ, both growth factors expanded the constitutively proliferative PSA-NCAM+ population and favored their differentiation toward the neuronal and oligodendroglial cell fate. The adult central nervous system thus harbors a focal reservoir of FGF-2 and PDGF(AB)-responsive cells which are able to generate substantial amounts of myelin-forming oligodendrocytes in vivo, opening a new prospective area for therapy in demyelinating diseases.     

A role for CXCR4 signaling in survival and migration of neural and oligodendrocyte precursors

Oligodendrocyte development is controlled by a number of survival and migratory factors. The present study shows that signaling of CXCR4 receptor by the chemokine CXCL12 regulates survival and migration of neural precursors (NP) as well as oligodendrocyte progenitors (OP). CXCR4 is expressed by E14 striatal NP and OP generated by neurospheres. In CXCR4-defective mice, the number of NP in neurosphere outgrowth was twofold less than in wild-type (WT) mice; NP radial cell migration was also decreased. In contrast, the addition of CXCL12 to WT NP increased radial migration from the sphere in a dose-dependent manner with a maximal response at 200 nM. When oligodendrocytes differentiated in neurosphere outgrowth, CXCR4 was downregulated. OP isolated from newborn brain coexpressed CXCR4 with platelet-derived growth factor receptor-alpha (PDGFR alpha) or chondroitin sulfate proteoglycan; receptor expression also decreased during differentiation in vitro. Neonatal OP showed a peak migratory response to 20 nM of CXCL12 in chemotactic chambers, a migration inhibited by a CXCR4 antagonist and anti-CXCL12 antibody. In the embryonic spinal cord, the number of OP-expressing PDGFR alpha was reduced more than twofold in CXCR4-defective mice compared with WT and the ratio of ventral to dorsal OP was significantly increased. This indicates a defect in OP survival and their dorsal migration from the ventral cord region, probably because CXCR4(-/-) OP are unable to respond to CXCL12 made by vascular endothelia and the pia mater. We propose that CXCR4 signaling regulate survival and outward chemotactic migration of OP during embryonic and postnatal CNS development.     

High-level expression of functional chemokine receptor CXCR4 on human neural precursor cells

Neural precursor cells (NPCs) are self-renewing, multipotent progenitors that give rise to neurons, astrocytes and oligodendrocytes in the central nervous system (CNS). Fetal NPCs have attracted attention for their potential use in studying normal CNS development. Several studies of rodent neural progenitors have suggested that chemokines and their receptors are involved in directing NPC migration during CNS development. In this study, we established a consistent system to culture human NPCs and examined the expression of chemokine receptors on these cells. NPCs were found to express the markers nestin and CD133 and to differentiate into neurons, astrocytes and oligodendrocytes at the clonal level. Flow cytometry and RNase protection assay (RPA) indicated that NPCs express high levels of CXCR4 and low levels of several other chemokine receptors. When examined using a chemotaxis assay, NPCs were able to respond to CXCL12/SDF-1alpha, a ligand of CXCR4. Treatment with anti-CXCR4 antibody or HIV-1 gp120 abolished the migratory response of NPCs towards CXCL12/SDF-1alpha. These findings suggest that CXCR4 may play a significant role in directing NPC migration during CNS development.     

GRO-alpha and CXCR2 in the human fetal brain and multiple sclerosis lesions

Chemokines, small proinflammatory cytokines, are involved in migration of inflammatory cells, but also have a role in normal central nervous system development. One chemokine, growth-related oncogene-alpha (GRO-alpha) and its receptor CXCR2, are involved in proliferation and migration of oligodendrocyte progenitors in rats. Here we studied the regional and cell type-specific expression of GRO-alpha and CXCR2 in the human telencephalon at midgestation, the time that oligodendrocytes are being generated in the human brain. Our results showed that both GRO-alpha and CXCR2 are predominately expressed by oligodendrocyte progenitors and activated microglial cells in the highly proliferative subventricular zone. This cellular and regional localization suggests that GRO-alpha/CXCR2 may play a role in human oligodendrocyte proliferation and subsequent migration. We also studied the expression of GRO-alpha and CXCR2 in brain sections of multiple sclerosis (MS) patients. Consistent with their role in the inflammatory process of MS, both GRO-alpha and CXCR2 were expressed in activated microglia localized on the border of MS lesions. However, neither GRO-alpha nor CXCR2 were present in early oligodendrocyte progenitors, a finding that may partially explain why remyelination is not more efficient in MS.     

Some glial progenitors in the neonatal subventricular zone migrate through the corpus callosum to the contralateral cerebral hemisphere

The great majority of glial cells of the mammalian forebrain are generated in the perinatal period from progenitors in the subventricular zone (SVZ). We investigated the migration of progenitors from the neonatal (postnatal day 0, P0) rat forebrain SVZ by labeling them in vivo with a green fluorescence protein (GFP) retrovirus and monitoring their movements by time-lapse video microscopy in P3 slices. We identified a small number of progenitors that migrated tangentially within the corpus callosum (CC) and crossed the midline. These cells retained a relatively uniform morphology: the leading process was extended toward the contralateral side but showed no process branching or turning away from the migratory direction. Net migration requires the elongation of the leading process and nuclear translocation, and the migrating cells in the CC showed both modes. We confirmed the presence of unmyelinated axon bundles within the P3 CC, but failed to detect any radially directed glial processes (vimentin- or GLAST-immunolabeled fibers) spanning through the CC. Confocal images showed a close proximity between neurofilament-immunolabeled axons and the leading process of the GFP-expressing progenitors in the CC. The destination of the callosal fibers was examined by applying DiI to the right cingulum; the labeled fibers ran throughout the CC and reached the left cingulate and motor areas. The distribution and final fates of the retrovirus-labeled cells were examined in P28 brains. A small proportion of the labeled cells were found in the contralateral hemisphere, where, as oligodendrocytes and astrocytes, they colonized predominantly the cortex and the underlying white matter of the cingulate and secondary motor areas. The distribution pattern appears to coincide well with the projection direction of the callosal fibers. Thus, glial progenitors migrate across the CC, presumably in conjunction with unmyelinated axons, to colonize the contralateral hemisphere.     

Stroke induces ependymal cell transformation into radial glia in the subventricular zone of the adult rodent brain.

Adult ependymal cells are postmitotic and highly differentiated. Radial glial cells are neurogenic precursors. Here, we show that stroke acutely stimulated adult ependymal cell proliferation, and dividing ependymal cells of the lateral ventricle had genotype, phenotype, and morphology of radial glial cells in the rat. The majority of radial glial cells exhibited symmetrical division about the cell cleavage plane, and a radial fiber was maintained throughout each stage of cell mitosis. Increases of radial glial cells parallel expansion of neural progenitors in the subventricular zone (SVZ). Furthermore, after stroke radial glial cells derived from the SVZ supported neuron migration. These results indicate that adult ependymal cells divide and transform into radial glial cells after stroke, which could function as neural progenitor cells to generate new neurons and act as scaffolds to support neuroblast migration towards the ischemic boundary region.     

Modulation of rat oligodendrocyte precursor cells by the chemokine CXCL12

Migration, proliferation, and differentiation of oligodendrocyte precursor cells are essential for the assembly of myelin in the central nervous system. Knowledge on the regulation of these precursor cells is therefore of great importance for the understanding of developmental myelination and remyelination in demyelinating diseases. Here, we show that primary rat oligodendrocyte precursor cells express the chemokine receptor CXCR4. Stimulation with the ligand CXCL12 (SDF-1 alpha) leads to intracellular Ca elevation. Furthermore, 10 ng/ml CXCL12 augmented differentiation of precursors into mature oligodendrocytes. Migration toward growth factor conditioned medium was inhibited by CXCL12, while proliferation was only slightly modulated. The effect of CXCL12 on both migration and differentiation was blocked using a G protein antagonist. These data suggest a role for CXCL12 and oligodendroglial CXCR4 receptors during developmental myelination and repair in demyelinating diseases of the central nervous system.     

Porcine neural progenitors require commitment to the oligodendrocyte lineage prior to transplantation in order to achieve significant remyelination of demyelinated lesions in the adult CNS

Glial cell transplantation is a potential therapy for human demyelinating disease, though obtaining large numbers of oligodendrocyte precursors from nonrodent species is currently problematic. Culturing of multipotent neural progenitors may provide a solution to this problem, because these cells can be expanded in vitro whilst retaining the ability to differentiate into both neurons and glial cells. In order to investigate the myelinating capability of multipotent neural progenitors, we isolated cells from the porcine subventricular zone, a region rich in neural progenitors, and transplanted them into areas of persistent demyelination in the spinal cord of immunosuppressed rats, created by the injection of ethidium bromide and subsequent exposure to 40 Gy X-irradiation. Porcine SVZ cells were transplanted either within 12 h of isolation or after 7 days in B104-conditioned medium. Freshly isolated cells did not mature into myelinating oligodendrocytes following transplantation and instead remained as clusters of undifferentiated progenitors. However, cells exposed to B104-conditioned medium prior to transplantation were able to effect complete remyelination of the demyelinated axons. This suggests that neural progenitors must be manipulated in vitro for commitment to the oligodendrocyte lineage prior to transplantation if significant remyelination is to be achieved.     

PDGF stimulates the massive expansion of glial progenitors in the neonatal forebrain

Platelet‐derived growth factor (PDGF) plays a major role in regulating migration, proliferation, and differentiation of glial progenitors during normal brain development and in the abnormal proliferation and dispersion that drives the formation of malignant gliomas. To further explore the relationship between PDGF's effects on normal glial progenitors and its role in the formation of gliomas, we infected progenitor cells in the subventricular zone (SVZ) of the lateral ventricle of neonatal rat pups with a retrovirus that expresses PDGF and green fluorescent protein (GFP). At 3 days post‐injection (dpi), a proliferation of PDGFRα+ progenitors was seen in the SVZ and white matter around the injection site and by 10 dpi the animals had large diffusely infiltrating tumors that resembled glioblastomas. The tumors contained a massive proliferation of both infected and uninfected PDGFRα+ progenitors, suggesting that PDGF was driving tumor formation via both autocrine and paracrine signaling. Rats co‐injected with two retroviruses (one that expresses PDGF‐IRES‐DSRED and one that expresses only GFP) formed tumors that contained a mixture of DSRED+ cells (PDGF producers) and GFP+ cells (recruited progenitors). Time‐lapse microscopy of slice cultures confirmed that both DSRED+ and GFP+ cells were highly migratory and proliferative. Furthermore, adding exogenous PDGF to slice cultures generated from nontumor‐bearing brains (injected with control GFP retrovirus only) stimulated the migration and proliferation of GFP+ progenitors. These findings reveal the inherent growth factor responsiveness and tumorigenic potential of PDGFRα+ progenitors and highlight the importance of paracrine signaling in stimulating glioma growth and infiltration. © 2009 Wiley‐Liss, Inc.     

Progenitor cells of the adult mouse subventricular zone proliferate, migrate and differentiate into oligodendrocytes after demyelination

Identifying a source of cells with the capacity to generate oligodendrocytes in the adult CNS would help in the development of strategies to promote remyelination. In the present study, we examined the ability of the precursor cells of the adult mouse subventricular zone (SVZ) to differentiate into remyelinating oligodendrocytes. After lysolecithin-induced demyelination of the corpus callosum, progenitors of the rostral SVZ (SVZa) and the rostral migratory pathway (RMS), expressing the embryonic polysialylated form of the neural cell adhesion molecule (PSA-NCAM), increased progressively with a maximal expansion occurring after 2 weeks. This observation correlated with an increase in the proliferation activity of the neural progenitors located in the SVZa and RMS. Moreover, polysialic acid (PSA)-NCAM-immunoreactive cells arizing from the SVZa were detected in the lesioned corpus callosum and within the lesion. Tracing of the constitutively cycling cells of the adult SVZ and RMS with 3H-thymidine labelling showed their migration toward the lesion and their differentiation into oligodendrocytes and astrocytes but not neurons. These data indicate that, in addition to the resident population of quiescent oligodendrocyte progenitors of the adult CNS, neural precursors from the adult SVZ constitute a source of oligodendrocytes for myelin repair.     

Expression of the chemokine receptors CXCR1 and CXCR2 in rat oligodendroglial cells

Chemokines are small proteins that act as chemoattractants and activators in leukocytes during physiological and inflammatory processes. In the CNS chemokine receptors have been shown to be expressed on neurons, astrocytes and microglia but their function in the CNS is poorly understood. CXCR1 and CXCR2 are receptors for ELR-positive CXC chemokines which include growth-regulated oncogene alpha (GRO-alpha) and interleukin-8 (IL-8). GRO-alpha is considered to influence proliferation of cultured oligodendrocyte progenitors (OLPs). Using RT-PCR we show here that the oligodendrocyte precursor cell line CG-4 expresses both CXCR1 and CXCR2. Furthermore we demonstrate that both CG-4 cells and primary cultures of rat OLPs are immunoreactive for CXCR2, the potential receptor for GRO-alpha. This finding demonstrates that the chemokine/chemokine receptor system is probably also involved in the regulation of oligodendroglial cells during developmental processes and may even have implications for inflammatory demyelinating diseases like multiple sclerosis.