A population of human brain parenchymal cells express markers of glial, neuronal and early neural cells and differentiate into cells of neuronal and glial lineages

Glial fibrillary acidic protein (GFAP)-positive cells derived from the neurogenic areas of the brain can be stem/progenitor cells and give rise to new neurons in vitro and in vivo. We report here that a population of GFAP-positive cells derived from fetal human brain parenchyma coexpress markers of early neural and neuronal cells, and have neural progenitor cell characteristics. We used a monolayer culture system to expend and differentiate these cells. During the initial proliferative phase, all cells expressed GFAP, nestin and low levels of betaIII-tubulin. When these cells were cultured in serum and then basic fibroblast growth factor, they generated two distinct progenies: (i) betaIII-tubulin- and nestin-positive cells and (ii) GFAP- and nestin-positive cells. These cells, when subsequently cultured in serum-free media without growth factors, ceased to proliferate and differentiated into two major neural cell classes, neurons and glia. In the cells of neuronal lineage, nestin expression was down-regulated and betaIII-tubulin expression became robust. Cells of glial lineage differentiated by down-regulating nestin expression and up-regulating GFAP expression. These data suggest that populations of parenchymal brain cells, initially expressing both glial and neuronal markers, are capable of differentiating into single neuronal and glial lineages through asymmetric regulation of gene expression in these cells, rather than acquiring markers through differentiation.     

Coexpression of nestin in neural and glial cells in the developing human CNS defined by a human-specific anti-nestin antibody

The presence of the intermediate filament protein nestin has been the predominant marker used to describe stem and progenitor cells in the mammalian CNS. In this study, a 998-bp fragment in the 3' region of the nestin mRNA was cloned from human fetal brain cells (HFBC). The nucleotide sequence of the cloned cDNA revealed 21 differences with the previously published human nestin sequence, resulting in 17 amino acid changes. A 150-amino-acid fragment derived from the cloned nestin cDNA was coupled to glutathione S-transferase and used as an immunogen to generate a rabbit polyclonal antiserum that selectively detects human nestin. HFBC that proliferated in response to basic fibroblast growth factor incorporated 5-bromo-2'-deoxyuridine into their nuclei and immunostained for nestin, indicating nestin expression in proliferating CNS progenitor cells. In all cell cultures, nestin costained with the neuroepithelial cell marker vimentin. A small subset of nestin-stained cells (1-2%) immunostained with neuronal marker MAP-2 during the first week and after 4 weeks in culture. However, during the first week in culture, approximately 10-30% of the total cell population of HFBC stained for the glial cell marker GFAP, and nearly all coimmunostained for nestin. After 4 weeks in culture, a subset of GFAP-positive cells emerged that no longer costained with nestin. These results describe nestin expression not only in CNS progenitor cells but also in the cells which were in transition from a progenitor stage to glial differentiation. Collectively, these data suggest a differential temporal regulation of nestin expression during glial and neuronal cell differentiation.     

Isolation and characterization of neural progenitor cells from post-mortem human cortex

Post-mortem human brain tissue represents a vast potential source of neural progenitor cells for use in basic research as well as therapeutic applications. Here we describe five human neural progenitor cell cultures derived from cortical tissue harvested from premature infants. Time-lapse videomicrography of the passaged cultures revealed them to be highly dynamic, with high motility and extensive, evanescent intercellular contacts. Karyotyping revealed normal chromosomal complements. Prior to differentiation, most of the cells were nestin, Sox2, vimentin, and/or GFAP positive, and a subpopulation was doublecortin positive. Multilineage potential of these cells was demonstrated after differentiation, with some subpopulations of cells expressing the neuronal markers beta-tubulin, MAP2ab, NeuN, FMRP, and Tau and others expressing the oligodendroglial marker O1. Still other cells expressed the classic glial marker glial fibrillary acidic protein (GFAP). RT-PCR confirmed nestin, SOX2, GFAP, and doublecortin expression and also showed epidermal growth factor receptor and nucleostemin expression during the expansion phase. Flow cytometry showed high levels of the neural stem cell markers CD133, CD44, CD81, CD184, CD90, and CD29. CD133 markedly decreased in high-passage, lineage-restricted cultures. Electrophysiological analysis after differentiation demonstrated that the majority of cells with neuronal morphology expressed voltage-gated sodium and potassium currents. These data suggest that post-mortem human brain tissue is an important source of neural progenitor cells that will be useful for analysis of neural differentiation and for transplantation studies.     

Neuronal lineage-specific induction of phospholipase Cepsilon expression in the developing mouse brain

Phospholipase C is a key enzyme of intracellular signal transduction in the central nervous system. We and others recently discovered a novel class of phospholipase C, phospholipase Cepsilon, which is regulated by Ras and Rap small GTPases. As a first step toward analysis of its function, we have examined the spatial and temporal expression patterns of phospholipase Cepsilon during mouse development by in situ hybridization and immunohistochemistry. Around embryonic day 10.5, abundant expression of phospholipase Cepsilon is observed specifically in the outermost layer of the neural tube. On embryonic day 12 and later, it is observed mainly in the marginal zone of developing brain and spinal cord as well as in other regions undergoing neuronal differentiation, such as the retina and olfactory epithelium. The phospholipase Cepsilon-expressing cells almost invariably express microtubule-associated protein 2, but hardly express nestin or glial fibrillary acidic protein, indicating that the expression of phospholipase Cepsilon is induced specifically in cells committed to the neuronal lineage. The expression of phospholipase Cepsilon persists in the terminally differentiated neurons and exhibits no regional specificity. Further, an in vitro culture system of neuroepithelial stem cells is employed to show that abundant expression of phospholipase Cepsilon occurs in parallel with the loss of nestin expression as well as with the induction of microtubule-associated protein 2 expression and neuronal morphology. Also, glial fibrillary acidic protein-positive glial lineage cells do not exhibit the high phospholipase Cepsilon expression. These results suggest that the induction of phospholipase Cepsilon expression may be a specific event associated with the commitment of the neural precursor cells to the neuronal lineage.     

Nestin-containing cells express glial fibrillary acidic protein in the proliferative regions of central nervous system of postnatal developing and adult mice

We are interested in the expression patterns of nestin, an embryonic intermediate filament that represent a neural precursor marker, in the mammalian central nervous system. With an immunohistochemical approach, distribution of nestin-containing cells and their colocalization with glial fibrillary acidic protein (GFAP) or neuronal nuclear specific protein (NeuN) were studied in adult and postnatal days 2-30 (P2-30) mice. Nestin-immunoreactivity was predominately distributed in certain proliferative regions, such as cerebral cortex, hippocampus, hypothalamus, subfornical organ, cerebellar cortex, area postrema, midline raphe glial structures, as well as ependymal and subependymal zones of the brain and spinal cord. The majority of nestin-immunoreactive cells, characterized by astroglial profiles of multiple and radial processes, showed a partial overlapping distribution with that of GFAP-immunoreactive astroglial cells. Double immunofluorescence confirmed that about 77% of these nestin-immunoreactive cells exhibited GFAP-immunoreactivity, indicating that a large percentage of nestin-expressing cells may have committed to astroglial cells. In developing mice, down-regulation of nestin expression was observed between P7 and P14. Although co-expression of nestin and NeuN occurred in cortical neurons of P2-7 mice, nestin-containing cells showing NeuN-immunoreactivity disappeared in CNS in older animals. Our results reveal the distribution pattern of nestin-containing neural precursors in the postnatal CNS and provide evidence on their differentiation fate to neurons and astrocytes, suggesting that nestin-containing glial cells may play an important role in remodeling and repairing in the postnatal and adult central nervous system.     

Specific characteristic of radial glia in the human fetal telencephalon

Phenotypic characteristics of cells in the developing human telencephalic wall were analyzed using electron microscopy and immunocytochemistry with various glial and neuronal cell markers. The results suggest that multiple defined cell types emerge in the neocortical proliferative zones and are differentially regulated during embryonic development. At 5-6 weeks gestation, three major cell types are observed. Most proliferating ventricular zone (VZ) cells are labeled with radial glial (RG) markers such as vimentin, glial fibrillary acidic protein (GFAP), and glutamate astrocyte-specific transporter (GLAST) antibodies. A subpopulation of these RG cells also express the neuronal markers beta III-tubulin, MAP-2, and phosphorylated neurofilament SMI-31, in addition to the stem cell marker nestin, indicating their multipotential capacity. In addition, the presence of VZ cells that immunoreact only with neuronal markers indicates the emergence of restricted neuronal progenitors. The number of multipotential progenitors in the VZ gradually decreases, whereas the number of more restricted progenitors increases systematically during the 3-month course of human corticogenesis. These results suggest that multipotential progenitors coexist with restricted neuronal progenitors and RG cells during initial corticogenesis in the human telencephalon. Since the multipotential VZ cells disappear during the major wave of neocortical neurogenesis, the RG and restricted neuronal progenitors appear to serve as the main sources of cortical neurons. Thus, the diversification of cells in human VZ and overlying subventricular zone (SVZ) begins earlier and is more pronounced than in rodents.     

Nestin mRNA expression correlates with the central nervous system progenitor cell state in many,but not all,regions of developing central nervous system

Nestin is a recently discovered intermediate filament (IF) gene. Nestin expression has been extensively used as a marker for central nervous system (CNS) progenitor cells in different contexts, based on observations indicating a correlation between nestin expression and this cell type in vivo. To evaluate this correlation in more detail nestin mRNA expression in developing and adult mouse CNS was analysed by in situ hybridization. We find that nestin is expressed from embryonic day (E) 7.75 and that expression is detected in many proliferating CNS regions; at E10.5 nestin is expressed in cells of both the rostral and caudal neural tube, including the radial glial cells; at E15.5 and postnatal day (P) 0 expression is observed largely in the developing cerebellum and in the ventricular and subventricular areas of the developing telencephalon. Furthermore, the transition from a proliferating to a post-mitotic cell state is accompanied by a rapid decrease in nestin mRNA for motor neurons in the ventral spinal cord and for neurons in the marginal layer of developing telencephalon. In contrast to these data we observe two proliferating areas, the olfactory epithelium and the precursor cells of the hippocampal granule neurons, which do not express nestin at detectable levels. Thus, nestin mRNA expression correlates with many, but not all, regions of proliferating CNS progenitor cells. In addition to its temporal and spatial regulation nestin expression also appears to be regulated at the level of subcellular mRNA localization: in columnar neuroepithelial and radial glial cells nestin mRNA is predominantly localized to the pial endfeet.     

MAP2 and nestin co-expression in dysembryoplastic neuroepithelial tumors

BACKGROUND: The ontogeny and maturity of neurons and oligodendroglia-like cells (OLC) found in dysembryoplastic neuroepithelial tumors (DNT) remains controversial. A developmental origin has been proposed based on the close association to cortical dysplasia and the benign microscopic and clinical course. Our goal was to characterize the expression of nestin, a neuroepithelial precursor/stem cell antigen in DNT, along with other pathological and clinical features of this entity. METHODS: The clinical and operative features of 13 surgical specimens meeting the histological criteria for DNT were reviewed. Nestin, microtubule-associated protein 2 (MAP2), neurofilament (NF) and glial fibrillary acidic protein (GFAP) were examined by immunohistochemistry and confocal scanning laser microscopy. RESULTS: Select neuronal cells in all cases demonstrated strong MAP2 immunoreactivity. Nestin-positive cells of neuronal morphology were found in 6 cases. OLC demonstrated frequent selective staining for MAP2, GFAP and nestin. Confocal microscopy demonstrated numerous examples of cells co-expressing nestin and MAP2. CONCLUSIONS: Our study suggests that OLCs represent a united population of immature neuronal (nestin + MAP2) and glial (GFAP) phenotypes. Larger, morphologically recognizable neurons also showed occasional co-expression of nestin and MAP2, suggesting a degree of dysmaturity in common with their OLC counterparts. The apparent mixed lineage of OLCs lends support to theories suggesting that DNTs arise from pluripotent neuroepithelial cells.     

Transplanted human embryonic germ cell-derived neural stem cells replace neurons and oligodendrocytes in the forebrain of neonatal mice with excitotoxic brain damage

Stem cell therapy is a hope for the treatment of some childhood neurological disorders. We examined whether human neural stem cells (hNSCs) replace lost cells in a newborn mouse model of brain damage. Excitotoxic lesions were made in neonatal mouse forebrain with the N-methyl-D-aspartate (NMDA) receptor agonist quinolinic acid (QA). QA induced apoptosis in neocortex, hippocampus, striatum, white matter, and subventricular zone. This degeneration was associated with production of cleaved caspase-3. Cells immunopositive for inducible nitric oxide synthase were present in damaged white matter and subventricular zone. Three days after injury, mice received brain parenchymal or intraventricular injections of hNSCs derived from embryonic germ (EG) cells. Human cells were prelabeled in vitro with DiD for in vivo tracking. The locations of hNSCs within the mouse brain were determined through DiD fluorescence and immunodetection of human-specific nestin and nuclear antigen 7 days after transplantation. hNSCs survived transplantation into the lesioned mouse brain, as evidenced by human cell markers and DiD fluorescence. The cells migrated away from the injection site and were found at sites of injury within the striatum, hippocampus, thalamus, and white matter tracts and at remote locations in the brain. Subsets of grafted cells expressed neuronal and glial cell markers. hNSCs restored partially the complement of striatal neurons in brain-damaged mice. We conclude that human EG cell-derived NSCs can engraft successfully into injured newborn brain, where they can survive and disseminate into the lesioned areas, differentiate into neuronal and glial cells, and replace lost neurons. (c) 2005 Wiley-Liss, Inc.     

Altered nestin expression in the cerebrum with periventricular leukomalacia

Nestin is a cytoskeletal protein expressed by neural stem cells, and by immature neurons and glial cells. In an effort to explore the potential of the infant brain for repair and plasticity, we immunohistochemically studied nestin expression in the human cerebral cortex of control subjects and of patients with periventricular leukomalacia. During normal development, nestin immunoreactivity of the cortical gray and white matter was detectable throughout the fetal period, and disappeared around birth. In brain with periventricular leukomalacia, nestin expression was altered in a time- and space-dependent manner. In the cortical gray matter, neuronal immunoreactivity was often reduced in the subacute stage, but was increased in chronic and remote stages. In the white matter near a lesion of periventricular leukomalacia, glial immunoreactivity was increased in all stages. In many cases, neurons and axons far from a lesion also showed an altered expression of nestin. These findings indicate that in brain with periventricular leukomalacia, neurons and glial cells may recapitulate nestin expression in response to ischemic brain injury, suggesting functional relevance in repair and plasticity.     

Increase of nestin-immunoreactive neural precursor cells in the dentate gyrus of pediatric patients with early-onset temporal lobe epilepsy.

A considerable potential for neurogenesis has been identified in the epileptic rat hippocampus. Here, we explore this feature in human patients suffering from chronic mesial temporal lobe epilepsy. Immunohistochemical detection of the neurodevelopmental antigen nestin was used to detect neural precursor cells, and cell-type specific markers were employed to study their histogenetic origin and potential for neuronal or glial differentiation. The ontogenetic regulation of nestin-positive precursors was established in human control brains (week 19 of gestation-15 years of age). A striking increase of nestin-immunoreactive cells within the hilus and dentate gyrus could be observed in a group of young patients with temporal lobe epilepsy (TLE) and surgical treatment before age 2 years compared to adult TLE patients and controls. The cellular morphology and regional distribution closely resembled nestin-immunoreactive granule-cell progenitors transiently expressed during prenatal human hippocampus development. An increased Ki-67 proliferation index and clusters of supragranular nestin-immunoreactive cells within the molecular layer of the dentate gyrus were also noted in the group of young TLE patients. Confocal studies revealed colocalization of nestin and the betaIII isoform of tubulin, indicating a neuronal fate for some of these cells. Vimentin was consistently expressed in nestin-immunoreactive cells, whereas cell lineage-specific markers, i.e., glial fibrillary acidic protein, MAP2, neurofilament protein, NeuN, or calbindin D-28k failed to colocalize. These findings provide evidence for increased neurogenesis in pediatric patients with early onset of temporal lobe epilepsy and/or point towards a delay in hippocampal maturation in a subgroup of patients with TLE.     

Early neuronal and glial determination from mouse E10.5 telencephalon embryonic stem cells: an in vitro study

We report here a novel in vitro model for differentiating neuronal and glial cells from mouse embryonic day 10 telencephalon stem cells. At this developmental stage, the telencephalon consists of a single layer of neuroepithelial stem cells. We used various markers of proliferation and differentiation (Ki-67, nestin, BrdU, Tuj-1 and GFAP) to follow proliferative progenitors and to identify neuronal and glial derivatives. Neuronal derivatives were obtained from nestin+ progenitors. GFAP+ astrocytic derivatives were detected after only 72 h of culture. Both neuronal and glial derivatives were generated close to nestin-positive aggregates. In addition, we were able to manipulate neuronal determination of telencephalon stem cells by gene transient transfection as demonstrated by RP42 gene overexpression. These observations suggest that this in vitro model is of potential use for studying early steps in neuronal or glial determination from embryonic stem cells, an issue of key importance for adult brain cell therapy approaches.     

Nestin‐expressing cell types in the temporal lobe and hippocampus: Morphology,differentiation, and proliferative capacity

Nestin is expressed in immature neuroepithelial and progenitor cell types and transiently upregulated in proliferative neuroglial cells responding to acute brain injury, including following seizures. In 36 temporal lobe (TLobe) specimens from patients with TLobe epilepsy (age range 8–60 years) we studied the number, distribution and morphology of nestin‐expressing cells (NEC) in the pes, hippocampus body, parahippocampal gyrus, amygdala, temporal cortex and pole compared with post mortem control tissues from 26 cases (age range 12 gestational weeks to 76 years). The proliferative fraction of NEC was evaluated in selected regions, including recognized niches, using MCM2. Their differentiation was explored with neuronal (DCX, mushashi, βIII tubulin, NeuN) and glial (GFAP, GFAPdelta, glutamine synthetase, aquaporin4, EAAT1) markers, both in sections or following culture. Findings were correlated with clinical parameters. A stereotypical pattern in the distribution and morphologies of NEC was observed, reminiscent of patterns in the developing brain, with increased densities in epilepsy than adult controls (p < .001). Findings included MCM2‐positive radial glial‐like cells in the periventricular white matter and rows of NEC in the hippocampal fimbria and sulcus. Nestin cells represented 29% of the hippocampal proliferative fraction in epilepsy cases; 20% co‐expressed βIII tubulin in culture compared with 28% with GFAP. Significant correlations were noted between age at surgery, memory deficits and nestin populations. TLobe NEC with ongoing proliferative capacity likely represent vestiges of developmental migratory streams and resident reactive cell populations of potential relevance to hippocampal epileptogenesis, TLobe pathology, and co‐morbidities, including memory decline.     

Nestin expression persists in astrocytes of organotypic slice cultures from rat cortex

Nestin is an intermediate filament protein typical for neural precursor cells that is down-regulated in the post-natal rodent brain. Re-expression of nestin has been observed in reactive astrocytes after injury. In this study, organotypic slice cultures from rat cortex were examined for expression of nestin and glial fibrillary acidic protein between 2 and 8 weeks in culture. Immunoreactivity for nestin and glial fibrillary acidic protein was seen in astrocytes which persisted throughout the observation period. Immunofluorescence double labeling showed widespread co-localization of nestin and glial fibrillary acidic protein. Image analysis revealed that levels of nestin-immunoreactivity plateaued after 5 weeks in culture. By comparison nestin immunoreactivity was absent from glial cells of the cortex in mature rats. These immunohistochemical findings of a persistent expression of nestin in glial cells of organotypic slice culture of the rat cortex indicate a different time course of glial maturation in vitro. This difference could be related to the altered trophic stimulation in vitro; differences in neuronal maturation, activity or survival; slow degeneration of the vasculature; or intrinsic properties of astrocytes.     

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.     

Heterogeneity in progenitor cell subtypes in the ventricular zone of the zebrafish adult telencephalon

The zebrafish has become a new model for adult neurogenesis, owing to its abundant neurogenic areas in most brain subdivisions. Radial glia‐like cells, actively proliferating cells, and label‐retaining progenitors have been described in these areas. In the telencephalon, this complexity is enhanced by an organization of the ventricular zone (VZ) in fast and slow‐dividing domains, suggesting the existence of heterogeneous progenitor types. In this work, we studied the expression of various transgenic or immunocytochemical markers for glial cells (gfap:gfp, cyp19a1b:gfp, BLBP, and S100β), progenitors (nestin:gfp and Sox2), and neuroblasts (PSA‐NCAM) in cycling progenitors of the adult zebrafish telencephalon (identified by expression of proliferating cell nuclear antigen (PCNA), MCM5, or bromodeoxyuridine incorporation). We demonstrate the existence of distinct populations of dividing cells at the adult telencephalic VZ. Progenitors of the overall slow‐cycling domains express high levels of Sox2 and nestin:gfp as well as all glial markers tested. In contrast, domains with an overall fast division rate are characterized by low or missing expression of glial markers. PCNA‐positive cells in fast domains further display a morphology distinct from radial glia and co‐express PSA‐NCAM, suggesting that they are early neuronal precursors. In addition, the VZ contains cycling progenitors that express neither glial markers nor nestin:gfp, but are positive for Sox2 and PSA‐NCAM, identifying them as committed neuroblasts. On the basis of the marker gene expression and distinct cell morphologies, we propose a classification for the dividing cell states at the zebrafish adult telencephalic VZ. © 2010 Wiley‐Liss, Inc.     

Nestin expression defines both glial and neuronal progenitors in postnatal sympathetic ganglia

Sympathetic ganglia are primarily composed of noradrenergic neurons and satellite glial cells. Although both cell types originate from neural crest cells, the identities of the progenitor populations at intermediate stages of the differentiation process remain to be established. Here we report on the identification in vivo of glial and neuronal progenitor cells in postnatal sympathetic ganglia, by using mouse superior cervical ganglia as a model system. There are significant levels of cellular proliferation in mouse superior cervical ganglia during the first 18 days after birth. A majority of the proliferating cells express both nestin and brain lipid-binding protein (BLBP). Bromodeoxyuridine (BrdU) fate-tracing experiments demonstrate that these nestin and BLBP double-positive cells represent a population of glial progenitors for sympathetic satellite cells. The glial differentiation process is characterized by a marked downregulation of nestin and upregulation of S100, with no significant changes in the levels of BLBP expression. We also identify a small number of proliferating cells that express nestin and tyrosine hydroxylase, a key enzyme of catecholamine biosynthesis that defines sympathetic noradrenergic neurons. Together, these results establish nestin as a common marker for sympathetic neuronal and glial progenitor cells and delineate the cellular basis for the generation and maturation of sympathetic satellite cells.