Cortical radial glial cells in human fetuses: depth-correlated transformation into astrocytes

In the human brain, the transformation of radial glial cells (RGC) into astrocytes has been studied only rarely. In this work, we were interested in studying the morphologic aspects underlying this transformation during the fetal/perinatal period, particularly emphasizing the region-specific glial fiber anatomy in the medial cortex. We have used carbocyanine dyes (DiI/DiA) to identify the RGC transitional forms and glial fiber morphology. Immunocytochemical markers such as vimentin and glial fibrillary acidic protein (GFAP) were also employed to label the radial cells of glial lineage and to reveal the early pattern of astrocyte distribution. Neuronal markers such as neuronal-specific nuclear protein (NeuN) and microtubule-associated protein (MAP-2) were employed to discern whether or not these radial cells could, in fact, be neurons or neuronal precursors. The main findings concern the beginning of RGC transformation showing loss of the ventricular fixation in most cases, followed by transitional figures and the appearance of mature astrocytes. In addition, diverse fiber morphology related to depth within the cortical mantle was clearly demonstrated. We concluded that during the fetal/perinatal period the cerebral cortex is undergoing the final stages of radial neuronal migration, followed by involution of RGC ventricular processes and transformation into astrocytes. None of the transitional or other radial glia were positive for neuronal markers. Furthermore, the differential morphology of RGC fibers according to depth suggests that factors may act locally in the subplate and could have a role in the process of cortical RGC transformation and astrocyte localization. The early pattern of astrocyte distribution is bilaminar, sparing the cortical plate. Few astrocytes (GFAP+) in the upper band could be found with radial processes at anytime. This suggests that astrocytes in the marginal zone could be derived from different precursors than those that differentiate from RGCs during this period.     

Radial glial cell development and transformation are disturbed in reeler forebrain

Radial glia are among the earliest cell types to differentiate in the developing mammalian forebrain. Glial fibers span the early cortical wall, forming a dense scaffold; this persists throughout corticogenesis, providing a cellular substrate which supports and directs the migration of young neurons. Although the mechanisms regulating radial glial cell development are poorly understood, a secreted cortical radial glial differentiation signal was recently identified in the embryonic mouse forebrain. This signal is abundant at the time radial glia function to support neuronal migration, and down-regulated perinatally, when radial glia are known to undergo transformation into astrocytes. Therefore, it seems that this signal functions as a radial glial maintenance factor, the availability of which regulates the phenotype of cortical astroglia. Here the differentiation signal is further characterized as RF60, a protein with a molecular weight of approximately 60 kD. In addition, the neurologic mutant mouse reeler provides a genetic model for analysis of RF60 function. Radial glia in reeler cortex are shown to be poorly differentiated and the radial scaffold is shown to be maintained for a shorter time than normal. Furthermore, although astroglial cells from normal cortex are induced to elaborate a radial phenotype by RF60, reeler astroglia show an impaired differentiation response to this. These findings suggest that an intrinsic defect in glial differentiation contributes to the phenotype of abnormal cortical lamination seen in reeler mouse, and indicate that RF60 may play a critical role in normal cortical patterning. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 459–472, 1997     

Oxidative stress‐induced attenuation of thrombospondin‐1 expression in primary rat astrocytes

Astrocytes, the major glial population in the central nervous system (CNS), can secrete thrombospondin (TSP)‐1 that plays the role in synaptogenesis and axonal sprouting during CNS development and tissue repair. However, little is known about the regulation of TSP‐1 expression in astrocytes under oxidative stress condition. Here, a hypoxic mimetic reagent, cobalt chloride (CoCl₂), was used to initiate hypoxia‐induced oxidative stress in primary rat astrocytes. CoCl₂ at the concentration range of 0.1–0.5 mM was found to cause no significant cell death in primary rat astrocytes. However, CoCl₂ at 0.2–0.5 mM increased intracellular reactive oxygen species (ROS) levels and glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) gene expression that is known as a hallmark for oxidative damage. We further found that TSP‐1 mRNA expression in astrocytes was inhibited dose‐ and time‐dependently by CoCl₂. TSP‐1 mRNA levels were increased in CoCl₂‐exposed astrocytes in the presence of the inhibitors (U0126 and PD98059) of mitogen‐activated protein kinase/extracellular signal‐regulated kinases (MAPK/ERK), when compared to that detected in the culture only exposed to CoCl₂. Moreover, the inhibition in TSP‐1 mRNA expression by CoCl₂ was blocked by the addition of the potent antioxidant, N‐acetylcysteine (NAC). Thus, we conclude that CoCl₂ inhibits TSP‐1 mRNA expression in astrocytes via a ROS mechanism possibly involving MAPK/ERK. This inhibition may occur after CNS injury and impair the supportive function of astrocytes on neurite growth in the injured CNS tissues. J. Cell. Biochem. 112: 59–70, 2011. © 2010 Wiley‐Liss, Inc.     

A cyp19a1b‐gfp (aromatase B) transgenic zebrafish line that expresses GFP in radial glial cells

Aromatase is an enzyme that catalyzes the synthesis of estrogen in gonads and brain. Teleost fish express aromatase (AroB) strongly in the brain facilitating its detailed examination. To understand the function of AroB in the brain, we generated transgenic zebrafish that expresses green fluorescent protein (GFP) driven by the brain aromatase cyp19a1b promoter. GFP was found in the radial glial cells of transgenic larvae and adult fish that overlap with AroB immunoreactivity in the correct temporal and spatial pattern. GFP was also coexpressed with radial cell marker BLBP, but was not in neurons. In addition, GFP expression in the radial glial cells was stimulated by estrogen, same as endogenous AroB expression. Thus, this transgenic line faithfully mimics the regulation of AroB expression in radial glial cells. It provides a powerful tool to further characterize progenitor radial cells in adult and developing fish and to evaluate estrogenic activities of xenoestrogens and phytoestrogens. genesis 47:67–73, 2009. © 2008 Wiley‐Liss, Inc.     

Differentiation,polarization, and migration of human induced pluripotent stem cell-derived neural progenitor cells co-cultured with a human glial cell line with radial glial-like characteristics

Here we established a unique human glial cell line, GDC90, derived from a human glioma and demonstrated its utility as a glial scaffold for the polarization and differentiation of human induced pluripotent stem cell-derived neural progenitor cells (iPSC-NPCs). When co-cultured with GDC90 cells, iPSC-NPCs underwent rapid polarization and neurite extension along the radially spreading processes of the GDC90 cells, and showed migratory behavior. This method is potentially useful for detailed examination of neurites or for controlling neurites behavior for regenerative medicine.     

Roles of P2 receptors in glial cells: focus on astrocytes

Central nervous system glial cells release and respond to nucleotides under both physiological and pathological conditions, suggesting that these molecules play key roles in both normal brain function and in repair after damage. In particular, ATP released from astrocytes activates P2 receptors on astrocytes and other brain cells, allowing a form of homotypic and heterotypic signalling, which also involves microglia, neurons and oligodendrocytes. Multiple P2X and P2Y receptors are expressed by both astrocytes and microglia; however, these receptors are differentially recruited by nucleotides, depending upon specific pathophysiological conditions, and also mediate the long-term trophic changes of these cells during inflammatory gliosis. In astrocytes, P2-receptor-induced gliosis occurs via activation of the extracellular-regulated kinases (ERK) and protein kinase B/Akt pathways and involves induction of inflammatory and anti-inflammatory genes, cyclins, adhesion and antiapoptotic molecules. While astrocytic P2Y₁ and P2Y_2,4 are primarily involved in short-term calcium-dependent signalling, multiple P2 receptor subtypes seem to cooperate to astrocytic long-term changes. Conversely, in microglia, exposure to inflammatory and immunological stimuli results in differential functional changes of distinct P2 receptors, suggesting highly specific roles in acquisition of the activated phenotype. We believe that nucleotide-induced activation of astrocytes and microglia may originally start as a defence mechanism to protect neurons from cytotoxic and ischaemic insults; dysregulation of this process in chronic inflammatory diseases eventually results in neuronal cell damage and loss. On this basis, full elucidation of the specific roles of P2 receptors in these cells may help exploit the beneficial neuroprotective features of activated glia while attenuating their harmful properties and thus provide the basis for novel neuroprotective strategies that specifically target the purinergic system.     

Astrocyte‐like cells differentiated from a novel population of CD45‐positive cells in adult human peripheral blood

We have previously reported a novel CD45‐positive cell population called peripheral blood insulin‐producing cells (PB‐IPCs) and its unique potential for releasing insulin in vitro. Despite the CD45‐positive phenotype and self‐renewal ability, PB‐IPCs are distinguished from hemopoietic and endothelial progenitor cells (EPCs) by some characteristics, such as a CD34‐negative phenotype and different culture conditions. We have further identified the gene profiles of the embryonic and neural stem cells, and these profiles include Sox2, Nanog, c‐Myc, Klf4, Notch1 and Mash1. After treatment with all‐trans retinoic acid (ATRA) in vitro, most PB‐IPCs exhibited morphological changes that included the development of elongated and branched cell processes. In the process of induction, the mRNA expression of Hes1 was robustly upregulated, and a majority of cells acquired some astrocyte‐associated specific phenotypes including anti‐glial fibrillary acidic protein (GFAP), CD44, Glutamate‐aspartate transporter (GLAST) and S100β. In spite of the deficiency of glutamate uptaking, the differentiated cells significantly relaxed the regulation of the expression of brain‐derived neurotrophic factor (BDNF) mRNA. This finding demonstrates that PB‐IPCs could be induced into a population of astrocyte‐like cells and enhanced the neurotrophic potential when the state of proliferation was limited by ATRA, which implies that this unique CD45+ cell pool may have a protective role in some degenerative diseases of the central nervous system (CNS).     

Differential regulation by fucoidan of IFN‐γ‐induced NO production in glial cells and macrophages

Fucoidan has shown numerous biological actions; however, the molecular bases of these actions have being issued. We examined the effect of fucoidan on NO production induced by IFN‐γ and the molecular mechanisms underlying these effects in two types of cells including glia (C6, BV‐2) and macrophages (RAW264.7, peritoneal primary cells). Fucoidan affected IFN‐γ‐induced NO and/or iNOS expression both in macrophages and glial cells but in a contrast way. Our data showed that in C6 glioma cells both JAK/STAT and p38 signaling positively regulated IFN‐γ‐induced iNOS, which were inhibited by fucoidan. In contrast, in RAW264.7 cells JAK/STAT is a positive regulator whereas p38 is a negative regulator of NO/iNOS production. In RAW264.7 cells, fucoidan enhanced p38 activation and induced TNF‐α production. We also confirmed the dual regulation of p38 in BV‐2 microglia and primary peritoneal macrophages. From these results, we suggest that fucoidan affects not only IFN‐γ‐induced NO/iNOS production differently in brain and peritoneal macrophages due to the different roles of p38 but the effects on TNF‐α production in the two cell types. These novel observations including selective and cell‐type specific effects of fucoidan on IFN‐γ‐mediated signaling and iNOS expression raise the possibility that it alters the sensitivity of cells to the p38 activation. J. Cell. Biochem. 111: 1337–1345, 2010. © 2010 Wiley‐Liss, Inc.     

Differentiation of human umbilical cord Wharton's jelly‐derived mesenchymal stem cells into germ‐like cells in vitro

Recent studies have demonstrated that mesenchymal stem cells could differentiate into germ cells under appropriate conditions. We sought to determine whether human umbilical cord Wharton's jelly‐derived mesenchymal stem cells (HUMSCs) could form germ cells in vitro. HUMSCs were induced to differentiate into germ cells in all‐trans retinoic acid, testosterone and testicular‐cell‐conditioned medium prepared from newborn male mouse testes. HUMSCs formed “tadpole‐like” cells after induction with different reagents and showed both mRNA and protein expression of germ‐cell‐specific markers Oct4 (POUF5), Ckit, CD49_f (α6), Stella (DDPA3), and Vasa (DDX4). Our results may provide a new route for reproductive therapy involving HUMSCs and a novel in vitro model to investigate the molecular mechanisms that regulate the development of the mammalian germ lineage. J. Cell. Biochem. 109: 747–754, 2010. © 2010 Wiley‐Liss, Inc.     

Aging results in copper accumulations in glial fibrillary acidic protein‐positive cells in the subventricular zone

Analysis of rodent brains with X‐ray fluorescence (XRF) microscopy combined with immunohistochemistry allowed us to demonstrate that local Cu concentrations are thousands of times higher in the glia of the subventricular zone (SVZ) than in other cells. Using XRF microscopy with subcellular resolution and intracellular X‐ray absorption spectroscopy we determined the copper (I) oxidation state and the sulfur ligand environment. Cu K‐edge X‐ray absorption near edge spectroscopy is consistent with Cu being bound as a multimetallic Cu‐S cluster similar to one present in Cu‐metallothionein. Analysis of age‐related changes show that Cu content in astrocytes of the SVZ increases fourfold from 3 weeks to 9 months, while Cu concentration in other brain areas remain essentially constant. This increase in Cu correlates with a decrease in adult neurogenesis assessed using the Ki67 marker (both, however, can be age‐related effects). We demonstrate that the Cu distribution and age‐related concentration changes in the brain are highly cell specific.     

GLAST/EAAT1‐induced Glutamine release via SNAT3 in Bergmann glial cells: evidence of a functional and physical coupling

Glutamate, the major excitatory transmitter in the vertebrate brain, is removed from the synaptic cleft by a family of sodium‐dependent glutamate transporters profusely expressed in glial cells. Once internalized, it is metabolized by glutamine synthetase to glutamine and released to the synaptic space through sodium‐dependent neutral amino acid carriers of the N System (SNAT3/slc38a3/SN1, SNAT5/slc38a5/SN2). Glutamine is then taken up by neurons completing the so‐called glutamate/glutamine shuttle. Despite of the fact that this coupling was described decades ago, it is only recently that the biochemical framework of this shuttle has begun to be elucidated. Using the established model of cultured cerebellar Bergmann glia cells, we sought to characterize the functional and physical coupling of glutamate uptake and glutamine release. A time‐dependent Na⁺‐dependent glutamate/aspartate transporter/EAAT1‐induced System N‐mediated glutamine release could be demonstrated. Furthermore, D‐aspartate, a specific glutamate transporter ligand, was capable of enhancing the co‐immunoprecipitation of Na⁺‐dependent glutamate/aspartate transporter and Na⁺‐dependent neutral amino acid transporter 3, whereas glutamine tended to reduce this association. Our results suggest that glial cells surrounding glutamatergic synapses may act as sensors of neuron‐derived glutamate through their contribution to the neurotransmitter turnover.     

Heterogeneity of glial progenitor cells during the neurogenesis-to-gliogenesis switch in the developing human cerebral cortex

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Development of depolarization‐induced calcium transients in insect glial cells is dependent on the presence of afferent axons

Changes in the intracellular Ca²⁺ concentration ([Ca²⁺]_i) induced by depolarization have been measured in glial cells acutely isolated from antennal lobes of the moth Manduca sexta at different postembryonic developmental stages. Depolarization of the glial cell membrane was elicited by increasing the external K⁺ concentration from 4 to 25 mM. At midstage 5 and earlier stages, less than 20% of the cells responded to 25 mM K⁺ (1 min) with a transient increase in [Ca²⁺]_i of approximately 40 nM. One day later, at late stage 5, 68% of the cells responded to 25 mM K⁺, the amplitude of the [Ca²⁺]_i transients averaging 592 nM. At later stages, all cells responded to 25 mM K⁺ with [Ca²⁺]_i transients with amplitudes not significantly different from those at late stage 5. In stage 6 glial cells isolated from deafferented antennal lobes, i.e., from antennal lobes chronically deprived of olfactory receptor axons, only 30% of the cells responded with [Ca²⁺]_i transients. The amplitudes of these [Ca²⁺]_i transients averaged 93 nM and were significantly smaller than those in normal stage 6 glial cells. [Ca²⁺]_i transients were greatly reduced in Ca²⁺‐free, EGTA‐buffered saline, and in the presence of the Ca²⁺ channel blockers cadmium and verapamil. The results suggest that depolarization of the cell membrane induces Ca²⁺ influx through voltage‐activated Ca²⁺ channels into antennal lobe glial cells. The development of the depolarization‐induced Ca²⁺ transients is rapid between midstage 5 and stage 6, and depends on the presence of afferent axons from the olfactory receptor cells in the antenna. © 2002 Wiley Periodicals, Inc. J Neurobiol 52: 85–98, 2002     

Massive culture of human liver cancer cells in a newly developed radial flow bioreactor system: Ultrafine structure of functionally enhanced hepatocarcinoma cell lines

Summary With a view to initiating clinical trials, cell morphology and function for a newly developed artificial liver support system employing highly functional human liver cell line, FLC-7, cultured in a radial flow bioreactor were compared to cells grown in a conventional monolayer culture. The radial flow bioreactor consists of a vertically extended cylindrical matrix comprised of porous glass bead microcarriers through which liquid medium flows from the periphery in toward the central axis generating a beneficial concentration gradient of oxygen and nutrients, while preventing excessive shear stresses or buildup of waste products. The three-dimensional culture system supports high-density (1.1 × 10⁸ cells/ml-matrix), large scale cultures (4.4 × 10¹⁰ cells/400 ml-bioreactor) with long-term viability. Scanning and transmission electron microscopy (SEM and TEM) revealed that cells cultured in a monolayer system were flattened and extended with numerous cytoplasmic projections. Cells in the three-dimensional culture were spherical and covered with microvillilike processes resembling liver cells in vivo. The cells were solidly attached on the surfaces and within the pores of the microcarriers in highly dense colonies. The spherical cells remained in close contact with adjacent cells, while circulation of liquid medium flowed freely through spaces between cells. FLC-7 cells produced albumin at a rate of 6.41 μg/24 h/10⁶ cells. Alpha-fetoprotein (AFP) production dropped nearly threefold in comparison to monolayer cultures. Results demonstrated that the new artificial liver support systems (ALSS) provides a superior three-dimensional culture environment that allows cells to perform at naturally functioning levels.