Acutely isolated astrocytes as models to probe astrocyte functions

Neuroscientists have become increasingly aware and accepting of the concept that astrocytes likely have many important functions in the CNS. One limitation in establishing these functions is the usual problem of what constitutes suitable experimental approaches. A major experimental step for functional studies of astrocytes has been the widespread use of primary astrocyte cultures, an approach that Leif Hertz pioneered. However, it is now becoming clear that, building on this work, an experimental paradigm shift is now needed. Namely, to increasingly study preparations corresponding to in situ conditions, such as slices. An alternative experimental system where the cells have some of the technical advantages of primary astrocyte cultures is freshly isolated astrocytes. Recent experiments from our laboratory have shown metabotropic glutamate receptor expression by such cells. Examples are given of how functional receptor studies and channel activity measured by patch clamp electrophysiology can be combined with single cell RT-PCR to define further the receptor or channel type are described to illustrate the uses of such preparations.     

Isolation and Culture of Mouse Cortical Astrocytes

Astrocytes are an abundant cell type in the mammalian brain, yet much remains to be learned about their molecular and functional characteristics. In vitro astrocyte cell culture systems can be used to study the biological functions of these glial cells in detail. This video protocol shows how to obtain pure astrocytes by isolation and culture of mixed cortical cells of mouse pups. The method is based on the absence of viable neurons and the separation of astrocytes, oligodendrocytes and microglia, the three main glial cell populations of the central nervous system, in culture. Representative images during the first days of culture demonstrate the presence of a mixed cell population and indicate the timepoint, when astrocytes become confluent and should be separated from microglia and oligodendrocytes. Moreover, we demonstrate purity and astrocytic morphology of cultured astrocytes using immunocytochemical stainings for well established and newly described astrocyte markers. This culture system can be easily used to obtain pure mouse astrocytes and astrocyte-conditioned medium for studying various aspects of astrocyte biology.     

Determinants of regional and local diversity within the astroglial lineage of the normal central nervous system

Astrocytes are a major component of the resident non-neuronal glial cell population of the CNS. They are ubiquitously distributed throughout the brain and spinal cord, where they were initially thought to function in both structural and homeostatic capacities, providing the framework and environment in which neurons performed their parenchymal duties. However, this stroma-like view of astrocytes is no longer satisfactory. Mounting evidence particularly within the last decade indicates that astrocytes do not simply support neuronal activity but directly contribute to it. Congruent with this evolving view of astrocyte function in information processing is the emergent notion that these glial cells are not a homogeneous population of cells. Thus, astrocytes in various anatomically distinct regions of the normal CNS possess unique phenotypic characteristics that may directly influence the particular neuronal activities that define these regions. Remarkably, regional populations of astrocytes appear to exhibit local heterogeneity as well. Many phenotypic traits of the astrocyte lineage are responsive to local environmental cues (i.e., are adaptable), suggesting that plasticity contributes to this diversity. However, compelling evidence suggests that astrocytes arise from multiple distinct progenitor pools in the developing CNS, raising the intriguing possibility that some astrocyte heterogeneity may result from intrinsic differences between these progenitors. The purpose of this review is to explore the evidence for and mechanistic determinants of regional and local astrocyte diversity.     

Blood-brain barrier modeling with co-cultured neural progenitor cell-derived astrocytes and neurons

In vitro blood-brain barrier (BBB) models often consist of brain microvascular endothelial cells (BMECs) that are co-cultured with other cells of the neurovascular unit, such as astrocytes and neurons, to enhance BBB properties. Obtaining primary astrocytes and neurons for co-culture models can be laborious, while yield and heterogeneity of primary isolations can also be limiting. Neural progenitor cells (NPCs), because of their self-renewal capacity and ability to reproducibly differentiate into tunable mixtures of neurons and astrocytes, represent a facile, readily scalable alternative. To this end, differentiated rat NPCs were co-cultured with rat BMECs and shown to induce BBB properties such as elevated trans-endothelial electrical resistance, improved tight junction continuity, polarized p-glycoprotein efflux, and low passive permeability at levels indistinguishable from those induced by primary rat astrocyte co-culture. An NPC differentiation time of 12 days, with the presence of 10% fetal bovine serum, was found to be crucial for generating NPC-derived progeny capable of inducing the optimal response. This approach could also be extended to human NPC-derived astrocytes and neurons which similarly regulated BBB induction. The distribution of rat or human NPC-derived progeny under these conditions was found to be a roughly 3 : 1 mixture of astrocytes to neurons with varying degrees of cellular maturity. BMEC gene expression analysis was conducted using a BBB gene panel, and it was determined that 23 of 26 genes were similarly regulated by either differentiated rat NPC or rat astrocyte co-culture while three genes were differentially altered by the rat NPC-derived progeny. Taken together, these results demonstrate that NPCs are an attractive alternative to primary neural cells for use in BBB co-culture models.     

Transplantation of specific human astrocytes promotes functional recovery after spinal cord injury

Repairing trauma to the central nervous system by replacement of glial support cells is an increasingly attractive therapeutic strategy. We have focused on the less-studied replacement of astrocytes, the major support cell in the central nervous system, by generating astrocytes from embryonic human glial precursor cells using two different astrocyte differentiation inducing factors. The resulting astrocytes differed in expression of multiple proteins thought to either promote or inhibit central nervous system homeostasis and regeneration. When transplanted into acute transection injuries of the adult rat spinal cord, astrocytes generated by exposing human glial precursor cells to bone morphogenetic protein promoted significant recovery of volitional foot placement, axonal growth and notably robust increases in neuronal survival in multiple spinal cord laminae. In marked contrast, human glial precursor cells and astrocytes generated from these cells by exposure to ciliary neurotrophic factor both failed to promote significant behavioral recovery or similarly robust neuronal survival and support of axon growth at sites of injury. Our studies thus demonstrate functional differences between human astrocyte populations and suggest that pre-differentiation of precursor cells into a specific astrocyte subtype is required to optimize astrocyte replacement therapies. To our knowledge, this study is the first to show functional differences in ability to promote repair of the injured adult central nervous system between two distinct subtypes of human astrocytes derived from a common fetal glial precursor population. These findings are consistent with our previous studies of transplanting specific subtypes of rodent glial precursor derived astrocytes into sites of spinal cord injury, and indicate a remarkable conservation from rat to human of functional differences between astrocyte subtypes. In addition, our studies provide a specific population of human astrocytes that appears to be particularly suitable for further development towards clinical application in treating the traumatically injured or diseased human central nervous system.     

Versatile and Simple Approach to Determine Astrocyte Territories in Mouse Neocortex and Hippocampus

Background

Besides their neuronal support functions, astrocytes are active partners in neuronal information processing. The typical territorial structure of astrocytes (the volume of neuropil occupied by a single astrocyte) is pivotal for many aspects of glia–neuron interactions.

Methods

Individual astrocyte territorial volumes are measured by Golgi impregnation, and astrocyte densities are determined by S100β immunolabeling. These data are compared with results from conventionally applied methods such as dye filling and determination of the density of astrocyte networks by biocytin loading. Finally, we implemented our new approach to investigate age-related changes in astrocyte territories in the cortex and hippocampus of 5- and 21-month-old mice.

Results

The data obtained by our simplified approach based on Golgi impregnation were compared to previously published dye filling experiments, and yielded remarkably comparable results regarding astrocyte territorial volumes. Moreover, we found that almost all coupled astrocytes (as indicated by biocytin loading) were immunopositive for S100β. A first application of this new experimental approach gives insight in age-dependent changes in astrocyte territorial volumes. They increased with age, while cell densities remained stable. In 5-month-old mice, the overlap factor was close to 1, revealing little or no interdigitation of astrocyte territories. However, in 21-month-old mice, the overlap factor was more than 2, suggesting that processes of adjacent astrocytes interdigitate.

Conclusion

Here we verified the usability of a simple, versatile method for assessing astrocyte territories and the overlap factor between adjacent territories. Second, we found that there is an age-related increase in territorial volumes of astrocytes that leads to loss of the strict organization in non-overlapping territories. Future studies should elucidate the physiological relevance of this adaptive reaction of astrocytes in the aging brain and the methods presented in this study might be a powerful tool to do so.     

Purified astrocytes promote the in vitro division of a bipotential glial progenitor cell.

Optic nerves of neonatal rats contain a bipotential glial progenitor cell which can be induced by tissue culture conditions to differentiate into either an oligodendrocyte (the myelin-forming cell of the CNS) or a type 2 astrocyte (an astrocyte population found only in the myelinated tracts of the CNS). In our previous studies most oligodendrocyte-type 2 astrocyte (O-2A) progenitor cells differentiated within 3 days in vitro with relatively little division of the progenitors or their differentiated progeny. We have now found that the O-2A progenitors are stimulated to divide in culture by purified populations of type 1 astrocytes, another glial cell-type found in the rat optic nerve. This cell-cell interaction appears to be mediated by a soluble factor(s) and results in the production of large numbers of both progenitor cells and oligodendrocytes. As type 1 astrocytes are the major glial cell-type in the optic nerve when oligodendrocytes first begin to be produced in large numbers in vivo, our results suggest that this astrocyte subpopulation may play an important role in expanding the oligodendrocyte population during normal development.     

JC virus induces nonapoptotic cell death of human central nervous system progenitor cell-derived astrocytes

JC virus (JCV), a human neurotropic polyomavirus, demonstrates a selective glial cell tropism that causes cell death through lytic infection. Whether these cells die via apoptosis or necrosis following infection with JCV remains unclear. To investigate the mechanism of virus-induced cell death, we used a human central nervous system progenitor-derived astrocyte cell culture model developed in our laboratory. Using in situ DNA hybridization, immunocytochemistry, electron microscopy, and an RNase protection assay, we observed that astrocytes support a progressive JCV infection, which eventually leads to nonapoptotic cell death. Infected astrocyte cell cultures showed no difference from noninfected cells in mRNA expression of the caspase family genes or in any ultrastructural features associated with apoptosis. Infected cells demonstrated striking necrotic features such as cytoplasmic vacuolization, watery cytoplasm, and dissolution of organelles. Furthermore, staining for caspase-3 and terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling were not detected in infected astrocyte cultures. Our findings suggest that JCV-induced cell death of these progenitor cell-derived astrocytes does not utilize an apoptosis pathway but exhibits a pattern of cell destruction consistent with necrotic cell death.     

Identification of genetic networks involved in the cell growth arrest and differentiation of a rat astrocyte cell line RCG-12

The purpose of the present study is to establish and characterize a conditionally immortalized astrocyte cell line and to clarify the genetic networks responsible for the cell growth arrest and differentiation. A conditionally immortalized astrocyte cell line, RCG-12, was established by infecting primary cultured rat cortical glia cells with a temperature-sensitive simian virus 40 large T-antigen. At a permissive temperature of 33 degrees C, the large T-antigen was expressed and cells grew continuously. On the other hand, the down-regulation of T-antigen at a non-permissive temperature of 39 degrees C led to growth arrest and differentiation. The cells expressed astrocyte-expressed genes such as glial fibrillary acidic protein. Interestingly, the differentiated condition induced by the non-permissive temperature significantly elevated the expression levels of several astrocyte-expressed genes. To identify the detailed mechanisms by which non-permissive temperature-induced cell growth arrest and differentiation, we performed high-density oligonucleotide microarray analysis and found that 556 out of 15,923 probe sets were differentially expressed 2.0-fold. A computational gene network analysis revealed that a genetic network containing up-regulated genes such as RB, NOTCH1, and CDKN1A was associated with the cellular growth and proliferation, and that a genetic network containing down-regulated genes such as MYC, CCNB1, and IGF1 was associated with the cell cycle. The established cell line RCG-12 retains some characteristics of astrocytes and should provide an excellent model for studies of astrocyte biology. The present results will also provide a basis for understanding the detailed molecular mechanisms of the growth arrest and differentiation of astrocytes.     

Astrocytes Enhance the Invasion Potential of Glioblastoma Stem-Like Cells

Glioblastomas (GBMs) are characterized as highly invasive; the contribution of GBM stem-like cells (GSCs) to the invasive phenotype, however, has not been completely defined. Towards this end, we have defined the invasion potential of CD133+ GSCs and their differentiated CD133− counterparts grown under standard in vitro conditions and in co-culture with astrocytes. Using a trans-well assay, astrocytes or astrocyte conditioned media in the bottom chamber significantly increased the invasion of GSCs yet had no effect on CD133− cells. In addition, a monolayer invasion assay showed that the GSCs invaded farther into an astrocyte monolayer than their differentiated progeny. Gene expression profiles were generated from two GSC lines grown in trans-well culture with astrocytes in the bottom chamber or directly in contact with astrocyte monolayers. In each co-culture model, genes whose expression was commonly increased in both GSC lines involved cell movement and included a number of genes that have been previously associated with tumor cell invasion. Similar gene expression modifications were not detected in CD133− cells co-cultured under the same conditions with astrocytes. Finally, evaluation of the secretome of astrocytes grown in monolayer identified a number of chemokines and cytokines associated with tumor cell invasion. These data suggest that astrocytes enhance the invasion of CD133+ GSCs and provide additional support for a critical role of brain microenvironment in the regulation of GBM biology.     

Gene expression patterns in in vivo normal adult astrocytes compared with cultured neonatal and normal adult astrocytes

This paper presents data on the basal gene expression patterns, determined by microarray analysis, for cultured neonatal and normal adult striatal astrocytes, and for comparison, for astrocytes isolated directly from adult rat striatum (in vivo adult astrocytes). Of the 1176 genes on the Clontech array, 1101 were expressed in one of the three types of astrocyte samples. Nineteen of the genes were expressed only in the astrocytes taken directly from adult rats (in vivo adult). The cultured neonatal astrocytes expressed many genes at a two-fold or greater level than their expression in cultured adult astrocytes, including genes in the adhesion, cytoskeleton, and extracellular matrix (ECM) family, signal transduction genes, and genes related to apoptosis, DNA-binding, and cell cycle regulation. Overall the results support the concept that cultured neonatal astrocytes are more "activated" than cultured adult cells, although the adult cells expressed higher levels of many metabolic enzyme and protease/protease inhibitor genes.     

Extracellular matrix-associated molecules collaborate with ciliary neurotrophic factor to induce type-2 astrocyte development

O-2A progenitor cells give rise to both oligodendrocytes and type-2 astrocytes in vitro. Whereas oligodendrocyte differentiation occurs constitutively, type-2 astrocyte differentiation requires extracellular signals, one of which is thought to be ciliary neurotrophic factor (CNTF). CNTF, however, is insufficient by itself to induce the development of stable type-2 astrocytes. In this report we show the following: (a) that molecules associated with the extracellular matrix (ECM) cooperate with CNTF to induce stable type-2 astrocyte differentiation in serum-free cultures. The combination of CNTF and the ECM-associated molecules thus mimics the effect of FCS, which has been shown previously to induce stable type-2 astrocyte differentiation in vitro. (b) Both the ECM-associated molecules and CNTF act directly on O-2A progenitor cells and can induce them to differentiate prematurely into type-2 astrocytes. (c) ECM-associated molecules also inhibit oligodendrocyte differentiation, even in the absence of CNTF, but this inhibition is not sufficient on its own to induce type-2 astrocyte differentiation. (d) Whereas the effect of ECM on oligodendrocyte differentiation is mimicked by basic fibroblast growth factor (bFGF), the effect of ECM on type-2 astrocyte differentiation is not. (e) The ECM-associated molecules that are responsible for inhibiting oligodendrocyte differentiation and for cooperating with CNTF to induce type-2 astrocyte differentiation are made by non-glial cells in vitro. (f) Molecules that have these activities and bind to ECM are present in the optic nerve at the time type-2 astrocytes are thought to be developing.     

Preferential Lentiviral Targeting of Astrocytes in the Central Nervous System

The ability to visualize and genetically manipulate specific cell populations of the central nervous system (CNS) is fundamental to a better understanding of brain functions at the cellular and molecular levels. Tools to selectively target cells of the CNS include molecular genetics, imaging, and use of transgenic animals. However, these approaches are technically challenging, time consuming, and difficult to control. Viral-mediated targeting of cells in the CNS can be highly beneficial for studying and treating neurodegenerative diseases. Yet, despite specific marking of numerous cell types in the CNS, in vivo selective targeting of astrocytes has not been optimized. In this study, preferential targeting of astrocytes in the CNS was demonstrated using engineered lentiviruses that were pseudotyped with a modified Sindbis envelope and displayed anti-GLAST IgG on their surfaces as an attachment moiety. Viral tropism for astrocytes was initially verified in vitro in primary mixed glia cultures. When injected into the brains of mice, lentiviruses that displayed GLAST IgG on their surface, exhibited preferential astrocyte targeting, compared to pseudotyped lentiviruses that did not incorporate any IgG or that expressed a control isotype IgG. Overall, this approach is highly flexible and can be exploited to selectively target astrocytes or other cell types of the CNS. As such, it can open a window to visualize and genetically manipulate astrocytes or other cells of the CNS as means of research and treatment.     

Cultured astrocytes from a syncytium after maturation

The formation of functional gap junctions between astrocytes was investigated during differentiation of these cells in culture. Precursor cells of GFA (glial fibrillary acidic) protein-positive astrocytes were cultured in a chemically defined medium as a homogeneous population. These cells were rarely coupled to one neighbour, as revealed by electrical and dye coupling and never formed a large syncytium, as investigated by injection and spread of Lucifer Yellow. Differentiation with respect to GFA protein accumulation can be induced in these cells by culturing in horse serum-containing medium. The formation of functional junctions developed within 2 weeks in about 20% of the cells. Coupled cells formed a large syncytium. When the astrocytes were co-cultured with primary cerebellar cells (consisting predominantly of small neurons) after the switch to serum-containing medium the percentage of coupled astrocytes increased to about 65%. Again the coupled cells formed a large syncytium. Since no physical contact was possible between the astrocyte cultures and the primary cerebellar cells the stimulation of coupling had to be signalized by soluble factor(s).     

Malignant transformation of p53-deficient astrocytes is modulated by environmental cues in vitro.

The early incidence of p53 mutation in astrocytomas suggests that it plays an important role in astrocyte transformation. Astrocytes isolated from homozygous p53 knockout mice grow rapidly, lack contact inhibition, and are immortal. Here we tested whether the loss of p53 is sufficient for progression to tumorigenicity of astrocytes. We grew primary astrocytes under three conditions for over 120 population doublings and assessed their antigenic phenotype, chromosome number, and expression of glioma-associated genes as well as their ability to form colonies in soft agarose and tumors s.c. and intracranially in nude mice. Under two conditions (10% FCS and 0.5% FCS plus 20 ng/ml EGF), cells acquired the ability to form colonies in soft agarose and tumors in nude mice, and this was accompanied by the expression of genes, including epidermal growth factor receptor, platelet-derived growth factor receptor alpha and beta, protein kinase Cdelta, and vascular endothelial growth factor, which are known to be aberrantly regulated in human astrocytomas. Under the third condition (0.5% FCS plus 10 ng/ml basic fibroblast growth factor), astrocytes gained the ability to form colonies in soft agarose and had abnormal chromosome numbers similar to cells in the first two conditions but did not form tumors in nude mice or overexpress glioma-associated genes. These data provide experimental evidence for the idea that the malignant progression initiated by the loss of p53 may be subject to modulation by extracellular environmental influences.     

Astrocyte ethanol exposure reveals persistent and defined calcium response subtypes and associated gene signatures

Astrocytes play a critical role in brain function, but their contribution during ethanol (EtOH) consumption remains largely understudied. In light of recent findings on the heterogeneity of astrocyte physiology and gene expression, an approach with the ability to identify subtypes and capture this heterogeneity is necessary. Here, we combined measurements of calcium signaling and gene expression to define EtOH-induced astrocyte subtypes. In the absence of a demonstrated EtOH receptor, EtOH is believed to have effects on the function of many receptors and downstream biological cascades that underlie calcium responsiveness. This mechanism of EtOH-induced calcium signaling is unknown and this study provides the first step in understanding the characteristics of cells displaying these observed responses. To characterize underlying astrocyte subtypes, we assessed the correlation between calcium signaling and astrocyte gene expression signature in response to EtOH. We found that various EtOH doses increased intracellular calcium levels in a subset of astrocytes, distinguishing three cellular response types and one nonresponsive subtype as categorized by response waveform properties. Furthermore, single-cell RNA-seq analysis of astrocytes from the different response types identified type-enriched discriminatory gene expression signatures. Combining single-cell calcium responses and gene expression analysis identified specific astrocyte subgroups among astrocyte populations defined by their response to EtOH. This result provides a basis for identifying the relationship between astrocyte susceptibility to EtOH and corresponding measurable markers of calcium signaling and gene expression, which will be useful to investigate potential subgroup-specific influences of astrocytes on the physiology and pathology of EtOH exposure in the brain.     

Heparin is a unique marker of progenitors in the glial cell lineage.

The oligodendrocyte-type-2 astrocyte progenitor cells (precursors of oligodendrocytes and type-2 astrocytes) are an excellent system in which to study differentiation as they can be manipulated in vitro. Maintenance of oligodendrocyte-type-2 astrocyte progenitor cells requires basic fibroblast growth factor, a growth factor whose action normally depends on a heparan sulfate coreceptor. Biochemical analysis revealed a most surprising result: that the oligodendrocyte-type-2 astrocyte progenitors did not synthesize heparan sulfate, the near ubiquitous N-sulfated cell surface polysaccharide, but the chemically related heparin in a form that was almost completely N- and O-sulfated. The heparin was detected in the pericellular fraction of the cells and the culture medium. In contrast the differentiated glial subpopulations (oligodendrocytes and type-2 astrocytes) synthesized typical heparan sulfate but with distinctive fine structural features for each cell type. Thus heparin is a unique differentiation marker in the glial lineage. Previously heparin has been found only in a subset of mature mast cells called the connective tissue mast cells. Its presence within the developing nervous system on a precise population of progenitors may confer specific and essential recognition properties on those cells in relation to binding soluble growth and/or differentiation factors and the extracellular matrix.     

Supportive or information-processing functions of the mature protoplasmic astrocyte in the mammalian CNS? A critical appraisal

It has been proposed that astrocytes should no longer be viewed purely as support cells for neurons, such as providing a constant environment and metabolic substrates, but that they should also be viewed as being involved in affecting synaptic activity in an active way and, therefore, an integral part of the information-processing properties of the brain. This essay discusses the possible differences between a support and an instructive role, and concludes that any distinction has to be blurred. In view of this, and a brief overview of the nature of the data, the new evidence seems insufficient to conclude that the physiological roles of mature astrocytes go beyond a general support role. I propose a model of mature protoplasmic astrocyte function that is drawn from the most recent data on their structure, the domain concept and their syncytial characteristics, of an independent rather than integrative functioning of the ends of each process where the activities that affect synaptic activity and blood vessel diameter will be concentrated.     

THE FINE STRUCTURE OF ASTROCYTES IN THE CEREBRAL CORTEX AND THEIR RESPONSE TO FOCAL INJURY PRODUCED BY HEAVY IONIZING PARTICLES

Normal and reactive astrocytes in the cerebral cortex of the rat have been studied with the electron microscope following focal alpha particle irradiation. The presence of glycogen and approximately 60-A fibrils identify astrocyte cytoplasm in formalin-perfused tissue. The glycogen particles facilitate the identification of small processes and subpial and perivascular end-feet. Both protoplasmic and fibrous astrocytes contain cytoplasmic fibrils and should be distinguished on the basis of the configuration of their processes and their distribution. Acutely reactive astrocytes are characterized by a marked increase in the number of glycogen granules and mitochondria from the first day after irradiation. These cells later hypertrophy and accumulate lipid bodies and increased numbers of cytoplasmic fibrils. The glial "scar" consists of a greatly expanded volume of astrocyte cytoplasm filled with fibrils and displays no signs of astrocyte death, reversion to primitive forms, or extensive multiplication.