Glial Properties Research Articles

In vitro properties of a newly established medulloblastoma cell line, MCD-1.

Medulloblastomas are poorly differentiated brain tumors believed to arise from primitive pleuripotential stem cells, and tend to express mixed neuronal and glial properties. In the present study, we examined immunohistochemical and neurotransmitter phenotypic properties in a newly established medulloblastoma cell line, MCD-1. MCD-1 cells were immortal, not contact-inhibited, but did not grow in soft agar. Immunohistochemical studies showed positive staining for neurofilament protein (NF), neuron-specific enolase (NSE), synaptophysin, MAP 2, tau, NCAM 180, vimentin, and S-100 protein. The cells expressed specific uptake of glutamate, serotonin, and choline, but not GABA or dopamine. A significant increase in process extension was seen in response to agents that enhance intracellular cyclic AMP, especially 3-isobutyl-1-methylxanthine (IBMX). Process formation induced by IBMX was associated with a decrease in cell proliferation as evidenced by a reduction in numbers of cells incorporating 5-bromo-2-deoxyuridine (BrdU). No increase in process extension was observed following exposure to NGF or retinoic acid. MCD-1 cells were shown to produce transforming growth factor beta (TGF beta), and were immunopositive for mutant p53. Transfection assays with the PG13-Luc reporter plasmid, which contains a p53-responsive enhancer element and a luciferase reporter gene, suggested MCD-1 cells are deficient in wild-type p53 and do not activate p53 on treatment with the anticancer agent adriamycin. The MCD-1 cell line is suggested to represent an abnormally differentiated cell type, which has some properties consistent with a multipotent neuronal phenotype while retaining some properties of immature cells of a glial lineage. The MCD-1 cell line can be used to provide a model of a medulloblastoma cell line that is resistant to growth-controlling and anticancer agents.

Human central neurocytoma cells show neuronal physiological properties in vitro

Central neurocytoma is a rare brain tumor composed of small round synaptophysin-positive cells, suggesting a neuronal origin of these tumor cells. Glial properties are inferred, however, from the observation that the tumor cells exhibit a strong morphological similarity to oligodendroglioma cells and show an astrocytic differentiation in vitro. To test for neuronal or glial physiological properties, we studied cultured neurocytoma cells derived from a surgical specimen from a 44-year-old man, employing the patch-clamp technique. Early primary cultures were composed of morphologically unique bi- or multipolar cells which were positive for synaptophysin and negative for the astrocyte marker glial fibrillary acidic protein. In the majority of these cells, whole-cell membrane current recordings revealed physiological properties of neurons, i.e., a high density of Na+ currents, the capacity to generate action potentials, and the expression of inotropic neurotransmitter receptors. Metabotropic neurotransmitter receptors could be demonstrated by Ca2+ imaging techniques. The remaining bi- or multipolar cells and almost all cells in later culture stages and in vitro passage lacked these neuronal properties and showed physiological features characteristic of glial cells. We conclude that the major population of neurocytoma cells shows physiological properties of neurons and that with time in culture this population is replaced by electrically passive cells.

Dual Immunologic Property of S-100 Protein in Normal Eyes and Eyes with Retinoblastoma: A Histopathological and Immunohistochemical Study of 88 Cases

To ascertain the cell types expressing S-100 protein in eyes with retinoblastoma, a follow-up study was made on 88 formalin-fixed and paraffin-embedded retinoblastoma specimens histopathologically and immunohistochemically, using antibodies against S-100 protein. In normal retinas and morphologically normal retinas with retinoblastoma, both astrocytes and ganglion cells were stained positively for S-100 protein. In the retinoblastomas, not only the reactive astrocytes in most cases, but also large tumor cells with large cell bodies, prominent nucleoli and well-differentiated figures were positive for S-100 proteins in 2 cases. These large tumor cells were thought to be neuronal and were called ganglion-like cells because they were morphologically and immunohistochemically compatible with the large ganglion cells in normal retina. The results indicate that in eyes with retinoblastoma, S-100 protein has both neuronal and glial immunologic properties and could have a limited value as a glial marker for the histogenesis study of retinoblastoma.

Comparative biochemical, morphological, and immunocytochemical studies between C‐6 glial cells of early and late passages and advanced passages of glial cells derived from aged mouse cerebral hemispheres

We have used C6 glial cells (2B clone), early and late passage, as well as advanced passages (8-17) of glial cells derived from aged (18-month-old) mouse cerebral hemispheres (MACH), as model systems for studying glial properties. In this study passages 20-24 were considered "early" and passages 73-90 were considered "late." Activities of glutamine synthetase (GS) and cyclic nucleotide phosphohydrolase (CNP) were used as biochemical markers for astrocytes and oligodendrocytes, respectively. Glial phenotypes were identified immunocytochemically using double staining for glial fibrillary acidic protein (GFAP) and A2B5 antigen (type 1 and type 2 astrocytes) or galactocerebroside (GalC) and A2B5 antigen (oligodendrocytes); cells positive for A2B5 and negative for both GFAP and GalC were considered to be precursor cells. Cultures were grown either in DMEM supplemented with 10% fetal bovine serum or in serum-free chemically defined medium (CDM) supplemented with insulin and transferrin. We report that early-passage C6 glial cells continue to be bipotential cells and when grown in the absence of serum express high GS and CNP activities correlating with the high number of GFAP- and GalC-positive cells, respectively. Late-passage cells continued to be committed to the type 2 astrocytic phenotype regardless of media composition (+/- serum). MACH cultures consist of protoplasmic type 1 astrocytes, differentiated type 2 astrocytes, and oligodendrocytes as well as glial progenitor cells. When these cultures were grown in CDM+transferrin, both GS and CNP activities increased, suggesting that transferrin has provided the signal for progenitor cells present in these cultures derived from aged brain to differentiate into type 2 astrocytes and oligodendrocytes.

Antisense nucleotides as a strategy for manipulating neural and glial properties

Antisense nucleotides as a strategy for manipulating neural and glial properties

Applications of immortalized cells in basic and clinical neurology

Immortalized cell lines can serve as model systems for studies of neuronal development and restoration of function in models of neurological disease. Cell lines which result from spontaneous or experimentally-induced tumors have been used for these purposes. More recently, the techniques of genetic engineering have resulted in the production of cell lines with specific desired characteristics. This has been accomplished by insertion of a desired gene into a pre-existing immortal cell or by immortalizing primary cells. The production of immortal cell lines using temperature-sensitive immortalizing genes offers an additional method of controlling gene expression, and thereby controlling cell proliferation and differentiation. In the nervous system, these techniques have produced immortal cell lines with neuronal and glial properties.

Regulation of the S100 protein and GFAP genes is mediated by two common mechanisms in RT4 neuro-glial cell lines

RT4-AC cells express both neuronal and glial properties and undergo cell-type conversion in culture to three distinct derivatives, described as either neuronal-like or glial-like. A coordinate induction of glial fibrillary acidic protein (GFAP) and S100 protein expression in RT4-AC cells occurs in response to either high cell density or elevated levels of intracellular cyclic AMP. RNA hybridization data indicate that S100 protein and GFAP gene expression is coordinately induced by cAMP. In addition, for the first time we provide direct evidence that the ability to express both the S100 and GFAP genes is conserved with cell-type conversion to the glial derivative cell types, but is coordinately lost with conversion to the neuronal derivative cell types. These results make it highly likely that the GFAP and S100 genes are regulated by two common mechanisms in RT4-AC cells: (1) cAMP-mediated control of gene expression; and (2) a mechanism that allows these two genes to be coordinately expressed or not expressed as a consequence of cell-type conversion.

Immortalization of precursor cells from the mammalian CNS

Recent studies show that the nervous system contains many molecularly distinct cell types. Clonal cell marking experiments demonstrate that different cell types in some areas of the CNS are products of a multipotential stem cell. The factors controlling the differentiation of vertebrate CNS precursor cells would be more accessible to molecular analysis if cell lines with precursor properties could be established. Here we show that cell lines expressing an antigenic marker specific for a major brain precursor cell population can be established from rat cerebellum. We demonstrate that cell lines express the precursor, neuronal or glial properties depending on the growth conditions. This work supports the view that brain precursor cells expressing the marker Rat 401 are multipotential and can differentiate into cells with either neuronal or glial properties. Cell lines capable of differentiation should be useful in defining the signaling systems generating the cell types of the brain.

Induction and segregation of glial intermediate filament expression in the RT4 family of peripheral nervous system cell lines.

We have found glial fibrillary acidic protein (GFAP), the major component of astrocyte intermediate filaments, to be expressed in cell lines of the RT4 peripheral neurotumor family. The RT4 family is a "stem-cell-like" cell line, RT4-AC, that spontaneously undergoes differentiation in culture to three derivative cell types. This process, termed cell-type conversion, results in a segregation among the derivative cell types of parental cell phenotypes that have been described as neuronal-like or glial-like. We have identified a 50-kDa GFAP-immunoreactive cytoskeletal protein and GFAP mRNA in continuous RT4-AC and RT4-D (glial-type derivative) cell lines, but not in two presumptive neuronal-type cell lines. This result suggests that GFAP gene expression is coordinately coupled with the expression of other glial properties during cell-type conversion. In addition, the RT4-AC and RT4-D sublines were found to significantly express GFAP only at high cell densities and not during logarithmic growth and to express GFAP precociously during morphological differentiation following treatment with 1 mM N6, O2'-dibutyryladenosine 3',5'-cyclic monophosphate. These observations closely reflect reports of glial filament expression in astrocyte cultures, suggesting that a common regulatory mechanism is employed by central and peripheral nervous system glia.

Cell-type-specific responses of RT4 neural cell lines to dibutyryl-cAMP: branch determination versus maturation.

This report describes the induction of cell-type-specific maturation, by dibutyryl-cAMP and testololactone, of neuronal and glial properties in a family of cell lines derived from a rat peripheral neurotumor, RT4. This maturation allows further understanding of the process of determination because of the close lineage relationship between the cell types of the RT4 family. The RT4 family is characterized by the spontaneous conversion of one of the cell types, RT4-AC (stem-cell type), to any of three derivative cell types, RT4-B, RT4-D, or RT4-E, with a frequency of about 10(-5). The RT4-AC cells express some properties characteristic of both neuronal and glial cells. Of these neural properties expressed by RT4-AC cells, only the neuronal properties are expressed by the RT4-B and RT4-E cells, and only the glial properties are expressed by the RT4-D cells. This in vitro cell-type conversion of RT4-AC to three derivative cell types is a branch point for the coordinate regulation of several properties and seems to resemble determination in vivo. In our standard culture conditions, several other neuronal and glial properties are not expressed by these cell types. However, addition of dibutyryl-cAMP induces expression of additional properties, in a cell-type-specific manner: formation of long cellular processes in the RT4-B8 and RT4-E5 cell lines and expression of high-affinity uptake of gamma-aminobutyric acid, by a glial-cell-specific mechanism, in the RT4-D6-2 cell line. These new properties are maximally expressed 2-3 days after addition of dibutyryl-cAMP. This indicates that conversion of RT4-AC to the derivative cell types is also a branch point for the regulation of cell-type-specific properties whose expression is responsive to cAMP. Thus, the potential for maturation in response to increased cAMP is a property that segregates in a cell-type-specific manner and is activated at the determinational level in this system.

Establishment of 'normal' nervous cell lines after transfer of polyoma virus and adenovirus early genes into murine brain cells.

Brain cells from murine embryos were transfected with the polyoma virus large T or the adenovirus 5 EIA gene and, simultaneously, with the phosphotransferase coding NeoR gene. The efficiently transfected cells were selected for their resistance to Geneticin (G418) and their ability to clone at low cell density. Subsequently, most of the selected cells could be sub-cloned and continuously grown for 6-18 months so far. Their doubling time varied between 18 and 72 h. From independent transfections, more than one hundred cell lines were established. They did not exhibit a transformed phenotype, but subsequent transfection with the polyoma middle T gene induced their oncogenic transformation. The maintenance and expression of the transferred genes were verified. Most of the analyzed cell lines retained glial properties. These results suggest that the lines obtained as well as a further extension of this in vitro system should be of interest for the study of nervous cell interactions, differentiation and functions.

S-100 protein in retinoblastoma revisited. An immunohistochemical study.

Fifty formalin-fixed and paraffin-embedded retinoblastoma specimens were studied for the presence of S-100 protein in malignant tumour cells and in reactive glial cells. Its distribution in four normal human eyeglobes enucleated because of an orbital tumour was also studied. The laboratory method was a sensitive immunohistochemical staining procedure, and sections from human brain served as controls. Three anti-S-100 protein antisera were used to determine whether previously reported discrepancies could be related to differences in antibody specificity, but all gave identical results. In morphologically normal human retina, a strong reaction for S-100 protein was seen in astrocytes, and a faint, inconsistent positivity was detected in Müller's cells. All intraocular retinoblastomata studied contained cells positive for S-100 protein, which seemed to be reactive glial cells derived from infiltrated retina. Undifferentiated malignant retinoblastoma cells, as well as differentiated tumour cells in Flexner-Wintersteiner rosettes were negative for S-100 protein. Differentiated tumours appeared to contain more S-100 protein-positive glial cells than undifferentiated tumours, but this may be related to their different growth rates. The results do not support the theory that retinoblastoma cells express glial properties.

Effects of transformation on the expression of laminin and fibronectin by neural cells.

We have studied laminin and fibronectin expression in a collection of rat cerebellar neural cell lines transformed with a mutant of Rous sarcoma virus which is temperature sensitive for transformation. We show that regardless of their neuronal or glial properties the cell lines produce both laminin and fibronectin. Laminin is expressed in similar amounts in cell lines grown at either the permissive or nonpermissive temperature for transformation, while fibronectin is generally expressed at higher levels in cells kept at the nonpermissive temperature. To provide further evidence that neural cells can produce laminin and fibronectin, double label immunofluorescence studies were conducted on primary cerebellar cultures. Both laminin and fibronectin were found to be present in the primary culture, and laminin was found to be associated with a subpopulation of astrocytes.

Chapter 15 On Neuronal and Glial Differentiation of a Pluripotent Stem Cell Line, RT4-AC: A Branch Determination

Publisher Summary The development of eukaryotes is the result of a series of hierarchical or nonhierarchical branchings of precursor cells (or pluripotent stem cells). Here, hierarchical branching implies that a predetermined order of segregation of cell types exists; nonhierarchical branching means that the order of segregation of cell types from stem cells is not fixed, but is more or less random (stochastic). Nonhierarchical branching has been observed in the branching of certain pluripotent stem cell systems. In this chapter, the term “maturation” is used to describe the cases in which terminal cells express overt differentiation properties in response to external agents, often without cell division. Under maturation conditions, the majority of a homogeneous population of precursor cells responds to an agent and expresses terminal differentiation properties. Detailed studies of neural as well as other characteristics reveal a clear-cut segregation of neuronal and glial properties and tumorigenicity among different derivative cell types upon cell type conversion. In this system, the stem cell type (RT4-AC) expresses some of both neuronal properties and glial properties. The systematic and consistent segregation of these properties is a remarkable and clear phenomenon that alludes to a fundamental aspect of cell differentiation (branch determination), and therefore is worthy of detailed studies on its mechanism. This chapter also observes differential maturational responses of the derivative cell types (RT4-B, RT4-D, and RT4-E) to dibutyryl-CAMP (dbcAMP). Based on the study of this system, the chapter discusses several points about the determination and maturation of neuronal and glial properties.

Clonal sublines of rat neurotumor RT4 and cell differentiation: V. Comparison of Na + influx, Rb + efflux, and action potential among stem-cell, neuronal, and glial cell types

A multipotential stem-cell-type cell line (RT4-AC) isolated from a rat peripheral neurotumor differentiates in culture into two neuronal-type cells (RT4-B and RT4-E) or into a glial-type cell (RT4-D). The neuronal classification of RT4-B and RT4-E cells is based on their positive response to veratridine in the tetrodotoxin-sensitive Na +-influx and Rb +-efflux assays and on the action potential observed upon hyperpolarized stimulation. In addition, these neuronal cell types do not synthesize two glial proteins, S100 protein (S100P) and glial fibrillary acidic protein (GFAP). The glial classification of RT4-D is based on the syntheses of S100P and GFAP. Additionally, RT4-D does not display veratridine-activated Na + influx and Rb + efflux nor action potential. The stem cell type, RT4-AC, expresses both neuronal and glial properties to a lesser degree. In the neuronal-type cell lines of the RT4 family (RT4-B and RT4-E), the large veratridine-activated Na + influx can further be stimulated by scorpion toxin. The Na + influx of the stem cell (RT4-AC), however, is only slightly stimulated by veratridine alone, but greatly stimulated by the addition of veratridine and scorpion toxin. These observations suggest that a progressive differentiation of voltage-dependent Na + channels may have occurred by the cell-type conversion from the stem cell type to the neuronal cell types. The exact nature of the change in Na + channels is currently not known.

Dual properties of cultured retinoblastoma cells: Immunohistochemical characterization of neuronal and glial markers

The dual properties of two human retinoblastoma cell lines, WERI-Rb1 and Y79, were investigated with immunohistochemistry. Two neuron-specific markers, dopamine- B-hydroxylase (DBH) and tetanus toxin, and an astrocyte-specific marker, the glial fibrillary acid protein (GFAP), were applied for immunohistochemical reactions. With peroxidase-antiperoxidase (PAP) and immunofluorescence techniques, all of the WERI-Rb1 and Y79 cells showed consistently positive results with both neuronal and glial markers. The findings demonstrate that cultured retinoblastoma cells WERI-Rb1 and Y79 have both neuronal and glial properties.

Conditioned medium from a clonal Rous Sarcoma virus transformed cerebellar cell line induces process extension in glial lines

A Rous Sarcoma virus transformed cerebellar cell line, BC6, secretes a factor which causes clonal glial cell lines to rapidly (1 h) extend processes. The factor shows a degree of specificity since only 3 out of 10 lines which exhibit either glial or combined glial and neuronal properties respond. The active factor appears to be a soluble protein since it remains in the supernatant after centrifugation at 100,000 g for 2 h and is trypsin-sensitive. When conditioned medium is fractionated on a Sephadex G-100 column, activity elutes in and just behind the void volume. By several criteria the factor is distinct from glia maturation factor.

Simultaneous expression of neuronal and glial properties by chick ciliary ganglion cells during development

Autoradiographic methods were used to show that non-neuronal cells from dissociated chick ciliary ganglia grown in cell culture for 1 day exhibit high affinity uptake for norepinephrine (NE) and/or specific receptors for nerve growth factor (NGF). Using immunofluorescence procedures, it was demonstrated that these cells reacted neither with the neuron-specific marker tetanus toxin nor with antibodies to the fibroblast marker fibronectin. The cells were, however, positive for 04 antigen, which is present on Schwann cells and oligodendrocytes and is recognized by a monoclonal antibody (Schachner, M., S. K. KIm, and R. Zehnle (1981) Dev. Biol. 83: 328-338). At all stages studied between embryonic day 6 (E6) and embryonic day 14 (E14), about 80% of the non-neuronal cells were positive for 04 antigen, the other non-neuronal cells being identified as fibroblasts or fibroblast-like cells by staining with antibodies to fibronectin. The proportion of cells with NGF receptors and cells with NE uptake decreased during development between E6 and E14. The percentage of 04-positive cells which have NGF receptors decreased from about 95% at E6 to about 35% at E14. The proportion of 04-positive cells with NE uptake decreased from about 57% at E6 to about 15% at E14. Thus, a considerable proportion of the non-neuronal cell population in embryonic ciliary ganglia displays neuronal properties. We suggest that those cells exhibiting biochemical properties of both differentiated glial cells and neurons are precursor cells which have the potential to develop either into glial cells or neurons.

Glial contribution to seizure: K + activation of (Na +, K +)-ATPase in bulk isolated glial cells and synaptosomes of epileptogenic cortex

Glial and neuronal (Na +, K +)-ATPase have dissimilar apparent affinities for K + ions. Glial (Na +, K +)-ATPase is maximally activated by 20 mM K + while neuronal (Na +, K +)-ATPase is maximally stimulated by 3–5 mM K +. Because this glial property may play an important role in the clearance of [K +] 0 during seizures, we investigated the K + activation of (Na +, K +)-ATPase within bulk isolated glial cells and synaptosomes isolated from epileptogenic brains. The primary focus (F), the homolateral brain area around the focus (PF) and the mirror (M) or secondary focus induced by freezing lesions were studied. Results show that both glial and synaptosomal enzyme activities in the epileptogenic state change in comparison with controls, i.e. sham-operated cats. In F and M., glial enzyme decreased reaction velocities between 3 and 18 mM K +. In PF, maximum velocities increased in glial (Na +, K +)-ATPase. These results indicate that in actively firing epileptogenic tissue (F, M), glial (Na +, K +)-ATPase decreased rate reactions while the catalytic activity was increased in cortical tissues surrounding the focus. These phenomena appeared early, i.e. 1–3 days after production of the freezing lesion, and was associated with a sharp decrease in absolute levels of enzyme activity. Synaptosomal (Na +, K +)-ATPase from controls always exhibited a saturation curve at 3–6 mM K +. Synaptosomal enzyme activities in the primary (F) lesion increased slightly 24 h after lesion production, then progressively decreased 3 days after lesion production. No significant changes were seen in M and PF.

Electron Microscopic Image Processing in the Classification and Quantitation of Human Glial Tumor Cell Properties

Cell lines derived from human glial tumors studied in vitro offer the opportunity for the study of a variety of the basic biological parameters of these cells as well as their specific reactions to different classes of chemotherapy and cellular and humoral immunological reagents. For example, it has been possible to show that there are both BCNU-resistant and sensitive glioma cell lines in vitro and that sensitive cells exhibit a series of shape and membrane changes not shown by resistant cells. In addition, use of human fibroblast interferon or superinduced-autologously-produced glioma cell interferon at 1CP - 104 units/ml causes a modest decrease in the growth rate of many glioma cell lines as well as more striking morphological changes suggestive of a tendency toward a more “differentiated” state.