Establishment of an epidermal growth factor-dependent, multipotent neural precursor cell line

Summary We have established a multipotent clonal cell line, named MEB5, from embryonic mouse forebrains after the infection of a retrovirus carrying E7 oncogene of human papillomavirus type 16. MEB5 cells proliferated in serum-free, epidermal growth factor (EGF)-supplemented medium. They expressed markers for neural precursor cells (nestin, A2B5, and RC1) and did not express markers for neurons (class III β-tubulin), astrocytes (glial fibrillary acidic protein), and oligodendrocytes (galactocerebroside). MEB5 cells were stably maintained in an undifferentiated state with a diploid karyotype in the presence of EGF. When they were deprived of EGF, about 50% of the cells died due apoptosis within 24 h. The remaining cells differentiated into neurons, astrocytes, or oligodendrocytes within 2 wk. The newly developed cells with neuronal morphology were immunoreactive for γ-aminobutyric acid and exhibited neuronal electrophysiological properties. When MEB5 cells were treated with leukemia inhibitory for 7 d, they were induced to differentiate exclusively into astrocytes. These results inducate that MEB5 is a cell line with characteristics of EGF-dependent, multipotent neural precursor cells. This cell line should provide a good model system to study the mechanisms of survival, proliferation, and differentiation of the multipotent precursor cells in the central nervous system.     

Partial expression of monoaminergic (serotoninergic) properties by the multipotent hypothalamic cell line F7. An example of learning at the cellular level

A serum-free medium supplemented with a glial conditioned medium, a brain extract from 8-to 10-day-old mice, hormones, and eye-derived growth factor has been devised which permitted the mouse primitive hypothalamic nerve cell line F7 to express some biochemical properties typical of monoaminergic neurons. Maximal expression was obtained when the culture conditions were applied for 2 days. Most (90–95%) cells then synthesized [³H]serotonin from [⁴H]5-hydroxytryptophan (but not from [³H]tryptophan). No synthesis was detected in the presence of carbidopa (20 μM), therefore suggesting the involvement of l-aromatic-amino-acid decarboxylase in this process. In addition, F7 cells cultured in such serum-free medium exhibited the capacity of accumulating exogenous serotonin by an ouabain-sensitive mechanism. These data further supported that active molecules in the cell environment can induce, in a primitive cell line, some of the enzymatic activities associated with monoaminergic neurons. Since other well-defined culture conditions can promote the differentiation of the same clone into oligodendrocytes (De Vitry et al., 1983), it can be concluded that the F7 cell has the properties of an embryonic stem cell of the CNS which, depending on external signals, may switch into different alternative developmental neural pathways. We postulate that the stabilization of neuron-like properties due to repetitive cell stimulation by active signals in the environment may represent an example of learning at the cellular level.     

Inhibition of p38 mitogen-activated protein kinase-induced apoptosis in cultured mature oligodendrocytes using SB202190 and SB203580

p38 Mitogen-activated protein kinase (p38 MAPK) is expressed in the oligodendrocyte lineage, and its activity has been implicated in the proliferation and transition of early progenitors into late progenitors. Although p38 MAPK expression has been found in the myelin sheath, however, its role in mature oligodendrocytes remains unknown. In the present study, in order to address the role of p38 MAPK in mature oligodendrocytes, selective inhibitors of p38 MAPK, SB202190, and SB203580 were added to primary cultures of mature oligodendrocytes. After 24h of exposure to the inhibitors, the appearance, and number of A2B5-positive progenitors were unchanged. However, the 2',3'-cyclic nucleotide-3'-phosphohydrolase-positive mature oligodendrocytes disappeared, and the numbers of living cells decreased in comparison to the control cells treated with SB202474, a negative analog of SB203580. Increases in the number of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive nuclei and in the activity of caspase-3/7 were detected 16 h after exposure to the inhibitors, thus causing the mature oligodendrocytes to die due to apoptosis. Similar results were obtained using a differentiated rat oligodendrocyte precursor cell (OPC) line, central glia-4 (CG-4). These findings indicate that p38 MAPK is vital for mature oligodendrocyte survival.     

大鼠脑神经干细胞系（RNSC—FMU 1）的建立和鉴定

A neural stem cell line (RNSC-FMU 1) from rat brain have been established successfully by isolating and culturing neural stem cells from newborn SD rat brain in vitro with free-serum medium and passaging with mechanical division. The cell line cultured can continuously generate in vitro for long-term and it is 21 months (>100 passages) so far. These cells keep the feature of neural stem cell and normal karyotype. These neural stem cells can be induced to differentiate into neurons, astrocytes and oligodendrocytes. The cells have an extensive self-renewal capacity; its doubling time of proliferation is about 20 h. The cells are also cryopreservable. Tumor formation is not observed in nude mice that explanted with the cells. This cell line is a good tool for research of neural stem cell.     

大鼠脑神经干细胞系(RNSC-FMU 1)的建立和鉴定

采用无血清培养液分离和培养新生SD 大鼠脑的神经干细胞,以机械分散的方法传代,成功地建立了大鼠脑神经干细胞系(RSNC-FMU 1)。该细胞系可在体外长期传代,至今已在体外连续生长超过21个月(>100代),保持了神经干细胞的特性和正常的核型,经诱导可分化成为神经元、星形胶质细胞和少突胶质细胞,具有较旺盛的自新能力,倍增时间约为20h,并可冷冻保存,裸鼠体内移植证实其不具有成瘤性。该细胞系为神经干细胞研究提供了一个良好的工具。     

Do oligodendrocytes divide?

Remyelination occurs in the adult central nervous system following a wide variety of experimental and naturally occurring demyelinating conditions, including multiple sclerosis. Remyelination is preceded by the appearance of new oligodendrocytes. These new cells may be generated from glial precursor cells, or from pre-existing differentiated oligodendrocytes that re-enter the cell cycle, which may first dedifferentiate, or both processes may occur. The evidence for the source of new oligodendrocytes following toxic or immune-mediated lesions is reviewed. Good evidence exists that fully differentiated oligodendrocytes can incorporate [³H]thymidine but this may be a rare event. Most of the evidence points towards glial precursor cells as the source of new oligodendrocytes in the adult, but definitive experiments have not yet been done. Research strategies, using our current knowledge and techniques, are outlined for solving this problem. Special issue dedicated to Dr. Marion E. Smith.     

Hyperforin promotes mitochondrial function and development of oligodendrocytes

St. John's wort has been found to be an effective and safe herbal treatment for depression in several clinical trials. However, the underlying mechanism of its therapeutic effects is unclear. Recent studies show that the loss and malfunction of oligodendrocytes are closely related to the neuropathological changes in depression, which can be reversed by antidepressant treatment. In this study, we evaluated the effects of hyperforin, a major active component of St. John's wort, on the proliferation, development and mitochondrial function of oligodendrocytes. The study results revealed that hyperforin promotes maturation of oligodendrocytes and increases mitochondrial function without affecting proliferation of an oligodendrocyte progenitor cell line and neural stem/progenitor cells. Hyperforin also prevented mitochondrial toxin-induced cytotoxicity in an oligodendrocyte progenitor cell line. These findings suggest that hyperforin may stimulate the development and function of oligodendrocytes, which could be a mechanism of its effect in depression. Future in vitro and in vivo studies are required to further characterize the mechanisms of hyperforin.     

Characterization of a mouse tet-on glia precursor cell line in vitro and in vivo using the electrophysiological measurement.

We report here a partial characterization of a "tet-on" glia O2A precursor cell line established from the reverse tetracycline-transactivator (rtTA)-SV40 T antigen (Tag) double transgenic mice. In culture, withdrawal of doxycycline prevents proliferation and the cell line undergoes apoptosis. Importantly, differentiation into type-2-astrocytes and oligodendrocytes can be induced when the cell line is cultured, in the absence of doxycycline, and with epithelial stem cell lines secreting hIL3 or hIL6. In contrast, no maturation into progeny was observed when a hCNTF-secreting cell line was used as the co-culture partner under the same condition. In order to address the question of whether the morphological distinct cells-spindle and stellar shaped cells are of a similar or different cell types, we have performed cell size analysis of these cells by FACS and electro-physiology measurement by the patch clamping technique. They are of a similar cell size, but poses distinct electrophysiological properties-spindle cells are less mature than the stellar cells. These tet-on glia O2A precursor cells were implanted to sites of transected sciatic nerve of adult mice and kept in the precursor stage by feeding mice with doxycycline containing drinking water. The toe movement of injured foot was measured every 3 weeks and the electrophysiological property of motor neuron was determined three months after the operation. Preliminary data have shown that these tet-on glia precursor cells are not toxic to the implanted hosts and can enhance the recovery of damaged motor nerves.     

Getting a GR(i)P on oligodendrocyte development

One of the most extensively studied of mammalian cells is the oligodendrocyte, the myelin-forming cell of the central nervous system. The ancestry and development of this cell have been studied with every approach utilized by developmental biologists. Such detailed efforts have the potential of providing paradigms of relevance to those interested in analyzing the ancestry and development of any cell type.One of the striking features of studies on the development of oligodendrocytes is that different analytical approaches have led to strikingly different theoretical views regarding the ancestry of these cells. On one extreme is the hypothesis that the steps leading to the generation of oligodendrocytes begin with the generation of a glial-restricted precursor (GRP) cell from neuroepithelial stem cells. GRP cells are thought to be capable of giving rise to all glial cells (including oligodendrocytes and multiple astrocyte populations), but not to neurons, a process that appears to require progression through further stages of greater lineage restriction. On the other extreme is the hypothesis that oligodendrocytes are derived from a precursor cell that generates only motor neurons and oligodendrocytes, with astrocytes being generated through a separate lineage. In this review, we critically consider the various contributions to understanding the ancestry of oligodendrocytes, with particular attention to the respective merits of the GRP cell vs. the motor neuron-oligodendrocyte precursor (MNOP) cell hypothesis. We draw the conclusion that, at present, the strengths of the GRP cell hypothesis outweigh those of the MNOP hypothesis and other hypotheses suggesting oligodendrocytes are developmentally more related to motor neurons than to astrocytes. Moreover, it is clear from existing data that, following the period of motor neuron generation, the major glial precursor cell in the embryonic spinal cord is the GRP cell, and that multiple previous studies on the earliest stages of oligodendrocyte generation in the developing spinal cord have been focused on a differentiation stage of GRP cells.     

Accumulation of the cyclin-dependent kinase inhibitor p27/Kip1 and the timing of oligodendrocyte differentiation.

Many types of vertebrate precursor cells divide a limited number of times before they stop and terminally differentiate. In no case is it known what causes them to stop dividing. We have been studying this problem in the proliferating precursor cells that give rise to postmitotic oligodendrocytes, the cells that make myelin in the central nervous system. We show here that two components of the cell cycle control system, cyclin D1 and the Cdc2 kinase, are present in the proliferating precursor cells but not in differentiated oligodendrocytes, suggesting that the control system is dismantled in the oligodendrocytes. More importantly, we show that the cyclin-dependent kinase (Cdk) inhibitor p27 progressively accumulates in the precursor cells as they proliferate and is present at high levels in oligodendrocytes. Our findings are consistent with the possibility that the accumulation of p27 is part of both the intrinsic counting mechanism that determines when precursor cell proliferation stops and differentiation begins and the effector mechanism that arrests the cell cycle when the counting mechanism indicates it is time. The recent findings of others that p27-deficient mice have an increased number of cells in all of the organs examined suggest that this function of p27 is not restricted to the oligodendrocyte cell lineage.     

Estimating the life-span of oligodendrocytes from clonal data on their development in cell culture

This paper presents a new method to analyze clonal data on oligodendrocyte development in cell culture. The process of oligodendrocyte generation from precursor cells is modelled as a multi-type Bellman-Harris branching process as suggested in an earlier paper [K. Boucher, A. Zorin, A.Y. Yakovlev, M. Mayer-Proschel, M. Noble, An alternative stochastic model of generation of oligodendrocytes in cell culture, J. Math. Biol. 43 (2001) 22]. This model has been extended to allow for death of oligodendrocytes as well as a dissimilar distribution of the first mitotic cycle duration as compared to the subsequent cycles of precursor cells, which lengths are assumed to be independent and identically distributed random variables. Since the time-span of oligodendrocytes is not directly observable in clonal data, plausible parametric assumptions are invoked to make estimation problems tractable. In particular, the time to cell death follows a two-parameter gamma distribution, while the lapse of time between the event of cell death and the event of cell disintegration is assumed to be exponentially distributed. A simulated pseudo maximum likelihood method for estimation of model parameters has been developed using simulation-based approximations of the expected numbers and variance-covariance matrices for different types of cells. Finite sample properties of the estimation procedure are studied by computer simulations. The proposed method is illustrated with an analysis of the clonal development of O-2A progenitor cells isolated from the rat optic nerve and the corpus callosum.     

The F9-EC cell line as a model for the analysis of differentiation.

The teratocarcinoma stem cell line F9 has been widely used as a model for the analysis of molecular mechanisms associated with differentiation. This cell line has been considered to be nullipotent and able to differentiate into endodermal-like derivatives upon treatment with retinoic acid. Nevertheless, under definite culture conditions, F9 cells are able to differentiate into derivatives of all three germ layers. The F9 cells express characteristics of early mouse embryonal cells and possess all repression factors known to be present in cells of the early mouse embryogenesis. Induction of differentiation can be achieved not only by adding chemical agents to the culture medium but also by transfection of several oncogenic sequences. In somatic cell genetic experiments, immortalized, differentiated F9-like cells have been shown to express dominantly genes responsible for the appearance of the differentiated phenotype.     

Hypomyelination Phenotype Caused by Impaired Differentiation of Oligodendrocytes in Emx1-cre Mediated Cdk5 Conditional Knockout Mice

Cyclin-dependent kinase 5 (Cdk5) plays a pivotal role in neuronal migration and differentiation, and in axonal elongation. Although many studies have been conducted to analyze neuronal functions of Cdk5, its kinase activity has also been reported during oligodendrocyte differentiation, which suggests Cdk5 may play an important role in oligodendrocytes. Here, we describe a hypomyelination phenotype observed in Emx1-cre mediated Cdk5 conditional knockout (cKO) mice (Emx1-cKO), in which the Cdk5 gene was deleted in neurons, astrocytes and oligodendrocyte -lineage cells. In contrast, the Cdk5 gene in CaMKII cKO mice was deleted only in neurons. Because the development of mature oligodendrocytes from oligodendrocyte precursor cells is a complex process, we performed in situ hybridization using markers for the oligodendrocyte precursor cell and for the differentiated oligodendrocyte. Our results indicate that hypomyelination in Emx1-cKO is due to the impaired differentiation of oligodendrocytes, rather than to the proliferation or migration of their precursors. The present study confirmed the in vivo role of Cdk5 in oligodendrocyte differentiation.     

Identification of two novel glial-restricted cell populations in the embryonic telencephalon arising from unique origins

Background  

Considerably less attention has been given to understanding the cellular components of gliogenesis in the telencephalon when compared to neuronogenesis, despite the necessity of normal glial cell formation for neurological function. Early proposals of exclusive ventral oligodendrocyte precursor cell (OPC) generation have been challenged recently with studies revealing the potential of the dorsal telencephalon to also generate oligodendrocytes. The identification of OPCs generated from multiple regions of the developing telencephalon, together with the need of the embryonic telencephalon to provide precursor cells for oligodendrocytes as well as astrocytes in ventral and dorsal areas, raises questions concerning the identity of the precursor cell populations capable of generating macroglial subtypes during multiple developmental windows and in differing locations.     

Cloning and characterization of the cDNA encoding a novel human pre-B-cell colony-enhancing factor.

A novel gene coding for the pre-B-cell colony-enhancing factor (PBEF) has been isolated from a human peripheral blood lymphocyte cDNA library. The expression of this gene is induced by pokeweed mitogen and superinduced by cycloheximide. It is also induced in the T-lymphoblastoid cell line HUT 78 after phorbol ester (phorbol myristate acetate) treatment. The predominant mRNA for PBEF is approximately 2.4 kb long and codes for a 52-kDa secreted protein. The 3' untranslated region of the mRNA has multiple TATT motifs, usually found in cytokine and oncogene messages. The PBEF gene is mainly transcribed in human bone marrow, liver tissue, and muscle. We have expressed PBEF in COS 7 and PA317 cells and have tested the biological activities of the conditioned medium as well as the antibody-purified protein in different in vitro assays. PBEF itself had no activity but synergized the pre-B-cell colony formation activity of stem cell factor and interleukin 7. In the presence of PBEF, the number of pre-B-cell colonies was increased by at least 70% above the amount stimulated by stem cell factor plus interleukin 7. No effect of PBEF was found with cells of myeloid or erythroid lineages. These data define PBEF as a novel cytokine which acts on early B-lineage precursor cells.     

Phosphorylation and sulfation of arylsulfatase A accompanies biosynthesis of the enzyme in normal and carcinoma cell lines

Arylsulfatase A (arylsulfate sulfohydrolase, EC 3.1.6.1), a mammalian lysosomal enzyme, is initially synthesized as a 69, 67 and 64 kDa precursor polypeptide in a prostate carcinoma cell line PC-3SF12, in HeLa cells and in a normal human embryonic lung cell line WI-38, respectively. These precursor polypeptides are secreted into the medium or processed to mature enzymes of apparent molecular mass 66, 64 or 62 kDa in PC-3SF12, HeLa or WI-38 cells, respectively. The precursor and mature polypeptides in WI-38 cells are phosphorylated, and the phosphate is lost upon treatment with endo-beta-hexosaminidase H. Arylsulfatase A is also shown to be sulfated in WI-38 cells. The presence of castanospermine, an inhibitor of sulfation of the second N-acetylglucosamine residue of the chitobiose core, does not reduce the extent of sulfation of arylsulfatase A, suggesting that either terminal sugars or the protein is sulfated. Sulfation may have a protective function similar to that of terminal sialic acid residues in glycoproteins. Although the subcellular location of arylsulfatase A is identical in PC-3SF12 and in WI-38 cells, pulse-chase experiments indicate that arylsulfatase A protein has a slower turnover in the prostate carcinoma cell line than it does in the normal human lung cell line. The differences in the apparent molecular weights of arylsulfatase A in the normal and carcinoma cell lines are shown to be due to variations in the carbohydrate content of the enzyme. The apparent molecular mass of the polypeptide chain obtained after endo-beta-hexosaminidase H treatment is 59 kDa, a value which is identical for all three cell lines studied here. These results suggest the possibility of an enhanced activity of terminal glucosyltransferase enzymes in carcinoma cell lines and in tumor tissues. Arylsulfatase A may be a useful marker for studying transformation-related processes in human cell lines.     

PDGF receptors on cells of the oligodendrocyte-type-2 astrocyte (O-2A) cell lineage

It has been shown previously that cultures of rat optic nerve contain three types of macroglial cells--oligodendrocytes and two types of astrocytes. Type-1 astrocytes develop from their own precursor cells beginning before birth, while oligodendrocytes and type-2 astrocytes develop postnatally from a common bipotential precursor called the O-2A progenitor cell. Proliferating O-2A progenitor cells give rise to postmitotic oligodendrocytes beginning around birth, and to type-2 astrocytes beginning in the second postnatal week. Studies in vitro have suggested that platelet-derived growth factor (PDGF), secreted by type-1 astrocytes, plays an important part in timing oligodendrocyte development: PDGF seems to keep O-2A progenitor cells proliferating until an intrinsic clock in the progenitor cells initiates the process leading to oligodendrocyte differentiation. The clock apparently determines when a progenitor cell becomes unresponsive to PDGF, at which point the cell stops dividing and, as a consequence, automatically differentiates into an oligodendrocyte. Here we have used radiolabelled PDGF to show that O-2A progenitor cells have PDGF receptors, suggesting that these cells respond directly to PDGF. The receptors resemble the type A PDGF receptor previously described on human fibroblasts and are initially retained when progenitor cells stop dividing and develop in vitro into oligodendrocytes. The latter finding indicates that receptor loss is not the reason that progenitor cells initially become mitotically unresponsive to PDGF.     

Stability and covalent modification of P-glycoprotein in multidrug-resistant KB cells

An antipeptide antibody (P7) to P-glycoprotein has been produced by immunizing rabbits with a synthetic peptide. Antibody P7 is directed against the amino-terminal region of P170 (residues 28-35). The antibody immunoprecipitates a 170-kDa P-glycoprotein from extracts of drug-resistant KB-V1 cells that is not present in the drug-sensitive cell line KB-3-1. Antibody P7 was used to quantitate the amount of P-glycoprotein present in drug-resistant KB lines at various levels of resistance and to demonstrate the presence of P-glycoprotein in NIH 3T3 cells transfected with a cloned MDR1 cDNA or human genomic DNA encoding MDR1. Pulse-chase labeling experiments demonstrated that P-glycoprotein is synthesized as a 140-kDa precursor which is slowly converted over 2-4 h to a 170-kDa glycoprotein. Tunicamycin treatment blocks the conversion of the precursor to the mature form, and removal of N-linked oligosaccharides with Endo F reduces the relative molecular weight of P-glycoprotein to 140K. The mobility of mature P-glycoprotein is unaffected by treatment with neuraminidase and Endo H. These data indicate that P-glycoprotein is N-glycosylated and contains little or no neuraminic acid. P-Glycoprotein is also phosphorylated, and the extent of phosphate incorporated is proportional to the amount of protein present in drug-resistant cells.     

Neuromodulin (GAP43): a neuronal protein kinase C substrate is also present in 0-2A glial cell lineage. Characterization of neuromodulin in secondary cultures of oligodendrocytes and comparison with the neuronal antigen

Neuromodulin (also called GAP43, G50, F1, pp46), a neural-specific calmodulin binding protein, is a major protein kinase C substrate found in developing and regenerating neurons. Here, we report the immunocytochemical characterization of neuromodulin in cultured 0-2A bipotential glial precursor cells obtained from newborn rat brain. Neuromodulin is also present in oligodendrocytes and type 2 astrocytes (stellate-shaped astrocytes), which are both derived from the bipotential glial 0-2A progenitor cells, but is absent of type 1 astrocytes (flat protoplasmic astrocytes). These results support the hypothesis of a common cell lineage for neurons and bipotential 0-2A progenitor cells and suggest that neuromodulin plays a more general role in plasticity during development of the central nervous system. The expression of neuromodulin in secondary cultures of newborn rat oligodendrocytes and its absence in type 1 astrocytes was confirmed by Northern blot analysis of isolated total RNA from these different types of cells using a cDNA probe for the neuromodulin mRNA and by Western blot analysis of the cell extracts using polyclonal antibodies against neuromodulin. The properties of the neuromodulin protein in cultured oligodendrocytes and neuronal cells have been compared. Although neuromodulin in oligodendrocytes is soluble in 2.5% perchloric acid like the neuronal counterpart it migrates essentially as a single protein spot on two-dimensional gel electrophoresis whereas the neuronal antigen can be resolved into at least three distinct protein spots. To obtain precise alignments of the different neuromodulin spots from these two cell types, oligodendrocyte and neuronal cell extracts were mixed together and run on the same two-dimensional gel electrophoresis system. Oligodendroglial neuromodulin migrates with a pI identical to the basic forms of the neuronal protein in isoelectric focusing gel. However, the glial neuromodulin shows a slightly lower mobility in the second dimensional lithium dodecyl sulfate-PAGE than its neuronal counterpart. As measured by 32Pi incorporation, neuromodulin phosphorylation in oligodendrocytes is dramatically increased after short-term phorbol ester treatments, which activate protein kinase C, and is totally inhibited by long-term phorbol ester treatments, which downregulates protein kinase C, thus confirming its probable specific in vivo phosphorylation by protein kinase C. In primary cultures of neuronal cells, two of the three neuromodulin spots were observed to be phosphorylated with an apparent preferential phosphorylation of the more acid forms.     

Telomerase and oligodendrocyte differentiation.

Myelin in the mammalian central nervous system (CNS) is produced by oligodendrocytes, most of which arise from oligodendrocyte precursor cells (OPCs) during late embryonic and early postnatal development. Both external and internal cues have been implicated in regulating OPC exit from the cell cycle and differentiation into oligodendrocytes. In this study, we demonstrate that differentiation of cultured OPCs into mature oligodendrocytes is associated with lower levels of activity of telomerase, the ribonucleoprotein that synthesizes telomeric DNA at the ends of chromosomes. Differentiation is also associated with lower levels of mRNA encoding the catalytic subunit of telomerase (TERT), whereas no difference is seen in the expression of its telomeric template RNA component (TR). These data suggest a possible role for telomerase during normal growth and differentiation of oligodendrocytes that may be relevant to the mechanism of myelination in the CNS.