In vitro differentiation of mouse teratocarcinoma cells monitored by intermediate filament expression

Teratocarcinoma differentiation has been studied using sera specific for each of the five intermediate filament (IF) classes. These antibodies distinguish cells of epithelial, muscle, neural, astrocytic, and mesenchymal origin. In embryoid bodies, derived from embryo transplants and obtained in the ascitic fluid by transplantation of teratocarcinoma, the cells of the inner cellular mass did not express any of these intermediate filament types while the outer cells expressed cytokeratin. Intermediate filament expression in the embryoid body thus appears analogous to that in the blastocyst and differs from that in embryonal carcinoma (EC) lines. Twelve EC lines have now been shown to express vimentin although in some EC lines not all cells express vimentin. Other established permanent differentiated cell lines, derived from EC lines in vitro or from tumors in vivo, have been characterized with respect to the type of IF they contain. The distribution of different IF types has been examined in EC cells induced to differentiate by addition of retinoic acid. The proportion of cells expressing each type of intermediate filament appears to depend on the EC cell line used, on the inducing agent, and on the length of treatment. Thus, for instance, F9 cells express cytokeratin, PCC3 derivatives express vimentin, many 1009 derivatives express either glial fibrillar acidic protein (GFA) or neurofilament proteins. Overall the results obtained are in excellent agreement with emerging principles of intermediate filament expression during embryonic differentiation, thus emphasizing the potential use of the various EC lines to study differentiation in culture.     

In vitro Differentiation of Mouse Teratocarcinoma Cells Monitored by Intermediate Filament Expression

Teratocarcinoma differentiation has been studied using sera specific for each of the five intermediate filament (IF) classes. These antibodies distinguish cells of epithelial, muscle, neural, astrocytic, and mesenchymal origin. In embryoid bodies, derived from embryo transplants and obtained in the ascitic fluid by transplantation of teratocarcinoma, the cells of the inner cellular mass did not express any of these intermediate filament types while the outer cells expressed cytokeratin. Intermediate filament expression in the embryoid body thus appears analogous to that in the blastocyst and differs from that in embryonal carcinoma (EC) lines. Twelve EC lines have now been shown to express vimentin although in some EC lines not all cells express vimentin. Other established permanent differentiated cell lines, derived from EC lines in vitro or from tumors in vivo, have been characterized with respect to the type of IF they contain. The distribution of different IF types has been examined in EC cells induced to differentiate by addition of retinoic acid. The proportion of cells expressing each type of intermediate filament appears to depend on the EC cell line used, on the inducing agent, and on the length of treatment. Thus, for instance, F9 cells express cytokeratin, PCC3 derivatives express vimentin, many 1009 derivatives express either glial fibrillar acidic protein (GFA) or neurofilament proteins. Overall the results obtained are in excellent agreement with emerging principles of intermediate filament expression during embryonic differentiation, thus emphasizing the potential use of the various EC lines to study differentiation in culture.     

Neurofilament Proteins in Cultured Chromaffin Cells

Antibodies were raised against the 200-kd, 145-kd, and 68-kd subunits of a rat neurofilament preparation. Immunoblots showed that each antibody was specific for its antigen and that it did not cross-react with any of the two other neurofilament polypeptides. Use of the three antibody preparations to stain bovine chromaffin cells in culture by the indirect immunofluorescence technique indicated that the three neurofilament polypeptides are present in chromaffin cells maintained in culture for 3 or 7 days. The three anti-neurofilament antibodies labelled the cells in a similar pattern: very thin filaments specifically localized around the nucleus were observed whereas neurites and growth cones, developed by cultured chromaffin cells, were generally not stained. Some fibroblasts were present in our cultures but they were never stained by any of the neurofilament antibodies. This indicated that the antibodies used do not react with vimentin, the major intermediate filament protein found in fibroblasts. The three neurofilament antibodies were also used to immunoprecipitate specifically three proteins of molecular weights 210 kd, 160 kd, 70 kd from solubilized extracts of cultured chromaffin cells that were radiolabelled with [35S]methionine. These proteins correspond in molecular weight to the neurofilament triplet found in bovine brain. Finally, the presence of neurofilaments in freshly isolated chromaffin cells was tested by immunoblotting using the 68-kd antibody. A 70-kd protein was specifically stained by this antibody, suggesting that neurofilaments are not only present in cultured chromaffin cells but also in the adrenal gland in vivo. It is concluded from these results that chromaffin cells contain completely assembled neurofilaments. This additional neuronal property again illustrates that chromaffin cells are closely related to neurons and therefore represent an attractive model system for the study of functional aspects of adrenergic neurons.     

Induction of NILE/L1 glycoprotein during neuronal differentiation of the embryonal carcinoma cell line EC 1003

Abstract. A new clone of the mouse embryonal carcinoma cell line 1003 (EC 1003.16) can be maintained in an undifferentiated state in serum-containing medium. Shifting these cells to serum-free, hormonally defined medium causes them to differentiate morphologically and acquire a number of molecular properties characteristic of neurons. Whereas undifferentiated cells lack the NILE/L1 glycoprotein, expression of this neuronal cell adhesion niolecule is induced in the differentiating cells. Message for NILE/L1 becomes detectable after 5 days in serum-free medium, and cell-surface NILE/L1 can first be seen at this same time. Changes in two other cell adhesion molecules occur in parallel with the induction of NILE/L1. Fibronectin receptor is present on un- differentiated cells, but is down-regulated by the differentiating neurons. The neural cell adhesion molecule (N- CAM) undergoes a shift from the very adhesive adult form to the less adhesive, highly sialylated embryonic form. These changes would appear to emphasize the role of NILE/L1 in adhesive interactions involving differentiating neurons. Some changes in ganglioside expression also occur during EC 1003.16 differentiation. Undifferentiated cells express the D 1.1 ganglioside but lack gangliosides that are reactive with the monoclonal antibody A,B, Differentiating cells lose D 1.1 and become A,B,-positive. Since D 1.1 is characteristic of undifferentiated neuroepithelial cells and A,B, reactivity is a marker for several types of differentiated neurons, these changes in vitro appear to mimic events that occur in vivo.     

Differentiation of monkey embryonic stem cells into neural lineages

Embryonic stem (ES) cells are self-renewing, pluripotent, and capable of differentiating into all of the cell types found in the adult body. Therefore, they have the potential to replace degenerated or damaged cells, including those in the central nervous system. For ES cell-based therapy to become a clinical reality, translational research involving nonhuman primates is essential. Here, we report monkey ES cell differentiation into embryoid bodies (EBs), neural progenitor cells (NPCs), and committed neural phenotypes. The ES cells were aggregated in hanging drops to form EBs. The EBs were then plated onto adhesive surfaces in a serum-free medium to form NPCs and expanded in serum-free medium containing fibroblast growth factor (FGF)-2 before neural differentiation was induced. Cells were characterized at each step by immunocytochemistry for the presence of specific markers. The majority of cells in complex/cystic EBs expressed antigens (alpha-fetal protein, cardiac troponin I, and vimentin) representative of all three embryonic germ layers. Greater than 70% of the expanded cell populations expressed antigenic markers (nestin and musashi1) for NPCs. After removal of FGF-2, approximately 70% of the NPCs differentiated into neuronal phenotypes expressing either microtubule-associated protein-2C (MAP2C) or neuronal nuclear antigen (NeuN), and approximately 28% differentiated into glial cell types expressing glial fibrillary acidic protein. Small populations of MAP2C/NeuN-positive cells also expressed tyrosine hydroxylase (approximately 4%) or choline acetyltransferase (approximately 13%). These results suggest that monkey ES cells spontaneously differentiate into cells of all three germ layers, can be induced and maintained as NPCs, and can be further differentiated into committed neural lineages, including putative neurons and glial cells.     

Schwann cells of the myelin-forming phenotype express neurofilament protein NF-M

Immature Schwann cells of the rat sciatic nerve can differentiate into myelin-forming or non-myelin-forming cells. The factors that influence this divergent development are unknown but certain markers such as galactocerebroside distinguish the two cell populations at an early stage of Schwann cell differentiation. Because myelination requires extensive changes in cell morphology, we have investigated the composition and structure of the Schwann cell cytoskeleton at a time when these cells become committed to myelination. Here we show that Schwann cells express a cytoskeletal protein of M(r) 145 before diverging into the myelin-forming path, i.e., before they acquire cell-surface galactocerobroside. The p145 protein has the characteristics of an intermediate filament (IF) protein and immunoelectron microscopy shows that it colocalizes with vimentin, which suggests that these two proteins can coassemble into IFs. Elevated intracellular cAMP levels, which can mimic some of the early effects of axons on Schwann cell differentiation, induced p145 synthesis, therefore, we conclude that myelin-forming Schwann cells express this protein at a very early stage in their development. Immunological comparisons with other IF proteins revealed a close similarity between p145 and the neurofilament protein NF-M; the identification of p145 as NF-M was confirmed by isolating and sequencing a full-length clone from a Schwann cell cDNA library. These data demonstrate that Schwann cells remodel their IFs by expressing NF-M before acquiring the myelin-forming phenotype and that IF proteins of the neurofilament-type are not restricted to neurons in the vertebrate nervous system.     

Induction of differentiation of the human histiocytic lymphoma cell line U937 in the absence of vimentin expression

We have studied the expression of vimentin in the human histiocytic lymphoma cell line U937, induced to differentiate along the monocyte/macrophage pathway. Normal monocytes possess a network of vimentin intermediate filaments (IFs) at all stages of maturation. The undifferentiated U937 leukemia cells contain very low amounts of vimentin, but express a conspicuous IF network when exposed to phorbol myristate acetate. In parallel, they acquire functional properties typical of cells of the monocyte lineage. These concomitant variations suggest that vimentin IFs could play a role in the process of differentiation. However, we observed that all-trans-retinoic acid and 1,25-dihydroxyvitamin D3 confer monocyte-like properties upon U937 cells without inducing vimentin expression. We obtained increased phenotypic changes, yet in the absence of a vimentin network, by combining the effects of both inducers. These results show that vimentin expression is not crucial for the acquisition of some of the functions characteristic of the monocyte/macrophage lineage.     

Immunocytochemical demonstration of vimentin in astrocytes and ependymal cells of developing and adult mouse nervous system

The occurrence of vimentin, a specific intermediate filament protein, has been studied by immunoflourescence microscopy in tissue of adult and embryonic brain as well as in cell cultures from nervous tissue. By double imminofluorescence labeling, the distribution of vimentin has been compared with that of subunit proteins of other types of intermediate filaments (glial fibrillary acidic [GFA] protein, neurofilament protein, prekeratin) and other cell-type specific markers (fibronectin, tetanus toxin receptor, 04 antigen). In adult brain tissue, vimentin is found not only in fibroblasts and cells of larger blood vessels but also in ependymal cells and astrocytes. In embryonic brain tissue, vimentin is detectable as early as embryonic day 11, the earliest stage tested, and is located in radial fibers spanning the neural tube, in ventricular cells, and in blood vessels. At all stages tested, oligodendrocytes and neurons do not express detectable amounts of vimentin. In primary cultures of early postnatal mouse cerebellum, a coincident location of vimentin and GFA protein is seen in astrocytes, and both types of filament proteins are included in the perinuclear aggregates formed upon exposure of the cells to colcemid. In cerebellar cell cultures of embryonic-day-13 mice, vimentin is seen in various cell types of epithelioid or fibroblastlike morphology but is absent from cells expressing tetanus toxin receptors. Among these embryonic, vimentin-positive cells, a certain cell type reacting neither with tetanus toxin nor with antibodies to fibronectin or GFA protein has been tentatively identified as precursor to more mature astrocytes. The results show that, in the neuroectoderm, vimentin is a specific marker for astrocytes and ependymal cells. It is expressed in the mouse in astrocytes and glial precursors well before the onset of GFA protein expression and might therefore serve as an early marker of glial differentiation. Our results show that vimentin and GFA protein coexist in one cell type not only in primary cultures in vitro but also in the intact tissue in situ.     

Expression of neuronal and glial markers in so-called oligodendroglial tumors induced by transplacental administration of ethyl-nitrosourea in the rat.

A series of 18 tumors with histological features of oligodendrogliomas, induced in the rat by means of transplacental ethyl-nitrosourea administration were studied for immunohistochemical demonstration of neuronal (synaptophysin and neurofilament protein) and glial (gliofibrillar acidic protein and vimentin) markers. Most of the tumors showed cells with strong positivity to synaptophysin and to a lesser degree, to neurofilament protein, suggesting the neuronal character of these neoplasms. In 10 tumors, cells with strong positivity to vimentin were found, and in three cases, tumoral cells expressed gliofibrillar acidic protein. The observation that ENU-induced oligodendroglial tumors express neuronal and, to a minor degree, glial markers, suggests their interpretation as primitive neuroectodermal tumors with clear neuronal differentiation.     

Synthesis and secretion of beta-nerve growth factor by mouse teratocarcinoma cell lines

Using a cDNA probe and a two-site enzyme immunoassay, beta-nerve growth factor (beta NGF) synthesis was monitored in several mouse teratocarcinoma cell lines. Trace amounts of NGF mRNA were detected in the embryonal carcinoma (EC) PCC4, F9 and 1003 clones, whereas the myocardial (PCD1), myogenic (1168) and adipogenic (1246) clones contained significantly higher levels of NGF mRNA and secreted mature beta NGF peptide in the culture medium. The 1003, 1168 and 1246 strains were derived from the same teratocarcinoma cell line and their ability or inability to synthesize the neurotrophic factor may reflect a developmental decision for divergent differentiation programs. Induction of NGF mRNA and protein synthesis was observed in a differentiated derivative of an SV40-transformed F9 clone which expresses the viral T antigen. Southern blot analysis of the genomic DNAs revealed no structural alterations of the NGF locus between teratocarcinoma cells that express the NGF gene and those that do not. Similar analysis of the DNA methylation pattern in C-C-G-G sequences using the Hpa II and Msp I isoschizomers indicated no methylation changes of the NGF gene in the teratocarcinoma DNAs. At least two, and probably all four, of the already mapped Msp I sites within the NGF gene are methylated in all teratocarcinoma DNAs examined, as well as in the male mouse submaxillary gland DNA, the organ richest in this factor.     

Comparative marker analysis of the ependymocytes of the subcommissural organ in four different mammalian species

Summary The subcommissural organ (SCO), classified as one of the circumventricular organs, is composed mainly of modified ependymal cells, attributable to a glial lineage. Nevertheless, in the rat, these cells do not possess glial markers such as glial fibrillary acidic protein (GFAP), protein S100, or the enzyme glutamine synthetase (GS). They receive a synaptic 5-HT input and show pharmacological properties for uptake of GABA resembling the uptake mechanism of neurons. In this study, we examine the phenotype of several mammalian SCO (cat, mouse, rabbit) and compare them with the corresponding features of the rat SCO. In all these species, the SCO ependymocytes possess vimentin as an intermediate filament, but never express GFAP or neurofilament proteins. They do not contain GS as do glial cells involved in GABA metabolism, and when they contain protein S100 (rabbit, mouse), its rate is low in comparison to classical glial or ependymal cells. Thus, these ependymocytes display characteristics that differentiate them from other types of glial cells (astrocytes, epithelial ependymocytes and tanycytes). Striking interspecies differences in the capacity of SCO-ependymocytes for uptake of GABA might be related to their innervation and suggest a species-dependent plasticity in their function.     

An immunofluorescence study of neurofilament protein expression by developing hippocampal neurons in tissue culture

We have studied the development of intermediate filament proteins in the neurons found in hippocampal cell cultures using single and double label immunofluorescence with both monoclonal and polyclonal antibodies. Neurons in these cultures are known to differentiate in a manner similar to their counterparts in situ: in particular they develop axonal and dendritic processes which differ from each other in form, in ultrastructure, and in synaptic polarity. During the first days in culture, developing neurons could not be stained with antibodies against any of the neurofilament proteins, although many cells reacted with anti-vimentin. Later in the first week, antibody staining revealed clearly filamentous staining for the L (68 000 daltons) and the M (145 000 daltons) neurofilament subunits, though M reactivity was much stronger at this earlier stage of development. Some neurofilament positive profiles in many cells could also be stained with vimentin, though the vimentin immunoreactivity became progressively less pronounced during further development, and disappeared after about two weeks in culture. Also at about two weeks in vitro we noted the first appearance of neurofilament H protein (200 000 daltons) immunoreactivity, which was localized to a subset of long neurites which could be identified on morphological grounds as axons. These processes lacked staining for microtubule associated protein 2 (MAP2), a dendritic marker. They tended to be close to islands of glial cells, suggesting that H induction may require complex neuron-glial interactions. These results are consistent with the suggestion that H protein immunoreactivity is a marker for axonal outgrowth. In addition to obvious filamentous staining, we were able to localize neurofilament antigens to an interesting class of small ring-like structures, found increasingly frequently as the cultures aged. We also present evidence that tyrosinated alpha-tubulin is present both within dendrites and axons of neurons in these cultures.     

Neural differentiation following culture of embryonal carcinoma cells in a serum-free defined medium

The embryonal carcinoma line C17-S₁ clone 1003 is multipotential in vivo. When the cells are grown in vitro in serum-containing medium most of them remain undifferentiated, while a few differentiate into a unique morphologic type of epithelioid cell. If 1003 cells are passaged into a defined medium containing insulin, transferrin, selenium, and fibronectin they grow for six to eight generations at the same rate as in serum-containing medium. During this time, all the cells of the culture differentiate into a limited number of phenotypes with neuroepithelial and neuronal cells predominating. Differentiation could be obtained in the defined medium at relatively low cell densities. Exogenous fibronectin is required for cell attachment to the substratum, and when absent the cells form aggregates in which differentiation still occurs. Low amounts of serum added to the defined medium allow multiplication and maintenance of cells of undifferentiated phenotype and prevent differentiation into neuronal cells.     

Conditional immortalization of normal and dysgenic mouse muscle cells by the SV40 large T antigen under the vimentin promoter control.

We have created new mouse muscle cell lines of an immortalized type, expressing normal differentiation at the myotube stage: sarcomeric organization, functional excitation-contraction coupling, and triadic differentiation. The DNA immortalizing recombinant utilizes a deletion mutant of the regulatory region of the human vimentin promoter controlling the expression of a SV40 thermosensitive large T antigen, in which the small t sequence has been deleted. Skeletal mouse replicative myoblasts synthesized predominantly vimentin. After myoblast fusion the vimentin gene is strongly repressed in multinucleated syncytia. Furthermore, the normal activity of the vimentin promoter in myoblasts is increased in the large T antigen-expressing cells. We observed that continuous and rapid division of myoblasts occurs at permissive temperature, suggesting that immortalization is achieved even though the small t antigen is absent. When fusion is induced by changing media conditions, large T antigen expression is totally repressed by the vimentin promoter. When the temperature is elevated to 39 degrees C, the preexisting large T antigen is inactivated. The resulting myotubes from normal mouse differentiate totally normally as indicated by their morphology, ultrastructure, and electrophysiological properties. Mutant (muscular dysgenesis) immortalized cells express the same properties as mutant primary counterparts with no contraction, no slow Ca2+ current, and no triadic differentiation. These immortalized cell lines are potentially very useful for further pharmacology, transplantation, and cell biology studies. The vimentin promoter control of immortalizing recombinant DNA can be used for any mammalian normal and mutant muscle cell lines.     

Synthesis and secretion of β-nerve growth factor by mouse teratocarcinoma cell lines

Using a cDNA probe and a two-site enzyme immunoassay, β-nerve growth factor (βNGF) synthesis was monitored in several mouse teratocarcinoma cell lines. Trace amounts of NGF mRNA were detected in the embryonal carcinoma (EC) PCC4, F9 and 1003 clones, whereas the myocardial (PCD1), myogenic (1168) and adipogenic (1246) clones contained significantly higher levels of NGF mRNA and secreted mature βNGF peptide in the culture medium. The 1003, 1168 and 1246 strains were derived from the same teratocarcinoma cell line and their ability or inability to synthesize the neurotrophic factor may reflect a developmental decision for divergent differentiation programs. Induction of NGF mRNA and protein synthesis was observed in a differentiated derivative of an SV40-transformed F9 clone which expresses the viral T antigen. Southern blot analysis of the genomic DNAs revealed no structural alterations of the NGF locus between teratocarcinoma cells that express the NGF gene and those that do not. Similar analysis of the DNA methylation pattern in C-C-G-G sequences using the Hpa II and Msp I isoschizomers indicated no methylation changes of the NGF gene in the teratocarcinoma DNAs. At least two, and probably all four, of the already mapped Msp I sites within the NGF gene are methylated in all teratocarcinoma DNAs examined, as well as in the male mouse submaxillary gland DNA, the organ richest in this factor.     

Differential spatial and temporal expression of two type III intermediate filament proteins in olfactory receptor neurons

Olfactory receptor neurons (ORNs) do not express the typical neuronal intermediate filament proteins (IFPs), the neurofilament triplet proteins. Immunocytochemical evidence shows that ORNs coexpress vimentin and peripherin but distribute them differently. Specifically, ORNs contain vimentin in dendrites, cell bodies, and axons, but not in terminals in glomeruli; peripherin is present in axons, but excluded from dendrites, cell bodies, and terminal glomeruli. In adult rats, ORN axon fascicles are variably stained with antisera for peripherin; in juvenile rats, staining of fascicles is uniform. Staining with antibody to vimentin is uniform in both adult and juvenile ORN axon fascicles. The unusual pattern of IFP expression and intracellular sorting may have implications for the unique plastic and regenerative capacities of these neurons.     

In vitro differentiation of human processed lipoaspirate cells into early neural progenitors

Human processed lipoaspirate (PLA) cells are multipotent stem cells, capable of differentiating into multiple mesenchymal lineages (bone, cartilage, fat, and muscle). To date, differentiation to nonmesodermal fates has not been reported. This study demonstrates that PLA cells can be induced to differentiate into early neural progenitors, which are of an ectodermal origin. Undifferentiated cultures of human PLA cells expressed markers characteristic of neural cells such as neuron-specific enolase (NSE), vimentin, and neuron-specific nuclear protein (NeuN). After 2 weeks of treatment of PLA cells with isobutylmethylxanthine, indomethacin, and insulin, about 20 to 25 percent of the cells differentiated into cells with typical neural morphologic characteristics, accompanied by increased expression of NSE, vimentin, and the nerve-growth factor receptor trk-A. However, induced PLA cells did not express the mature neuronal marker, MAP, or the mature astrocyte marker, GFAP. It was also found that neurally induced PLA cells displayed a delayed-rectifier type K+ current (an early developmental ion channel) concomitantly with morphologic changes and increased expression of neural-specific markers. The authors concluded that human PLA cells might have the potential to differentiate in vitro into cells that represent early progenitors of neurons and/or glia.     

Early segregation of a neuronal precursor cell line in the neural crest as revealed by culture in a chemically defined medium

This article addresses the problem of the segregation of cell lines during the development of peripheral nervous system components from the neural crest. We show here that committed precursors of peripheral neurons are present in the crest before the migration of its cells has started. If cultured in a serum-deprived medium, a subpopulation of the crest cells readily differentiates into neurons without dividing. Neuronal markers such as neurofilament proteins and receptor sites for tetanus toxin are not expressed in the committed neuronal precursors, but appear after a few hours in culture. They are coexpressed in neurons with the mesenchymal intermediate filament protein, vimentin, which is common to all neural crest cells regardless of their prospective fate. A strong inhibitory effect of serum factor(s) on neurite outgrowth is demonstrated. We show also that conditions stimulating proliferation of crest cells are incompatible with promotion of neuronal differentiation and vice-versa.