Directed differentiation of mouse P19 embryonal carcinoma cells to neural cells in a serum- and retinoic acid-free culture medium (original) (raw)
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Neurons derived from P19 embryonal carcinoma cells have varied morphologies and neurotransmitters
Neuroscience, 1994
Treatment of PI9 embryonal carcinoma ceils with retinoic acid induces their differentiation into a population of cells consisting of neurons and other cell types normally derived from neuroectoderm. We used immunohistological and histochemical techniques to identify some of the neurotransmitters in the Pl9-derived neurons. The majority of neurons contained GABA, glutamic acid decarboxylase, and GABA-transaminase. Neuropeptide Y and somatostatin were less frequently found and both were partially co-expressed with GABA and with one another. Smaller numbers of cells were positive for tyrosine hydroxylase, DOPA decarboxylase, serotonin, calcitonin gene-related peptide, galanin and substance P. The variety and proportions of cells with different transmitter types were reprodu~ble from one experiment to the next and varied very little over 40 days in culture except for cells containing enkephalin, which were abundant only in mature cultures of 32 days or more. Synapses formed between neurons and some contained both small clear and large dense-core vesicles within the presynaptic bouton. Because GABA, neuropeptide Y and somatostatin are abundant in Pl9-derived neurons as well as in embryonic neurons in rostra1 regions of the mammalian CNS, we suggest that the developmental events occurring in PI9 cell cultures closely resemble those of the embryonic neuroectoderm.
Molecular Brain Research, 1994
Mouse P19 embryonal carcinoma cells can be reproducibly differentiated into neurons and glial cells upon treatment with high concentration of retinoic acid (RA). In order to understand the molecular mechanisms that control early neural differentiation, we screened a eDNA library made from 24-h RA-treated P19 cells with subtracted eDNA probes. One clone was positive in the secondary screening and was designated as p205. This clone (1.1 kb) has an open reading frame of 317 amino acids with homology to G-protein/3 subunit. This protein sequence was identical to chicken and human genes previously identified as a major histocompatibility complex-associated gene. The complete conservation of its amino acid sequence between mouse, human and chicken provides strong evidence that the p205 protein fulfills a fundamental function. Developmental Northern blot analysis revealed that a p205 mRNA is expressed at high levels in the embryonic mouse brain, decreasing as development proceeds. In situ hybridization revealed that p205 mRNA is strongly and ubiquitously expressed in the embryonic and early postnatal mouse brain. This expression decreased during postnatal development and was localized in the dentate gyrus, habenula, piriform cortex, paraventricular nucleus of the hypothalamus and supraoptic nucleus of the adult brain. These results suggest that this protein plays an important role in the developing brain and neuronal differentiation.
Differentiation and maturation of embryonal carcinoma-derived neurons in cell culture
The Journal of neuroscience : the official journal of the Society for Neuroscience, 1988
We have previously shown that retinoic acid-treated cultures of the P19 line of embryonal carcinoma cells differentiate into neurons, glia, and fibroblast-like cells (Jones-Villeneuve et al., 1982). We report here that the monoclonal antibody HNK-1 reacts with the neurons at a very early stage of their differentiation and is, therefore, an early marker of the neuronal lineage. Cells in differentiated P 19 cultures synthesized acetylcholine but not catecholamines, suggesting that at least some of the neurons are cholinergic. The neurons also carry high-affinity uptake sites for GABA but not for serotonin. In long-term cultures, neuronal processes differentiated into axons and dendrites, which formed synapses. This biological system should prove valuable for examining the development and maturation of cholinergic neurons, since their differentiation occurs in cell culture.
Cellular and Molecular Neurobiology, 2004
1. Neurogenin1 (Ngn1) is a basic helix-loop-helix (bHLH) transcription factor that is expressed in neuronal precursors during development of the nervous system. 2. In the present work, we investigated a instructive potential of Ngn1 in pluripotent embryonal carcinoma P19 cells. Treatment with retinoic acid (RA) induced expression of Ngn1 as well as NeuroD in P19 cells in early period of neuronal differentiation. P19 cells contained endogenous E47, a heterodimeric partner of neurogenic bHLH factors, and overexpression of Ngn1 alone was sufficient to induce the maximum activation of the E-box-mediated gene expression. 3. Sustained expression of Ngn1 in the absence of RA was sufficient to induce substantial expression of neuronal markers. The data indicate that Ngn1 is able to commit pluripotent P19 cells to adopt a neural cell phenotype in the absence of RA, which may finally lead to enhanced neuronal differentiation. The results also suggest that RA may induce neuronal differentiation of P19 cells by promoting a bHLH cascade including Ngn1.
Alternatives to laboratory animals : ATLA, 2015
Serum is generally regarded as an essential component of many eukaryotic cell culture media, despite the fact that serum composition varies greatly and may be the source of a wide range of artefacts. The objective of this study was to assess serum-free growth conditions for the human embryonal carcinoma cell line, NT2/D1. These cells greatly resemble embryonic stem cells. In the presence of retinoic acid (RA), NT2/D1 cells irreversibly differentiate along the neuronal lineage. We have previously shown that the early phases of neural induction of these cells by RA involve the up-regulation of SOX3 gene expression. Our goal was to compare RA-induced differentiation of NT2/D1 cells in serum-containing and serum-free media, by using SOX3 protein levels as a marker of differentiation. We found that NT2/D1 cells can be successfully grown under serum-free conditions, and that the presence or absence of serum does not affect the level of SOX3 protein after a 48-hour RA induction. However, s...
A rapid and efficient method for neuronal induction of the P19 embryonic carcinoma cell line
Journal of Neuroscience Methods, 2014
h i g h l i g h t s • We establish the simple and efficient method for neural differentiation. • Only neural cells are differentiated without glial-and non-neural cells. • Functional neural cells are obtained within 4-6 days. • The cells are responsive to several neurotransmitters. • Synchronized activation indicates neuronal cells with functional synapses.
Retinoic acid-induced neural differentiation of embryonal carcinoma cells
… and cellular biology, 1983
We have previously shown that the P19 line of embryonal carcinoma cells develops into neurons, astroglia, and fibroblasts after aggregation and exposure to retinoic acid. The neurons were initially identified by their morphology and by the presence of neurofilaments within their cytoplasm. We have more fully documented the neuronal nature of these cells by showing that their cell surfaces display tetanus toxin receptors, a neuronal cell marker, and that choline acetyltransferase and acetyl cholinesterase activities appear coordinately in neuroncontaining cultures. Several days before the appearance of neurons, there is a marked decrease in the amount of an embryonal carcinoma surface antigen, and at the same time there is a substantial decrease in the volumes of individual cells. Various retinoids were able to induce the development of neurons in cultures of aggregated P19 cells, but it did not appear that polyamine metabolism was involved in the effect. We have isolated a mutant clone which does not differentiate in the presence of any of the drugs which are normally effective in inducing differentiation of P19 cells. This mutant and others may help to elucidate the chain of events triggered by retinoic acid and other differentiation-inducing drugs.
Journal of Tissue Engineering and Regenerative Medicine, 2018
Tissue engineering, as a novel transplantation therapy, aims to create biomaterial scaffolds resembling the extracellular matrix in order to regenerate the damaged tissues. Adding bioactive factors to the scaffold would improve cell-tissue interactions. In this study, the effect of chitosan poly vinyl alcohol nanofibers containing carbon nanotube scaffold with or without active bioglass (BG + /BG-), in combination with neonatal rat brain extract (NRBE) on cell viability, proliferation and neural differentiation of P19 embryonic carcinoma (EC) stem cells was investigated. To induce differentiation, the cells were cultured in α-MEM supplemented with NRBE on the scaffolds. The expression of undifferentiated stem cell markers as well as neuroepithelial and neural specific markers was evaluated and confirmed by real-time RT-PCR and immunofluorescence procedures. Finally, the three dimensional (3D) cultured cells were implanted into the damaged neural tubes of chick embryos and their fates were followed in ovo. Based on the histological and immunofluorescence observations, the transplanted cells were able to survive, migrate, and penetrate into the host embryonic tissues. Gene network analysis suggested the possible involvement of neurotransmitters as a downstream target of synaptophysin and tyrosine hydroxylase. Overall, the results of this study indicated that combining the effects of 3D cell culture and natural brain tissue extract can accelerate the differentiation of P19 EC cells into neuronal phenotype cells.
Physiological Research
In both embryonal carcinoma (EC) and embryonic stem (ES) cells, the differentiation pathway entered after treatment with retinoic acid (RA) varies as it is based upon different conditions of culture. This study employs mouse EC cells P19 to investigate the effects of serum on RA-induced neural differentiation occurring in a simplified monolayer culture. Cell morphology and expression of lineage-specific molecular markers document that, while non-neural cell types arise after treatment with RA under serum-containing conditions, in chemically defined serum-free media RA induces massive neural differentiation in concentrations of 10(-9) M and higher. Moreover, not only neural (Mash-1) and neuroectodermal (Pax-6), but also endodermal (GATA-4, alpha-fetoprotein) genes are expressed at early stages of differentiation driven by RA under serum-free conditions. Furthermore, as determined by the luciferase reporter assay, the presence or absence of the serum does not affect the activity of th...
Neuroscience, 1996
Almtraet-Retinoic acid treatment of P19 embryonal carcinoma cells induces their differentiation into cultures containing neurons and astrocytes. We present two lines of experimentation indicating that oligodendrocytes also develop from retinoic acid-treated PI9 cells. We isolated an immortal cell line from retinoic acid-treated P19 cell cultures whose proliferation is dependent upon epidermal growth factor. Upon removal of the growth factor these cells differentiate into both astrocytes and oligodendrocytes as determined by immunostaining with antibodies to the astrocyte marker glial fibrillar acidic protein and the oligodendrocyte markers, myelin associated glycoprotein and 2', T-cyclic nucleotide 3'-phosphodiesterase. This cell line appears to be a bi-potential glial precursor. We also found that oligodendrocytes developed directly from P19 cells when retinoic acid-treated cells were transplanted into the brains of neonatal rat pups. Cells that developed into oligodendrocytes migrated into fiber bundles up to several millimeters from the site of the graft. These P19derived oligodendrocytes appeared to myelinate axons from host neurons. Thus, retinoic acid-treated P19 cells differentiate into neurons, astrocytes and oligodendrocytes, the three cell types that normally develop from embryonic neuroectoderm, indicating that these cell cultures differentiate in a fashion closely resembling that of embryonic neuroectoderm.