Differentiation Of EC Cells Research Articles

In vitro differentiation induction of embryonal carcinoma stem cells into insulin-producing cells by Cichorium intybus L. leaf extract.

Medicinal herb Cichorium intybus L. (chicory) has been used traditionally for the treatment of various diseases, including diabetes. One of the promising therapeutic options to treat diabetes is replacing the degenerative pancreatic β cells by stem cell-derived IPCs (insulin-producing cells). By the combination of cell therapy as a modern approach and traditional medicine, the current study was designed to evaluate the effects of chicory leaf extract (LE) on the differentiation potential of P19 EC cells (an embryonal carcinoma stem cell line) into IPCs. The plant (voucher no. 4567) were collected and deposited in the herbarium of Shahrekord University. In vitro experiments were designed to compare the effects of various concentrations of LE on the differentiation potential of P19 EC cells. The differentiated cells showed morphological characteristics of pancreatic β cells. They could also synthesized and secreted insulin when exposed to glucose. Moreover, the cells expressed specific proteins and genes of mature pancreatic β cells. In conclusion, LE as a natural herbal extract was efficiently able to induce the differentiation of P19 EC cells into the clusters similar to pancreatic islets with the molecular, cellular and functional characteristics of mature β cells.

Effects of altered expression and localization of cyclophilin A on differentiation of p19 embryonic carcinoma cells.

1. The retinoblastoma susceptibility gene product, p105Rb (RB), is an important regulator in the control of cell proliferation, differentiation, and apoptosis. Several cellular factors that complex with RB and exert their cellular regulatory functions have been identified, such as the RB:cyclophilin A (CypA) complex. 2. CypA is a cytoplasmic immunophilin and known for its involvement in T-cell differentiation and proliferation. Although CypA has a pivotal role in the immune response, its function in other signaling pathways is largely unknown. 3. In this study, we used a model of neuronal differentiation to demonstrate that the nuclear translocation of CypA, the appearance of hypophosphorylated RB and the enhancement of RB: CypA complex formation correlates with retinoic acid induced neuronal differentiation. 4. Inhibition of CypA expression results in repression of both the hypophosphorylated RB and the neuron-specific differentiation marker, class III beta tubulin. 5. The evidence of enriched CypA and colocalization of RB with CypA in the nucleus of primary adult sensory neurons substantiated the important event of RB-mediated neuronal differentiation of p19 EC cells.

Ectopic Expression of Axin Blocks Neuronal Differentiation of Embryonic Carcinoma P19 Cells

Axin regulates Wnt signaling through down-regulation of beta-catenin. To test the role of Wnt signaling in neuronal differentiation, embryonal carcinoma P19 cells (P19 EC), which can be stimulated to differentiate into a neuron-like phenotype in response to retinoic acid (RA), were used. Reverse transcription-PCR and Western blot analysis showed that Axin is expressed in undifferentiated cells, whereas the level is clearly reduced during RA-induced neuronal differentiation. Interestingly, Axin levels were not reduced during endodermal differentiation of P19 EC cells and F9 EC cells by RA, suggesting that the reduction of the Axin level is a specific property of neuronal differentiation. Western analysis showed that the cytoplasmic level of beta-catenin increased during neuronal differentiation of P19 EC cells. Indirect immunofluorescence with beta-catenin antibody showed that the localization of beta-catenin was changed from membrane in undifferentiated cells to nuclei in neuronal P19 EC cells. Induced expression of Axin during endodermal and early neuronal differentiation, using the Tet-On system, did not block normal differentiation. However, maintenance of the Axin level blocked neuronal differentiation and inhibited expression of a neuron-specific marker protein, beta III-tubulin. Also, ectopic induction of a beta-catenin signaling inhibitor, ICAT, inhibited expression of beta III-tubulin. In contrast, addition of Wnt-3A-conditioned medium during the neuronal differentiation period enhanced the expression of beta III-tubulin. Overall, our data show that Wnt-3a/canonical beta-catenin signaling through the down-regulation of Axin may play an important role in neuronal differentiation.

Modulation of SOX2 and SOX3 gene expression during differentiation of human neuronal precursor cell line NTERA2.

The SOX genes comprise a family of transcriptional regulators implicated in the control of nervous system development. The developing brain is the major site of expression of many Sox genes. Sox2 and Sox3 genes are predominantly expressed in the immature, undifferentiated cells of the neural epithelium throughout the entire CNS. NTERA2 is a human embryonal carcinoma cell line that phenotypically represents undifferentiated, pluripotent embryonic stem cells. In the presence of retinoic acid, cells differentiate into mature neurons providing an in vitro model for studying human genes that promote and regulate neural differentiation. In this study it is shown for the first time that the retinoic acid-induced neuronal differentiation of NTERA2 cells is accompanied by down-regulation of SOX2 and up-regulation of SOX3 gene during early phases of induction. These data suggest that the effects of retinoic acid on neural differentiation of NTERA2 EC cells might be mediated by modulation of SOX2 and SOX3 gene expression.

Expression of glycoconjugates bearing the Lewis X epitope during neural differentiation of P19 EC cells

The Lewis X (Le x) bearing glycolipids were noticeably increased in amounts during the course of neural differentiation of P19 EC cells induced by retinoic acid (RA, all- trans form). Applying neoglycolipid technology and in situ TLC-LSIMS, the oligosaccharide chains of these scarce Le x bearing glycolipids were partially characterized after released by endoglycoceramidase and subsequent conversion into neoglycolipids. In order to understand the enzymatic basis for the expression of Le x bearing glycolipids, we measured glycolipid, glycoprotein and oligosaccharide fucosyltransferase (Fuc-T) activities using appropriate substrates in P19 EC cells with or without RA treatment. All three Fuc-Ts were increased after RA treatment and the highest activity was in the differentiated neural cells. We then investigated the two possible Fuc-T genes that might be responsible for these changes using RT-PCR analysis. Mouse Fuc-TIX (mFuc-TIX) transcript was detected in all cell types but it was only strongly expressed in RA-induced aggregates and neural cells. In the case of mouse Fuc-TIV (mFuc-TIV) gene, its transcript was only detectable in RA-induced aggregates and not found in either undifferentiated or RA-induced neural cells. These results strongly support that RA induces only a transient expression of the mFuc-TIV gene in cell aggregates but a more persistent expression of the mFuc-TIX gene at the transcription level throughout neural cell differentiation. The mFuc-TIX gene is probably the main cause for the increased expression of Le x glycoconjugates during neural differentiation of P19 EC cells.

Interferon-γ induces differentiation of ectoplacental cone cells to phenotypically distinct trophoblasts

Maturation of the murine ectoplacental cone results in the development of the placental tissue which essentially consists of two trophoblastic zones, the spongiotrophoblast and labyrinthine trophoblast. In this study we attempted to investigate the action of cytokines on ectoplacental cone cell differentiation to mature trophoblast cells. After determining the cellular composition of the ectoplacental cone cell suspensions based on the expression of cytokeratin, vimentin, Mac-1, class I and class II MHC antigens, the cells were exposed to the differentiation-inducing cytokines IL-3, GM-CSF, CSF-1 and IFN-γ. From the four factors employed, only IFN-γ increased the levels of cytokeratin-positive cells without inducing Mac-1 expression. IL-3 increased the percentages of cytokeratin as well as Mac-1- and vimentin-positive cells whereas GM-CSF and CSF-1 preferentially promoted an increase of the Mac-1 and vimentin markers. For further analysis, ectoplacental cone cells were negatively selected for Mac-1, class I and class II antigens to exclude non-trophoblastic contaminants and thereafter treated with the same cytokines. We show here that only IFN-γ leads the sorted ectoplacental cone cells to a pure trophoblastic population composed of 100% cytokeratin-positive cells. The specificity of IFN-γ on this differentiation pathway is strengthened by the fact that murine maternal serum on the day of EC formation contains high levels of this cytokine, suggesting that its natural presence supports — at least phenotypically — the in vivo differentiation of EC cells to trophoblasts.

T-Ag inhibits implantation by EC cell derived embryoid bodies.

When introduced into EC cells of a blastocyst, polyomavirus (Py) T-Ag results in mice mosaic for T-Ag but otherwise essentially normal. It had been reported that SV40 T-Ag does not inhibit differentiation of F9 EC cells, but did inhibit endogenous retrovirus (ERV) production. We therefore sought to determine if Py T-Ag had any affect on EC derived embryoid body implantation onto mouse placenta. F9 EC cells were selected for T-Ag maintenance. Like the SV40 transformed cells, we show that these Py T-Ag selected EC cells no longer express IAP transcripts following differentiation into embryoid bodies. Normal and Py T-Ag selected F9 cells were differentiated into embryoid bodies then implanted into pseudopregnant mice. We observe, that normal F9 derived embryoid bodies underwent the initial stages of implantation whereas the Py T-Ag selected embryoid bodied did not implant. The implications of this observation with respect to trophectoderm and ERV function are discussed. We examine the idea that ERVs may be a required element for normal embryo implantation.

Alteration of cGMP metabolism during chondrogenic differentiation of chondroprogenitor-like EC cells, ATDC5

Guanosine 3′,5′-cyclic monophosphate (cGMP) has been recently reported to be involved in bone formation. ATDC5 cells were used to investigate cGMP metabolism during chondrogenic differentiation. Natriuretic peptide receptor (NPR)-A and NPR-B coupled with guanylate cyclase (GC) mediate biological functions of NPs, whereas NPR-C uncoupled with GC is thought to be the clearance receptor for NPs. The amounts of NPR-A, NPR-B, and CNP transcripts were increased but the amount of NPR-C transcripts was decreased in association with the chondrogenic differentiation of ATDC5 cells. CNP, a specific ligand for NPR-B lets ATDC5 cells accumulate great amounts of cGMP, revealing NPR-B as a dominant biological receptor through differentiation. cGMP hydrolytic activities of PDE1 and PDE5 existed in ATDC5 cells, and the activity of PDE1, which is stimulated by Ca 2+ and calmodulin (CaM) was major of them. Total cGMP hydrolytic activities as well as the amounts of PDE1 and PDE5 transcripts were enhanced during chondrogenic differentiation. Therefore, cGMP production and hydrolysis, cGMP metabolism was considered to be activated in association with chondrogenic differentiation of ATDC5 cells. These observations may lead to a better understanding of cGMP in the chondrocytes where bone formation occurs.

Using EC and ES cell culture to study early development: recent observations on Indian hedgehog and Bmps.

Despite great technological advances in the study of mammalian development in the past two decades, certain problems in early development, such as how the extraembryonic lineages are established, have remained intractable. We suggest that teratocarcinoma (EC) and embryonic stem cells (ES) remain useful in vitro tools for studying some of these problems. We present a continuation of our studies on the role of IHH-based signaling in early development and demonstrate that the IHH N-peptide is expressed in the outer visceral endoderm cells of both the EC and ES-derived embryoid body. We also show that Bmp2 is upregulated and Bmp4 downregulated during the differentiation of F9 EC cells into embryoid bodies, whereas both genes are upregulated when J7 ES cells differentiate into embryoid bodies. We also examine the spatial localization of Ihh, Bmp2, and Bmp4 in day 6.5-7.0 and 7.5-8.0 embryos by in situ hybridization analysis. These data support the EC temporal expression data in that all 3 genes are expressed in visceral endoderm. Bmp4 expression appears to be limited to extraembryonic regions, where mesoderm as well as visceral endoderm are stained. Ihh and Bmp2 are expressed in extraembryonic tissues and the embryo proper. Functional roles for the observed expression patterns are discussed.

Apoptotic cell death in neuronal differentiation of P19 EC cells: cell death follows reentry into S phase.

Apoptotic cell death was observed during aggregate culture of the mouse embryonal carcinoma cell line P19 exposed to all-trans retinoic acid (tRA). This finding was confirmed by genomic DNA agarose gel electrophoresis and transmission electron microscopy. Apoptosis was associated with P19 cell neuronal differentiation; alternative causes of cell death, i.e., cavitation-related, cytotoxicity of tRA, or spontaneous cell death were excluded. Analysis by flow cytometry revealed that the apoptosis was likely to occur in multiplying cells that underwent to reentering into S phase. We therefore examined 5-bromo-2'-deoxyuridine (BrdU) incorporation and proliferating cell nuclear antigen (PCNA) expression and localization in the aggregates by immunofluorescent staining. Although the P19 cells in the aggregates exposed to tRA incorporated BrdU at an equivalent level to those not exposed to tRA, the cells showed diminished PCNA expression and nuclear accumulation. We propose that P19 apoptosis during neuronal differentiation is a model system in which programmed cell death occurs simultaneously with cell division leading to differentiation.

Apoptotic cell death in neuronal differentiation of P19 EC cells: Cell death follows reentry into S phase

Apoptotic cell death in neuronal differentiation of P19 EC cells: Cell death follows reentry into S phase

Scheduled perturbation in DNA during in vitro differentiation of mouse embryo-derived cells.

Studies of sister chromatid exchanges (SCE) and recombination rate of certain minisatellite DNAs have demonstrated that their levels are considerably higher during the preimplantation stage than in latest developmental stages of embryos. It appeared likely that single-strand DNA breaks (SSB) may be relevant to both events during early development. With this in mind, we estimated SSB during in vitro retinoic acid (RA)-induced and spontaneous differentiation of mouse teratocarcinoma (EC) and embryonic stem (ES) cells. Using the method of nucleoid sedimentation and single-cell DNA electrophoresis, we have observed a dramatic increase in the SSB during the first 2-4 mitoses after beginning of differentiation of EC cells, followed by a gradual return to the basal level characteristic of undifferentiated cells. The increase in the SSB was manifested as the appearance of mass nucleoids with slow sedimentation rates, as well as the low-weight mass fragments in DNA patterns of most cells. We concluded that not less than half of genomic DNA has been nicked at the early steps of differentiation. The decrease in SSB level was observed in spite of continuing differentiation, as judged by embryonic antigens and morphological criteria. Also, the increase in the SCE level coincided with that of SSB, possibly being its consequence. The scheduled "surge" of SSB may be the earliest event in commencing differentiation at steps without a phenotypic manifestation.

Wortmannin Enhances CPP32-like Activity during Neuronal Differentiation of P19 Embryonal Carcinoma Cells Induced by Retinoic Acid

P19EC cells undergoes apoptosis during neuronal differentiation induced by retinoic acid. Two CPP32-like proteases, CPP32 and Mch-3, are expressed in untreated and retinoic acid-treated P19 EC cells. CPP32-like activity is remarkably increased in apoptosis during neuronal differentiation of P19 EC cells. Inhibition of CPP32-like proteases prevents apoptosis, suggesting that activation of CPP32-like proteases play central roles in the apoptosis during neuronal differentiation of P19 EC cells. Wortmannin, PI-3K inhibitor, enhances the CPP32-like activity of the retinoic acid-treated P19 EC cells. PI-3K may be involved in the apoptosis during neuronal differentiation as negative regulator.

Heat Shock Induces Differentiation of Human Embryonal Carcinoma Cells into Trophectoderm Lineages

NCR-G3 cells were established from a testicular embryonal carcinoma and are highly multipotential, differentiating into trophectoderm cells upon exposure to retinoic acid. Differentiated NCR-G3 cells begin to produce human chorionic gonadotropin (hCG), a trophectoderm-specific hormone. We have previously isolated the up-regulated genes at the early stage of differentiation. One of them was found to be a heat shock protein gene. The heat shock protein gene (HSP90) is induced at the early stage of differentiation and decreases to the basal level or under the basal level at the later stage. We speculate that heat shock per se induces the differentiation of human EC cells. With exposure to heat, NCR-G3 cells began to express a series of differentiation markers such as cytokeratin and hCG. Heat, which is classically known to induce heat shock proteins, is able to differentiate an embryonal cell line into trophectoderm lineages, implying a new recognized function of a heat-like event in early differentiation.

Regulation of Oct-4 gene expression during differentiation of EC cells

The stem cell-specific factor Oct-4 is expressed in undifferentiated embryonal carcinoma and embryonic stem cells and is quickly down regulated upon RA-induced differentiation. Irrespective of the direction of differentiation, Oct-4 repression in P19 EC cells requires treatment with high doses of either all-trans or 9-cis RA. Unlike in P19 cells, no RA-induced down regulation of Oct-4 expression is observed in the P19-derived RA-resistant RAC65 cells. However, in these cells Oct-4 promoter repression can be rescued in a RA-dependent manner by cotransfection of RAR alpha 2 or RAR beta 2 but not RARr gamma 1, matching previously reported transactivation properties of these receptor types. In the vicinity of the transcription initiation site of the Oct-4 gene, three Hormone Response Element HRE half sites are present which are arranged as direct repeats with different spacing. In vitro translated RAR and RXR proteins bind to this HRE as heterodimers with low affinity, in such a way that all three HRE half sites contribute to complex formation. Although P19 EC cells contain weak binding activity interacting with the Oct-4 promoter HRE, strong binding activity is observed in nuclear extracts from RA-treated P19 cells. This binding activity was shown to correspond to COUP-TFs but not nuclear RA receptors. Moreover, the presence of these binding factors in nuclear extracts corresponds to silencing of Oct-4 expression. These results implicate RA and the action of its nuclear receptors in silencing Oct-4 expression upon differentiation of EC cells. The observed silencing is most likely not exerted by direct binding of RARs to the Oct-4 proximal promoter HRE. Our results support models in which different nuclear receptor complexes sequentially occupy different sites in the Oct-4 promoter HRE to silence Oct-4 expression during RA-induced differentiation.

Function of HSP90 and M-CSF/c-fms during differentiation of human EC cells into trophoectoderm

Function of HSP90 and M-CSF/c-fms during differentiation of human EC cells into trophoectoderm

Cell differentiation induced by poly(ADP-ribose) synthetase inhibitors

From a survey of about 400 compounds, we found a number of potent and specific inhibitors of poly(ADP-ribose) synthetase. Two new classes of inhibitors include vesnarinones and heterocyclic amines. Although the inhibitory activity was not very strong, several of these classes of inhibitors proved to induce most efficiently differentiation of murine teratocarcinoma EC cells in culture; for example, 70 μM benzylvesnarinone or 1 mM PhIP effected almost complete change in cell morphology in 5 to 7 days. Analyses of poly(ADP-ribose) synthesis and NAD concentrations in EC cells after treatment with a differentiation inducer suggested that poly(ADP-ribose) might play a role at an initiation stage of cell differentiation.

Differential expression of a novel murine non-receptor protein tyrosine phosphatase during differentiation of P19 embryonal carcinoma cells

Protein phosphorylation on tyrosine residues is one of the major mechanisms of cell signal transduction and is regulated by protein tyrosine kinases and protein tyrosine phosphatases. Here we report the molecular cloning of an additional member of the protein tyrosine phosphatase-family from differentiated murine P19 embryonal carcinoma cells. This non-receptor protein tyrosine phosphatase, P19-PTP, does not contain regulatory sequences, homologous to the ones found in other non-receptor PTPases. P19-PTP is differentially expressed during in vitro differentiation of P19 EC cells, in that P19-PTP mRNA could only be detected in embryoid bodies, derived from P19 cells.

Aggregation and cell cycle dependent retinoic acid receptor mRNA expression in P19 embryonal carcinoma cells

Differentiation of P19 EC cells along different pathways into derivatives resembling cells of the three embryonic germ layers is accompanied by characteristic differences in modulation of expression of each of the three retinoic acid receptor genes, RAR alpha, -beta and -gamma. Differentiation induced by addition of RA to P19 EC cells cultured in monolayer is accompanied by a rapid increase in expression of both RAR alpha and -beta. Induction of RAR beta occurs in a characteristic biphasic manner, suggesting that multiple factors and/or different mechanisms are involved in controlling its expression. RAR beta mRNA is induced to a far higher level during early aggregation in the presence of RA than during early differentiation in monolayer, suggesting that the direction of differentiation depends on the number and/or ratio of alpha and beta type of RA receptors. Aggregation of P19 EC cells in the presence of RA, but not DMSO, is accompanied by repression of RAR gamma, suggesting that the expression of RAR beta and RAR gamma during neuroectodermal differentiation is mutually exclusive. The effects of RA on RAR expression are significantly greater in G1 than in S-phase of the cell cycle. These results extend previous observations that commitment to differentiation is cell cycle dependent and indicates that critical target gene regulation in response to RA has to take place in G1 for differentiation to occur.

Phosphofructokinase and pyruvate kinase in mouse embryonal carcinoma P19 cells in relation to growth and differentiation

Abstract Two key enzymes of glycolysis, phosphofructokinase and pyruvate kinase, were studied in embryonal carcinoma cells (P19 EC cells) and three differentiated derivatives in relation to growth rate and differentiation state. The growth rates of P19 EC cells and its differentiated derivatives are positively correlated with both the specific activity of phosphofructokinase and the expression of the L-subunit of this enzyme. The specific activity of pyruvate kinase and its isozyme composition is not correlated with growth rate but seems to be correlated with the differentiation state of these cells. The decrease in specific activity of pyruvate kinase during differentiation of P19 EC cells induced by retinoic acid or dimethylsulfoxide preceded the shift from K- to M-type pyruvate kinase. In contrast to aggregates that were treated with dimethylsulfoxide, the specific activity of pyruvate kinase was reduced after aggregation in the presence of retinoic acid. Only after plating dimethylsulfoxide-treated aggregates again in the presence of dimethylsulfoxide, was a decrease in specific activity obtained. Both retinoic acid and dimethylsulfoxide are able to induce a K- to-M shift of pyruvate kinase.