Teratocarcinoma differentiation: plasminogen activator activity associated with embryoid body formation.

Changes in plasminogen activator activity have been examined as a clonal line of mouse embryonal carcinoma cells aggregate and differentiate to form cystic embryoid bodies in vitro. Within the first 10 days of study, the pluripotent embryonal carcinoma cells aggregate; a layer of endodermal cells appears on the outside of the aggregate forming an embryoid body; a basement membrane forms between the outer layer of endodermal cells and the internal cells; a cyst forms within the embryoid body; and the internal cells assume a columnar appearance along the inner portion of the basement membrane. After the formation of the endodermal layer, there is a rise in intracellular plasminogen activator activity. This rise continues for up to 25 days in culture, providing that the three-dimensional integrity of the embryoid bodies is maintained by culturing them on bacterial petri dishes. Selective removal of the outer endodermal layer of cells reduces the plasminogen activatory activity of the resulting embryoid body cores. Intracellular and secreted plasminogen activator activity of simple embryoid bodies composed of only two cell types can be increased by culturing the embryoid bodies in dbcAMP, theophylline, or cholera toxin. These results suggest that the embryoid body endodermal cells are the source of a cAMP-inducible plasminogen activator activity.     

H1 Histone and nucleosome repeat length alterations associated with the in vitro differentiation of murine embryonal carcinoma cells to extra-embryonic endoderm

The histone compositions and average distance between nucleosomes have been determined for F9.22 and PSA1 murine embryonal carcinoma cell lines, for primary extra-embryonic endoderm derived from the in vitro differentiation of PSA1 embryonal carcinoma cells, and for two long-term extra-embryonic endodermal cell lines. A change in the relative proportions of two forms of the H1 histones (H1A and H1B) was found to correlate with the extra-embryonic endodermal differentiated phenotype. The embryonal carcinoma cells had a ratio of H1A/H1B of 1.49 or greater. In contrast, extra-embryonic endoderm from either cell lines or freshly isolated from differentiating embryonal carcinoma cell cultures had a ratio of H1A/H1B of less than 0.9. Partial peptide mapping of gel purified H1A and H1B suggest the two proteins differ in primary structure. The nucleosome repeat length of the embryonal carcinoma cell lines was 196 bp of DNA. Primary extra-embryonic endoderm was found to have a value of 205 bp, but the long-term extra-embryonic endodermal cell lines had an average nucleosome repeat length of 187 bp. Since both freshly isolated primary endoderm and the long-term endodermal cell lines express differentiated functions (basement membrane glycoproteins and plasminogen activator activity), there appears to be no simple correlation between the nucleosome repeat length and the expression of these differentiated functions.     

RXRalpha-null F9 embryonal carcinoma cells are resistant to the differentiation, anti-proliferative and apoptotic effects of retinoids.

The F9 murine embryonal carcinoma (EC) cell line, a well established model system for the study of retinoic acid (RA)-induced differentiation, differentiates into cells resembling three types of extra-embryonic endoderm (primitive, parietal and visceral), depending on the culture conditions and RA concentration used. A number of previously identified genes are differentially expressed during this process and serve as markers for the different endodermal cell types. Differentiation is also accompanied by a decreased rate of proliferation and an apoptotic response. Using homologous recombination, we have disrupted both alleles of the retinoid X receptor (RXR) alpha gene in F9 cells to investigate its role in mediating these responses. The loss of RXRalpha expression impaired the morphological differentiation of F9 EC cells into primitive and parietal endoderm, but has little effect on visceral endodermal differentiation. Concomitantly the inducibility of most primitive and parietal endoderm differentiation-specific genes was impaired, while several genes upregulated during visceral endodermal differentiation were induced normally. We also demonstrate that RXRalpha is required for both the anti-proliferative and apoptotic responses in RA-treated F9 cells. Additionally, we provide further evidence that retinoic acid receptor (RAR)-RXR heterodimers are the functional units transducing the effects of retinoids in F9 cells.     

The induction of antigenic changes in a teratocarcinoma stem cell line (F9) by retinoic acid

Treatment of embryonal carcinoma cells F9 with retinoic acid results in the appearance of epithelioid cells resembling endoderm which synthesize basement membrane protein and plasminogen activator. Concomitant with the appearance of these properties of differentiated cells, the epithelial cells cease to express SSEA-1, an antigenic determinant characteristic of teratocarcinoma stem cells and early mouse embryos. Our evidence indicates that the phenotypic changes that accompany retinoic acid treatment of embryonal carcinoma cells are irreversible and a consequence of the differentiation of the cells into endoderm.     

Expression of EGF receptor and transferrin by F9 and PC13 teratocarcinoma cells

We document the time of appearance and the levels of two markers of differentiation during the formation of embryoid bodies by two embryonal carcinoma (EC) cell lines. Neither of these markers has been described before for EC cells differentiating in aggregate culture, and they further extend the identification and characterization of new cell types. Both F9 and PC13 EC cell lines form embryoid bodies (so-called because they resemble early mouse embryos) with an outer epithelial layer of visceral endoderm cells, after suspension culture in the presence of retinoic acid. However, the two cell lines differ in the procedures needed to initiate the differentiation process. Once floating aggregate cultures have been formed, the time course of the appearance of epidermal growth factor (EGF) receptors and of the secretion of transferrin are similar in both cell lines, although the levels differ. EGF receptors and transferrin are quantified by 125I-EGF binding assays and enzyme-linked immunosorbent assays (ELISA) using specific antibodies, respectively. The expression of EGF receptors increases about two fold while that of transferrin increases up to 40 fold after treating F9 aggregates with retinoic acid. The EGF receptors reach a maximum 4 days after adding retinoic acid and then decline, while transferrin only increases later from a low but detectable level. For PC13 cells, EGF receptors increase tenfold, and transferrin synthetic rate increases 40 fold during the time-course. Interestingly, unstimulated F9 cells in monolayer cultures also express low levels of these markers, while the levels in PC13 EC cells are barely detectable above background.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis and retention of fibronectin (LETS protein) by mouse teratocarcinoma cells.

Mouse teratocarcinoma cells in culture were examined for both the synthesis (by metabolic labelling) and surface accumulation (by indirect immunofluorescence) of fibronectin, a glycoprotein with subunits of molecular weight 220000 D known to form part of the extracellular matrix of many cells in vivo. Although lines of both pluripotent and nullipotent embryonal carcinoma cells synthesize the protein and release it into the medium, they do not retain it on their surfaces. Monolayers of the endoderm line PSA5-E both synthesize fibronectin and lay it down in an extracellular network. A line of PYS parietal endoderm cells does not retain surface fibronectin, although it does accumulate other extracellular matrix material. When pluripotent embryonal carcinoma cells differentiate into cystic embryoid bodies, fibronectin accumulates in a basement membrane below the outer endoderm cells (both visceral and parietal-like) and may play a transient role in organizing the inside cells into an epithelial layer.     

Expression of EGF receptor and transferrin by F9 and PC13 teratocarcinoma cells

Abstract. We document the time of appearance and the levels of two markers of differentiation during the formation of embryoid bodies by two embryonal carcinoma (EC) cell lines. Neither of these markers has been described before for EC cells differentiating in aggregate culture, and they further extend the identification and characterization of new cell types. Both F9 and PC13 EC cell lines form embryoid bodies (so-called because they resemble early mouse embryos) with an outer epithelial layer of visceral endoderm cells, after suspension culture in the presence of retinoic acid. However, the two cell lines differ in the procedures needed to initiate the differentiation process. Once floating aggregate cultures have been formed, the time course of the appearance of epidermal growth factor (EGF) receptors and of the secretion of transferrin are similar in both cell lines, although the levels differ. EGF receptors and transferrin are quantified by ¹²⁵I-EGF binding assays and enzyme-linked immunosorbent assays (ELISA) using specific antibodies, respectively. The expression of EGF receptors increases about two fold while that of transferrin increases up to 40 fold after treating F9 aggregates with retinoic acid. The EGF receptors reach a maximum 4 days after adding retinoic acid and then decline, while transferrin only increases later from a low but detectable level. For PCI 3 cells, EGF receptors increase tenfold, and transferrin synthetic rate increases 40 fold during the time-course. Interestingly, unstimulated F9 cells in monolayer cultures also express low levels of these markers, while the levels in PC13 EC cells are barely detectable above background. A variety of other teratocarcinoma EC cell lines either do not express these markers at detectable levels or express very low levels. One explanation of our finding is that F9 cells, unlike most other EC cell lines, are already partially differentiated along the pathway to endoderm.     

Cell position regulates endodermal differentiation in embryonal carcinoma cell aggregates

It has been suggested that cell position regulates endodermal differentiation in mouse embryo inner cell masses and in aggregates of embryonal carcinoma (EC) cells. This hypothesis states that cells at the interface between the cell mass and blastocoel fluid or culture medium differentiate into endoderm, whereas internally located cells follow alternative developmental pathways. To test the cell position hypothesis, pluripotent PSA-1 cells were aggregated with hypoxanthine phosphoribosyltransferase-deficient, parietal-like, endodermal cells. The resulting aggregates consisted of cores of PSA-1 cells surrounded by endodermal cells. Autoradiography was used to distinguish between endodermal cells that were the products of EC cell differentiation and the exogenous endoderm. Alkaline phosphatase staining was used to distinguish EC cells from endodermal cells. As predicted by the cell position hypothesis, the PSA-1 EC cells, all of which were internally located, did not differentiate into endodermal cells. Nonspecific inhibition of differentiation did not account for the lack of PSA-1-derived endoderm since the PSA-1 cells in such aggregates did differentiate into columnar ectodermal-like cells. Similar experiments were also conducted with F9 cells. In this case, aggregation cultures contained retinoic acid to induce F9 cells to differentiate into visceral endoderm. In cultures containing F9 cells surrounded by parietal-like endodermal cells, no F9-derived endoderm was detected either autoradiographically or by assaying for alpha-fetoprotein production, a visceral endoderm marker. Thus, retinoic acid-induced endodermal differentiation was also regulated by cell position. Collectively, the above results provide strong evidence for the hypothesis that cell position regulates endodermal differentiation in aggregates of EC cells.     

Paracrine promotion of cardiomyogenesis in embryoid bodies by LIF modulated endoderm

In the vertebrate embryo the heart is the first organ to form. Embryonic and extra-embryonic tissues are supposed to contribute to cardiac lineage commitment before and during gastrulation in a paracrine fashion. Evidence has accumulated that factors secreted by the anterior lateral endoderm and extra-embryonic endoderm contribute to cardiomyogenesis. Here we exploit in vitro differentiation of embryonic stem cells in embryoid bodies to study differentiation of the extraembryonic endodermal lineage, gastrulation-like processes, and the influence of endoderm on cardiomyogenesis. We demonstrate that in embryoid bodies primitive endoderm differentiates to visceral and parietal endoderm and that parietal endoderm influences onset of cardiomyogenesis in a concentration-dependent manner. Both increased concentrations of leukemia inhibitory factor and its absence in lif-/- embryoid bodies hampered parietal endoderm formation. Reduced differentiation of parietal endoderm correlated with an attenuation of cardiomyogenesis even in the presence of LIE These and previous results suggest that leukemia inhibitory factor is directly and indirectly, via endoderm formation, involved in the regulation of cardiomyogenesis. Increased proliferation of parietal endoderm in lifr -/- embryoid bodies and addition of conditioned lif -/- cell culture supernatant promoted cardiomyogenesis, demonstrating for the first time that parietal endoderm also contributes to cardiomyogenesis in embryoid bodies in a paracrine and leukemia inhibitory factor and its receptor independent pathway. New factors signaling independently of the leukemia inhibitory-factor receptor pathway may sustain cardiomyocyte cell proliferation and thus be a future target for gene therapy of cardiomyopathies and cell therapy of the myocardium.     

Neoplastic embryoid bodies of embryonal carcinoma C44 as a source of blastocele-like fluid

Abstract. There is a cytotoxic activity in blastocele fluid that kills embryonal carcinoma cells with trophectodermal potential but spares those with embryonic potential [26]. This activity is present when programmed cell death occurs in the inner cell mass (ICM), and the ICM loses its trophectodermal potential [5, 8–10]. Because of the paucity of blastocele fluid, cystic embryoid bodies of embryonal carcinoma C44 were examined ultrastructurally and in tissue culture to determine if they corresponded to late blastocysts and if their fluid corresponded to blastocele fluid. No troph-ectoderm was demonstrated in the embryoid bodies, but embryonal carcinoma and endoderm were present, leading to the conclusion that the embryonal carcinoma corresponded to late ICM that had expressed endodermal potential. As a result the cyst fluid might have contained the toxic activity of blastocele fluid. The cyst fluid of C44 embryoid bodies did contain a soluble, low-molecular-weight, cytotoxic activity that preferentially killed embryonal carcinoma cells with trophectodermal potential while sparing those with embryonic potential. Enough of this fluid was available to determine the chemical nature of this toxic activity.     

A teratocarcinoma-derived endoderm stem cell line (1H5) that can differentiate into extra-embryonic endoderm cell types

We investigated the ability of the teratocarcinoma-derived, epithelial-type cell line 1H5 to differentiate into either of the two pathways to primary endoderm, and tested the hypothesis that 1H5 represents a state similar to primitive endoderm in the late 4th-day blastocyst. Like other endodermal cell types, 1H5 cells mixed with embryonal-carcinoma cells sort out into "embryoid bodies" or structures that resemble 4th-day mouse embryos. The epithelial line conforms morphologically and biochemically to the few known characteristics typical of primitive endoderm. The present study demonstrates that the formation in vitro of overt visceral endoderm is readily achieved. The spontaneous arrangement of the cells into a cystic form is followed by the appearance of several markers of visceral endoderm, most notably alphafetoprotein, which is detected when 1H5 cells are cultured either in the presence of retinoic acid or when the cells interact with embryonal-carcinoma cells in a specific spatial arrangement after sorting out. However, some less specific properties of visceral endoderm are not expressed. Although 1H5 differentiates histologically into parietal-like endoderm in the tumor form, parietal cells cannot yet be identified with certainty in vitro because of the paucity of parietal-specific markers. The 1H5 cell line could provide a useful system for studying the characteristics and mechanisms underlying visceral-endoderm differentiation in vitro, since it has the distinct advantage that homogeneous cultures are produced, in contrast to other teratocarcinoma cell lines such as F9 which differentiate into a mixture of cell types.     

The induction of differentiation in teratocarcinoma stem cells by retinoic acid.

Embryonal carcinoma cells, the stem cells of teratocarcinomas, usually undergo extensive differentiation in vivo and in vitro to a wide variety of cell types. There exist, however, several embryonal carcinoma cell lines that have almost completely lost the capacity to differentiate, so that the cells are propagated primarily as the stem cells. Using one such cell line, F9, we have found that retinoic acid at concentrations as low as 10(-9) M induces multiple phenotypic changes in the cultures in vitro. These changes include morphological alteration at the resolution of the light microscope, elevated levels of plasminogen activator production, sensitivity to cyclic AMP compounds and increased synthesis of collagen-like proteins. The nature of these changes, as well as their independence of the continued presence of retinoic acid, are consistent with the proposition that retinoic acid induces differentiation of embryonal carcinoma cells into endoderm.     

Epithelial layer formation in differentiating aggregates of F9 embryonal carcinoma cells

F9 embryonal carcinoma (EC) cells, cultured in suspension in medium containing 5 X 10(-8) M retinoic acid, aggregate and differentiate into embryoid bodies with an outer layer of visceral endoderm cells that synthesize and secrete alphafetoprotein (AFP) (Hogan, B. L. M., A. Taylor, and E. Adamson, 1981, Nature (Lond.). 291:235-237). Here we analyze the formation of the outer layer of cells as a model for epithelial differentiation. Three morphological phases are described, but analyses of cell numbers and the synthetic rates of some proteins, as well as the appearance of markers of visceral endoderm and basement membrane, show that the formation of the outer layer occurs as an orderly progression of multiple events. The markers used to follow the ontogeny of epithelial layer formation include SSEA-1, l, and i blood group antigens, laminin, fibronectin, type IV collagen, cytoskeletal intermediate filament proteins (vimentin, Endo A, and B), and AFP. The onset of epithelium formation occurs between the third and fourth day of culture, but its function is maximally expressed only when it is well organized. We found the rate of AFP secretion to be a measure of the proper alignment and maturity of the epithelium which occurs at the seventh or eighth day. This model of epithelium formation may help to explain how similar processes occur during embryogenesis.     

Collagen Synthesis in Mouse Embryonal Carcinoma Cells: Effect of Retinoic Acid

Abstract. In five lines of mouse embryonal carcinoma cells, PCC3/A1, PCC4, PCC4/Aza-R1, PCC7-S/Aza-R1, and F9, collagen synthesis was examined by immunofluorescence reaction using specific antibodies directed against collagen. All the embryonal carcinoma cell lines showed type IV collagen, and PCC7-S/Aza-R1 revealed the additional presence of type III collagen. When the F9 and PCC3/A1 EC cells were treated with retinoic acid and dibutyryl-cAMP, they differentiated into morphologically different cellular types. These cellular types showed new types of collagen. Thus, in treated F9 cells, type I, type III, and type V collagen were detected and in treated PCC3/A1 cells, type III and type V collagen were detected.
In two established cellular strains, PYS-2 corresponding to parietal endoderm and 3TDM-1 corresponding to trophoblastoma, collagen was identified by immunological reaction and electrophoretic mobility. The trophoblastoma cell line was characterized by the production of type I, type III, and type IV collagen, whereas endodermal PYS-2 revealed type IV collagen.     

Cell surface antigens of clonal teratocarcinoma cells at various stages of differentiation

We have studied cell surface antigen expression of teratocarcinoma cells at various stages of differentiation. These cells can be maintained in the undifferentiated state or will differentiate in vitro in a manner which parallels the early development of the mouse embryo. Three antigens were studied: a stem cell antigen (C); the major histocompatibility alloantigens (H-2); and the alloantigen Thy-1.The stem cell antigen was recognized by an anti-serum raised against a pluripotent teratocarcinoma cell line. This antiserum was shown to label embryonal carcinoma cells and early mouse embryo cells. The activity of the antiserum against embryonal carcinoma cells could be adsorbed with brain, kidney, and sperm from adult mice.The phenotype of the undifferentiated embryonal carcinoma cells is C⁺, H-2⁻, Thy-1⁻ or C⁻, H-2⁻, Thy-1⁻. The first stage in the process of differentiation is the formation of simple embryoid bodies with a layer of endodermal cells surrounding an inner core of embryonal carcinoma cells. The endodermal cells are C⁻, H-2⁻, Thy-1⁻. Further differentiation of the embryoid bodies attached to a substratum is associated with the appearance of H-2⁺ and Thy-1⁺ cells in the cultures.     

Induction of the expression of retinol-binding protein and transthyretin in F9 embryonal carcinoma cells differentiated to embryoid bodies

Studies were conducted to determine if the expression of the gene for retinol-binding protein (RBP) and/or transthyretin (TTR) could be induced upon differentiation of F9 teratocarcinoma cells to either visceral endoderm or parietal endoderm. Both TTR mRNA and RBP mRNA were undetectable in the undifferentiated F9 stem cells and in F9 cells differentiated to parietal endoderm. However, TTR mRNA and RBP mRNA were both detected in F9 cell aggregates differentiated to embryoid bodies (which contain visceral endoderm-like cells) by treatment of the aggregates in suspension with retinoic acid. TTR mRNA was observed at 3 days, and RBP mRNA at 5 days, after treatment of the F9 cell aggregates with retinoic acid. Both TTR mRNA and RBP mRNA were found to be specifically localized by in situ hybridization in the outer layer of cells (the visceral endoderm-like cells) of the embryoid bodies. Finally, synthesis and secretion of both RBP and TTR by F9 cell embryoid bodies was demonstrated by specific immunoprecipitation of each newly synthesized protein from the culture medium. These data thus demonstrate the production and presence of RBP mRNA and TTR mRNA, and the synthesis and secretion of RBP and TTR, by F9 cell embryoid bodies (specifically by visceral endoderm-like cells). This finding suggests that these two proteins may be synthesized by rodent embryos extremely early in embryonic development.     

Localization of phosphatase activities on mouse teratocarcinoma embryoid bodies

Non-specific alkaline phosphatase and Mg²⁺-dependent adenosine triphosphatase activities were ultracytochemically investigated on embryoid bodies of murine teratocarcinomas, in order to find markers of endodermal cell differentiation of early embryonic cells. The former was localized mainly on the cell surface of inner embryonal carcinoma cells, as already shown by other workers, and weakly on the bound surface of outer endodermal cells of embryoid bodies. The latter, however, was found only on the outer free surface of endodermal cells and never on the surface of embryonal carcinoma cells. It suggests that Mg²⁺-dependent ATP activity might become the marker for early differentiation of embryonal carcinoma cells.     

Ras/MAPK pathway confers basement membrane dependence upon endoderm differentiation of embryonic carcinoma cells

The formation of extraembryonic endoderm is one of the earliest steps in the differentiation of pluripotent cells of the inner cell mass during the early stages of embryonic development. The primitive endoderm cells and the derived parietal and visceral endoderm cells gain the capacity to produce collagen IV and laminin. The deposition of these components results in the formation of basement membrane and epithelium of the endoderm, with polarized cells covering the inner surface of the blastocoels. We used retinoic acid-induced endoderm differentiation of stem cell-like F9 embryonic carcinoma cells to study the role of the Ras pathway and its regulation in the formation of the visceral endoderm. Upon endoderm differentiation of F9 cells induced by retinoic acid, c-Fos expression, the downstream target of the Ras pathway, is suppressed by uncoupling Elk-1 phosphorylation/activation to MAPK activity. However, attachment to matrix gel greatly enhances the activation of MAPK in endoderm cells but not in undifferentiated F9 cells. Enhanced MAPK activation as a result of contact with basement membrane is able to compensate for reduced Elk-1 phosphorylation and c-Fos expression. We conclude that endoderm differentiation renders the activation of the Ras pathway basement membrane dependent, contributing to the epithelial organization of the visceral endoderm.     

High molecular weight extracellular proteins synthesized by endoderm cells derived from mouse teratocarcinoma cells and normal extraembryonic membranes

Rabbit antiserum raised against teratocarcinoma embryoid bodies reacts with two extracellular, collagenase-resistant glycoproteins, PYS A and B, with molecular weights of approximately 350,000 and 220,000 daltons. The 220,000-dalton protein is distinguishable from fibronectin. The two proteins are synthesized and secreted into the medium in large amounts by the teratocarcinoma-derived parietal endoderm line PYS-1, and by normal parietal endoderm cells from the 10.5-day embryo. There was no detectable synthesis of PYS A and B by normal visceral endoderm cells isolated from the 10.5-day embryo, and only trace amounts of PYS A were synthesized by the teratocarcinoma-derived visceral endoderm line PSA5E and by mesodermal cells isolated from the visceral yolk sac. The two proteins therefore seem to be good biochemical markers for distinguishing parietal from visceral endoderm cells. Synthesis and secretion of PYS A and B could not be detected in undifferentiated embryonal carcinoma cells or in endoderm cells derived from them in the presence of retinoic acid.