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.     

Expression of c-src and c-abl in embryonal carcinoma cells and adult mouse tissues

We have studied the expression of c-src and c-abl proto-oncogenes in early mouse development using embryonal carcinoma (EC) cells as a model system, and compared this to the expression pattern in adult tissues. In all three EC lines tested (F9, PC13, and P19), c-src and c-abl mRNA can be detected. When F9 and PC13 are induced to differentiate they form endodermal cells characteristic of the early embryo, and we found no change in c-src or c-abl expression. In contrast, P19 cells showed increased levels of both mRNAs both mRNAs when induced to differentiate along the neural pathway by retinoic acid, whereas differentiation along the muscle pathway by dimethyl sulfoxide resulted in decreased levels of c-abl expression. These results are consistent with the idea that c-src and c-abl have important functions in the differentiation of the cell types of the later embryo, but not in those of the early embryo.     

The location and expression of fibroblast growth factor (FGF) in F9 visceral and parietal embryonic cells after retinoic acid-induced differentiation

It is well-established that fibroblast growth factors (FGFs) participate in mesoderm formation and patterning in the developing embryo. To identify cells in mammalian embryos that produce and/or respond to FGFs, we utilized the F9 teratocarcinoma cell system. Undifferentiated F9 cells resemble inner cell mass (ICM) cells of the mouse blastocyst by several criteria including having a characteristic high nuclear to cytoplasmic ratio and by their expression of stage-specific embryonic antigens. F9 stem cells differ from ICM cells by their low spontaneous rate of differentiation and their differentiation potential. ICM cells are heterogeneous with a proportion of the cells maintaining totipotency. In contrast, F9 stem cells appear capable of forming only endodermal derivatives. Retinoic acid (RA) treatment of F9 stem cells is required for them to differentiate, and under different culturing conditions the F9 cells will form either extraembryonic parietal or visceral endoderm. We have previously shown that FGF is synthesized by F9 parietal endoderm, but not by F9 stem cells. Our present study demonstrates that F9 aggregate cultures that contain visceral endoderm cells produce cell-associated-heparin-binding mitogens for 3T3 and endothelial cells, factors with characteristics of FGFs. Furthermore, our studies detect endothelial cell-mitogens within the extracellular matrix (ECM) of F9 parietal endoderm cells, not detected within F9 stem cell 'matrices'. Parietal endoderm cell matrix mitogens could be removed by prior treatment of the ECM with buffers containing heparin or 2 M NaCl, and could be neutralized by basic FGF antibodies.     

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.     

Human teratocarcinoma stem cells: glycolipid antigen expression and modulation during differentiation

Teratocarcinomas are germ cell tumors in which pluripotent stem cells, embryonal carcinoma (EC) cells, undergo differentiation along the pathways resembling those occurring during early embryogenesis. Human EC cell lines established in vitro provide a model for studying embryonic cellular differentiation in a way that is pertinent to early human development. The predominant glycolipid antigens expressed by EC cells of both humans and mice have globoseries core structures; in humans they are terminally modified to yield the monoclonal antibody-defined stage-specific embryonic antigens SSEA-3 and SSEA-4, and also globo-ABH antigens; in the mouse terminal modification yields the Forssman antigen rather than SSEA-3 and -4. These observations focus attention on the possible role of the P-blood group system, which regulates synthesis of globoseries oligosaccharides, in the behavior of cells in the early embryo and in teratocarcinomas. Marked changes in the core structures of the cell surface glycolipids occur as the EC cells differentiate; thus globoseries structures rapidly diminish and are replaced by lactoseries and then by ganglioseries glycolipids. During differentiation of the NTERA-2 line of pluripotent human EC cells into neurons and other cell types, the various subsets of differentiated cells that arise are distinguished by their differential expression of new glycolipid antigens, particularly ganglioside GT3 (recognized by antibody A2B5), and ganglioside 9-0-acetyl GD3 (recognized by antibody ME311). Neurons are found among the A2B5+/ME311- cells.     

The amount of a specific cellular protein (p53) is a correlate of differentiation in embryonal carcinoma cells

A specific cellular protein of molecular weight of 53–55,000 (p53) has been shown to be induced in all SV40 transformed cells. A similar protein has also been shown to be present in embryonal carcinoma cells and in midgestation murine embryo primary cells, which are not infected by SV40. In embryo cell primaries the amount of the protein was shown to decrease with the increase in the stage of embryo development. As differentiation or decrease in cell growth rate can account for this, and since the growth rate of embryo primary cells cannot be measured, we chose to investigate various embryonal carcinoma cells. We report that the p53 is present in a pluripotent embryonal carcinoma cell OTT6050, and in its differentiated parietal endoderm derivative, PYS-2 cells. The amount of p53 is higher in the undifferentiated EC stem cells than in the differentiated PYS-2 (parietal endoderm) cells. The amount of the protein decreases in F9 embryonal carcinoma cells induced to differentiate to a parietal endoderm cell type by treatment with retinoic acid, as it does following spontaneous differentiation of OTT6050 EC cells. To determine if a change in growth rate, rather than differentiation, might acount for the diminished levels of this protein, the amount ofp53 was measured in growing and in growth arrested cell populations. When the growth rate of F9 cells was reduced by treatment with 8-bromocyclic AMP there was no change in the amount of p53. The half life of the p53 was compared in the undifferentiated and the differentiated cell types to determine if a change in stability might account, in part, for the altered levels of this protein. The p53 is found to be most stable in the SV40 transformed established clonal cells. It is less stable in the fibroblast clonal cells which were not transformed by SV40. The results of these experiments indicate that a decrease in the amount of p53 primarily correlates with differentiation in the embryonal carcinoma cell lines studied and not with cell growth rate. Furthermore, the decrease appears to be related (in part) to the decreased stability of the p53.     

A fucosyltransferase in teratocarcinoma stem cells. Decreased activity accompanying differentiation to parietal endoderm cells

Teratocarcinoma stem cell F9 expressed a potent fucosyltransferase activity acting on asialofetuin. A majority of the product was susceptible to alpha-L-fucosidase I from almond emulsin, indicating that the linkage formed was mainly Fuc alpha 1 leads to 3GlcNAc. The specific activity of the transferase decreased when the stem cells were induced to differentiate into parietal endoderm cells by retinoic acid and dibutyryl cyclic AMP. Furthermore, PYS-2 cell, a parietal endoderm cell line virtually lacked the transferase. The change in the fucosyltransferase activity could be correlated with cell surface changes occurring during differentiation.     

Embryonal carcinoma cells adhere preferentially to fibronectin and laminin but their endodermal differentiation leads to a reduced adherence to laminin

F9 and PC13 embryonal carcinoma (EC) cells adhered rapidly to growth substrata coated with fibronectin or laminin. When F9 cells were induced to differentiate into visceral or parietal endoderm-like cells, their ability to adhere to laminin diminished, but their adherence to fibronectin remained unchanged. Correspondingly, permanently differentiated teratocarcinoma-derived endoderm cells (PYS-2 and PSA-5e) adhered markedly less efficiently to laminin than to fibronectin. F9 cells adhered to proteolytic fibronectin fragments containing the cell-binding site but not to fragments containing gelatin- or heparin-binding sites. They also adhered slowly to gelatin, but this adhesion was completely blocked by cycloheximide. The results show that the teratocarcinoma stem cells may have specific mechanisms mediating adhesion to fibronectin and laminin and that endodermal differentiation leads to a reduction in their capacity to adhere to laminin but not to fibronectin.     

High expression of stathmin in multipotential teratocarcinoma and normal embryonic cells versus their early differentiated derivatives

Stathmin is a ubiquitous cytoplasmic protein, phosphorylated in response to agents regulating the proliferation, the differentiation and the specialized functions of cells, in a way possibly integrating the actions of diverse concomitant regulatory signals. Its expression is also regulated in relation with cell proliferation and differentiation and reaches a peak at the neonatal stage. To assess the possible role of stathmin at earlier stages of development, we examined its expression and regulation in embryonal carcinoma (EC) and derived cell lines as well as in the early mouse embryo. Interestingly, stathmin is highly abundant in the undifferentiated, multipotential cells of the F9, 1003 and 1009 EC cell lines. Its high expression markedly decreased, both at the protein and mRNA levels, when F9 cells were induced to differentiate into endodermal-like cells with retinoic acid and dibutyryl-cAMP. Stathmin was also much less abundant in differentiated cell lines such as the trophectodermal line TDM-1, as well as in several F9- and 1003-derived cell lines committed to differentiate towards the mesodermal and neuroectodermal lineages but still proliferating. Therefore, the observed decrease of stathmin expression is not related to the reduced proliferation rate but rather to the differentiation of the multipotential EC cells. The immunocytochemical pattern of stathmin expression during early mouse development indicated that stathmin is also highly abundant in the multipotential cells of the inner cell mass of the blastula, whereas it is much lower in the differentiated trophectodermal cells. These results confirm the physiological relevance of the observations with EC cells, and suggest that stathmin, in addition to its high expression at later stages of development and in the adult nervous system, may be considered as a new marker of the multipotential cells of the early mouse embryo.     

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.     

Analysis of the differentiation-promoting potential of inducible c-fos genes introduced into embryonal carcinoma cells.

To investigate the differentiation-promoting potential of c-fos in embryonal carcinoma cells (EC cells) we have designed various human metallothionein promoter-mouse-c-fos gene constructs containing also the selectable SV40 promoter-driven neo gene. Upon transfection into F9 EC cells and selection for neo resistance, the following results were obtained. (i) With each of the constructs, colonies of morphologically altered and differentiated (i.e., TROMA-1 and TROMA-3 expressing) cells were identified. (ii) Expression of c-fos was required to affect the differentiation state of F9 cells to a significant extent, but a low level was sufficient; no enhancement of differentiation was noticeable even after 100-fold induction of c-fos expression by cadmium. (iii) F9 cell clones were isolated which, in spite of very high levels of exogenous c-fos expression, had stem cell morphology. These cells, however, continuously generated morphologically altered and differentiated cells upon subculturing. (iv) In other EC cell lines, which resemble stem cells more closely than the 'partially differentiated' F9 cells, c-fos expression showed either a less pronounced (P19 cells) or no differentiation-promoting effect at all (PC13 cells). Our results suggest that the c-fos gene product acts in concert with other, probably 'spontaneously' occurring events to promote differentiation of certain EC cell lines.     

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

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

Generation of insulin-expressing cells from mouse embryonic stem cells

The therapeutic potential of transplantation of insulin-secreting pancreatic beta-cells has stimulated interest in using pluripotent embryonic stem (ES) cells as a starting material from which to generate insulin secreting cells in vitro. Mature beta-cells are endodermal in origin so most reported differentiation protocols rely on the identification of endoderm-specific markers. However, endoderm development is an early event in embryogenesis that produces cells destined for the gut and associated organs in the embryo, and for the development of extra-embryonic structures such as the yolk sac. We have demonstrated that mouse ES cells readily differentiate into extra-embryonic endoderm in vitro, and that these cell populations express the insulin gene and other functional elements associated with beta-cells. We suggest that the insulin-expressing cells generated in this and other studies are not authentic pancreatic beta-cells, but may be of extra-embryonic endodermal origin.     

Inhibition of DNA synthesis causes stem cell differentiation: induction of teratocarcinoma F9 cell differentiation with nucleoside analogues of DNA-synthesis inhibitors and their inducing abilities counterbalanced specifically by normal nucleosides

Nucleoside analogues inhibiting DNA synthesis can induce cell differentiation in teratocarcinoma cells. We have examined how their abilities to induce F9 cell differentiation were specifically counterbalanced by their corresponding normal nucleosides. We have also compared the differentiation inducing ability of the wild type F9 cells with that of its thymidine kinase-less mutant using plasminogen activator, as a differentiation marker, which is expressed at a very early stage of endodermal cell differentiation and can be assayed quantitatively. The results obtained were clearly explainable by the conventionally accepted action mechanisms of the nucleoside analogues, thus strongly suggesting that their abilities to induce cell differentiation were direct consequences of the inhibition of DNA synthesis; thus this confirms the notion that a close association exists between the inhibition of DNA synthesis and the induction of teratocarcinoma stem cell differentiation.     

Human teratocarcinomas

Teratocarcinomas are one of the commonest forms of cancer in young adult men. Cell lines derived from these tumors, and particularly the cell lines composed of their embryonal carcinoma (EC) stem cells, may provide useful information concerning the development and subsequent pathology of teratocarcinomas in humans. In addition, it is likely that human EC cells resemble early embryonic cells and can be used as an in vitro counterpart of such cells from the human embryo. Several common properties of human EC cells have been identified, and a human EC cell line, TERA-2, that is capable of extensive somatic differentiation has been cloned. In nude mice, TERA-2 EC cells form tumors containing neural elements and glandular structures that resemble primitive gut. In culture, these EC cells can be induced to differentiate by exposure to retinoic acid and hexamethylenebisacetamide (HMBA). Differentiation is marked by the disappearance of several cell surface antigens characteristic of human EC cells, and the appearance of other antigens on the various subsets of differentiated derivatives. In retinoic acid-induced cultures, these differentiated derivatives include neurons and cells permissive for the replication of cytomegalovirus, a virus that can cause birth defects in humans. On the other hand, HMBA appears to activate an alternative pathway of differentiation for TERA-2 EC cells, although the identity of the resulting cells remains to be elucidated. In addition to providing a tool for analyzing the evolution of teratocarcinomas in human patients, the TERA-2 EC cells may provide us with insights into the mechanisms of cellular differentiation in the human embryo and a model in which to investigate how teratogenic agents such as HCMV can disrupt these processes.     

Effects of pulsed electromagnetic field on growth and differentiation of embryonal carcinoma cells

A murine embryonal carcinoma cell line (F9) was used to examine the effect of a pulsed electromagnetic field on the growth and differentiation of malignant cells. The cells can be induced to differentiate into parietal endodermal cells by treatment with retinoic acid. The pulsed electromagnetic field (1 Gauss and 10 Gauss) promoted the growth of embryonal carcinoma cells in both the presence and absence of retinoic acid. The pulsed electromagnetic field was also found to inhibit retinoic acid-induced differentiation, when the degree of differentiation was based on morphological criteria or on the production of plasminogen activator.     

An adhesion-defective variant of F9 embryonal carcinoma cells fails to differentiate into visceral endoderm

Adhesion-defective EC cells were isolated from a population of mutagenized F9 cells by serial transfer of cells that did not adhere to gelatin-coated dishes. The variant cells grew in suspension as multicellular clusters of loosely aggregated cells. The cells adhered to, but did not flatten on, fibroblast monolayers and extracellular matrix produced by parietal-like endoderm. Two different mutant cell lines exhibited increased sensitivity to the lectin abrin and decreased sensitivity to wheat germ agglutinin, suggesting that changes in cell surface glycosylation are associated with the mutant phenotype. These adhesion-defective mutants were used to study the relationship between cell-cell adhesion and endodermal differentiation. Unlike wild-type cells, when cultured with low concentrations of retinoic acid (RA) in suspension culture, the mutant cells did not form embryoid bodies but remained as loosely adhering strings of cells. Electron microscopic examination revealed that most of the differentiated variant cells resembled parietal endoderm, and this was confirmed by immunofluorescent staining for TROMA-3 marker. The levels of some of the markers that characterize the differentiative pathways were examined by immunoprecipitation and by enzyme-linked immunosorbent assay (ELISA). The variant line produced higher levels of laminin and type IV collagen compared to the wild-type cells. alpha-Fetoprotein (AFP) was produced at a significantly lower level by the variant compared to wild-type F9 cells during the differentiative process. The results show that variant cells differentiated toward parietal endoderm but have a very much restricted ability to differentiate to visceral endoderm. We conclude that aggregation and/or compaction provide some essential signals during the differentiation of F9 cells into epithelial layers of visceral endoderm.     

A strategy for immortalizing lines committed to endoderm, neuroectoderm or mesoderm from mouse teratocarcinoma

With the aim of immortalizing embryonic cells fixed at early embryonic stages, various plasmids carrying the SV40 early region were introduced into the mouse embryonal carcinomas (EC) F9 and 1003. Only the construction PK4, in which the SV40 oncogenes are placed under the control of the adenovirus E1A promoter, led to the immortalization of the cells at the onset of differentiation. Clones corresponding to committed precursors of each embryonic lineage (neuroectoderm, mesoderm and endoderm) were then selected with high efficiency according to the following strategy: selection of immature cells which: have lost EC cell markers, keep a stable phenotype, are immortalized by the expression of the SV40 oncogenes and are still able to differentiate along a restricted lineage in vitro or in vivo. Examples of an endodermal precursor (H7) which differentiates into extraembryonic and embryonic endoderm, of a neuroectodermic clone (ICII) committed to a serotoninergic differentiation, and of a mesodermal osteogenic clone (CI) which gives rise to bone in vivo and in vitro, are given.