Alterations in the developmental potential of embryonal carcinoma cells in mixed aggregates of nullipotent and pluripotent cells.

Pluripotent embryonal carcinoma cells can be triggered to differentiate in vitro by allowing them to form multicellular aggregates. Nullipotent embryonal carcinoma cells form aggregates, but further development is blocked. Pluripotent and nullipotent embryonal carcinoma cell lines were co-cultured to form mixed aggregates in order to determine whether a developmental signal produced by the pluripotent cell could induce the nullipotent cells to differentiate. Unlike pure pluripotent cell aggregates, aggregates from cultures initiated with a 1:1 mixture of pluripotent (PSA-1) and nullipotent (F9) cells formed endoderm but failed to differentiate further. The nullipotent cells did not produce a detectable soluble inhibitor of differentiation. A hypoxanthine phosphoribosyltransferase-deficient subclone of the nullipotent cell line was used so that the fate of both nullipotent and pluripotent cells could be followed in autoradiographs of histological sections of aggregates labeled with ³H-hypoxanthine. Seven day old aggregates of pure pluripotent cell cultures contained endoderm, ectoderm and embryonal carcinoma cells. On the other hand, in 7 day old mixed cell aggregates, almost all the pluripotent cells became endoderm located on the outer surface of the aggregate. The nullipotent cells in the mixed aggregates assumed an internal position and remained embryonal carcinoma cells. Following the efficiency of plating of pluripotential cells in pure and mixed aggregates as a function of time showed that viable pluripotent embryonal carcinoma cells were lost at a 10 fold greater rate in mixed cell aggregates than in pure pluripotent cell aggregates. We conclude that nullipotent embryonal carcinoma cells in mixed aggregates with pluripotent cells exert a limitation on the ability of these pluripotent cells to differentiate.     

The small heat shock protein hsp25 is accumulated in P19 embryonal carcinoma cells and embryonic stem cells of line BLC6 during differentiation

Murine embryonal carcinoma and embryonic stem cell lines were investigated with regard to the occurrence of the small heat shock protein hsp25 during cell growth and differentiation. In the embryonal carcinoma cell line F9 considerable constitutive levels of hsp25 were observed which could be slightly increased by treatment with retinoic acid. No hsp25 was found, however, in the embryonal carcinoma cell line PCC4. When analyzing the pluripotent embryonal carcinoma cell line P19 and the pluripotent embryonic stem cell line BLC6, both characterized by high differentiation capacity, no hsp25 was observed under cell culture conditions maintaining the undifferentiated state. Induction of differentiation caused by prolonged cell culture, retinoic acid treatment, or embryoid body formation, however, resulted in an increase of the level of hsp25. The finding that hsp25 is accumulated in a differentiation-dependent manner suggests that this protein is associated with processes involved in differentiation. Therefore, hsp25 can be regarded as a marker of differentiation in the investigated embryonal carcinoma cell line P19 and the embryonic stem cell line BLC6.     

Stimulation of the Clonal Growth and Differentiation of Feeder Layer Dependent Mouse Embryonal Carcinoma Cells by β-Mercaptoethanol

Embryonal carcinoma cells are the undetermined stem cells of teratocarcinomas. Supplementation of culture medium with β-mercaptoethanol permits the feeder layer independent clonal growth and differentiation of normally feeder layer dependent embryonal carcinoma cell lines. Differentiated cells within the clones appeared less than 6 days after plating and were distinguished from embryonal carcinoma cells by their morphology, lack of histochemically detectable alkaline phosphatase activity, and secretion of plasminogen activator. Over 70% of the colonies secreted plasminogen activator after 6 days.
In comparison, a different embryonal carcinoma cell line which has lost the potential for substantial differentiation, either in vitro or in vivo forms very few clones (< 1%) which secrete plasminogen activator. Embryonal carcinoma cells derived from the rare clones which secrete plasminogen activator have the same frequency of production of plasminogen activator secreting colonies as the parental cell line.     

Calcium-induced compaction and its inhibition in embryonal carcinoma cell aggregates

H6 embryonal carcinoma cells form aggregates of cells in culture medium which contains 2 mM calcium. These aggregates are described as uncompacted, indicating that the individual cells of the aggregate are spherical and are in limited contact with each other. In contrast, compaction of the aggregate, induced by increasing the calcium concentration, results in a tight mass of cells flattened against one another and connected by intercellular junctions. At least 85-97% of the aggregates undergo compaction in 7 mM calcium and are subsequently decompacted if removed to 2 mM calcium. Since calcium ionophore A23187 does not induce compaction, extracellular rather than intracellular calcium seems to be the limiting factor. We have demonstrated that this calcium-induced morphogenetic change is sensitive to inhibition by agents which also prevent the calcium-dependent compaction of the 8-cell mouse embryo. The cytoskeletal-binding drugs tetracaine HCl, colcemid, vinblastine, colchicine, and cytochalasin B each inhibit compaction of H6 aggregates. Interference at surface molecule sites by exposure to the lectins wheat germ agglutinin or concanavalin A or by interruption of glycosylation with exposure to tunicamycin, or by reaction with anti-H6 Fab or anti-F9, also prevent compaction. Since the mouse embryo and embryonal carcinoma cells share certain processes which are involved in initiating and maintaining compaction, these processes and their subsequent roles in differentiation may be examined using embryonal carcinoma cell aggregates.     

Metabolic cooperation in aggregates of embryonal carcinoma cells

Metabolic cooperation may be associated with the processes of compaction and subsequent differentiation in aggregates of embryonal carcinoma cells (ECC). To determine if the gap junctions present in loose and compacted aggregates of H6 ECC are active in metabolic cooperation, aggregates of each type containing a mixture of 5-bromodeoxyuridine- and 6-thioguanine-resistant H6 cells were exposed to HAT medium, 6-thioguanine, or [³H]thymidine. These three methods indicated that some crossfeeding occurred through the small clusters of gap junctions in loose aggregates and more crossfeeding occurred through the larger clusters of gap junctions in compacted aggregates.     

Junction formation in aggregated embryonal carcinoma cells

Under the action of supplemental calcium, H6 mouse embryonal carcinoma cell aggregates undergo compaction, a morphological phenomenon similar to mouse embryonic compaction. Formation of various types of cell junctions, especially gap junctions, is associated with compaction of the embryo and we sought to analyze the pattern of junction formation during aggregation and compaction of H6 cells. At 24 hr of aggregation, gap junctions were abundant in both uncompacted and compacted aggregates but quantitative analysis of freeze fracture replicas of these junctions showed a 20-fold increase in the size of the largest gap junctions in compacted aggregates. Such a difference in size could even be detected at 12 hr of aggregation. Tight junctions were not normally formed in 12 hr aggregates but initial stages of tight junction formation could be noticed in 12 hr compacted aggregates. More definitive tight junctions and desmosomes were evident only after 48 hr of aggregation. Thus we have observed that both uncompacted and compacted aggregates can form gap junctions at similar frequencies, suggesting that cell flattening, which contributes to the compacted morphology, is not a requisite for gap junctions. Likewise, generation of the compacted morphology seems to be independent of gap junction formation. This supports the idea that compaction in embryonal carcinoma cells results from calcium-induced cell flattening, probably through the mobilization of cytoskeletal elements. Calcium-dependent features of H6 cell aggregation and compaction enables the independent analysis of separate steps in compaction.     

Genetic activity of X chromosomes in pluripotent female teratocarcinoma cells and their differentiated progeny

We have induced teratocarcinomas from female embryos heterozygous for electrophoretic variants of the X-linked gene coding for phosphoglycerate kinase (PGK). An embryonal carcinoma cell line, P10, has been isolated from such a teratocarcinoma. It has a normal female karyotype and cultures contain both PGK isoenzymic forms. Clonal populations derived from P10 also contain both PGK electrophoretic variants. In addition, both X chromosomes in these cells replicate in synchrony with the autosomes during early S phase of the cell cycle. These data indicate that the undifferentiated P10 embryonal carcinoma cells contain two active X chromosomes. When cultured under the appropriate conditions, the P10 cells differentiate to form a variety of tissue types. At least some of these differentiated cells contain an inactive X chromosome as determined by cytogenetic analysis. Apparently X chromosome inactivation accompanies the differentiation of these female embryonal carcinoma cells.     

Evidence for a dynamic role of the linker histone variant H1x during retinoic acid-induced differentiation of NT2 cells

The dynamics of chromatin structure are tightly regulated by multiple epigenetic mechanisms such as histone modifications and incorporation of histone variants. In the current work, differentiation of an embryonal carcinoma cell line, NT2, was induced by retinoic acid, and total histone proteins were compared throughout this process. The results showed a significant change in expression level of a variant of H1 histone named H1x. Chromatin immunoprecipitation coupled with real-time PCR analysis demonstrated a preferential incorporation of this protein in the regulatory region of Nanog, a marker gene of stemness that is significantly suppressed in differentiated cells. This finding reveals a dynamic role of H1x in differentiation, and implies a repressive role for this histone variant.     

Aggregation-deficient embryonal carcinoma cells: defects in peanut agglutinin (PNA) receptors

The components involved in cell adhesion were studied using the H6 line of embryonal carcinoma cells. H6 cells are especially suitable for studies on cell interactions, since genetic mutants can be selected, and various processes of cell adhesion can be controlled by regulating the calcium concentration in the medium. Three aggregation-defective variants of H6 were isolated, all of which showed reduced binding of the lectin, peanut agglutinin (PNA). Quantitation of PNA receptors on the cell surface by immunoprecipitation of iodinated surface proteins indicated that these receptors were reduced on the variants by one-half to one-quarter. The separation of immunoprecipitated PNA receptors on sodium-dodecyl-sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated that one type of receptor, with an apparent molecular weight of 94 kilodaltons, was reduced. Parental and variant cells bind similar quantities of concanavalin A and soybean agglutinin, suggesting that there is no generalized effect on major glycoproteins. Thus, the defect in aggregation and the defect in the 94-kilodalton protein may be correlated, and this glycoprotein may have a role in the mediation of H6 cell-cell adhesion.     

Analysis of F9 embryonal carcinoma cell lactosaminoglycans in relation to their differential expression during induction of differentiation

Teratocarcinoma stem cells can be used to study the events related to early differentiation, and many cell surface changes have been described which correlate with the different stages of early embryogenesis. In this work we analyze the [3H]galactose-labeled glycopeptides derived from the mouse embryonal carcinoma cell line F9. We show that the high-molecular-weight glycopeptides typical of embryonal carcinoma cells are composed of two distinct molecular weight classes, namely H1 and H3, and that retinoic acid-induced differentiation determines a relative increase of the larger peak (H1) which is mainly due to a decrease in the expression of H3 species. We also show that, beside this decrease, there is a greater increase in the expression of lower-molecular-weight species. Furthermore, we present evidence that H1 and H3 species are polylactosaminoglycans N-linked to the peptidic backbone, and that induction of differentiation determines slight modifications in the structure of such species.     

The role of aggregation in embryonal carcinoma cell differentiation

Cultures of the P19 line of embryonal carcinoma cells differentiate into various cell types including cardiac muscle when aggregated and exposed to medium containing 1% dimethylsulfoxide (DMSO). DMSO-treated aggregates became completely covered with an epithelial cell type 3 to 4 days following drug exposure. This epithelial cell was tentatively identified as primitive extraembryonic endoderm by its ultrastructural appearance and its possession of cytokeratin intermediate filaments. Muscle cells developed within the interior of DMSO-treated aggregates. They first became apparent 5 to 6 days after DMSO exposure and were characterized by the presence of striated muscle-specific myosin, immature myofibrils, and intercalated discs. We determined the proportion of cells developing into epithelium and muscle in aggregates of various sizes and showed that the proportion of epithelium was highest in small aggregates whereas muscle cells developed only in aggregates of relatively large size. The muscle was usually associated with necrotic areas which developed within the interior of large aggregates. Our results suggest that cardiac muscle differentiation in the aggregates requires both the DMSO-induced formation of an epithelial cell coat and one other condition which may be the proximity to necrotic areas.     

Derivation of a nondifferentiating clone from multipotential PSA1 embryonal carcinoma cells

The ability of PSA1 embryonal carcinoma cells to differentiate when grown as clones on a monolayer of feeder cells was assessed using morphological criteria. The first appearance of a differentiated phenotype within a clone occurred at different times for individual clones after 10 days of culture, this being apparently unrelated to clone size or cell density. Those clones which showed no morphological evidence of differentiation after several weeks (about 5% of the clones observed) were selected and recloned with the aim of finding variant lines which were stably deficient in their differentiating ability. Undifferentiated clones - identified and selected after about 3 weeks of growth - were of three different types after recloning: those similar to control cultures of PSA1, those having delayed and reduced differentiation frequency, and those having variable frequencies of differentiation in replicate reclonings. The isolation of a variant with a more complete differentiation deficiency was accomplished by selecting ten nondifferentiating clones growing isolated in individual culture wells after 5 weeks of culture. One of these, T2H9, proved to be a stable, differentiation-deficient variant subline with less than 3% of its clones showing any morphological evidence of differentiation in five repeated reclonings. It was also determined that the frequency of undifferentiated clones in embryonal carcinoma cultures increased from 0.3% to 54% after 11 months of in vitro aging, i.e., approximately 200 cell doublings. The isolation of clonal embryonal carcinoma cell derivatives which are stable, heritable differentiation variants provides resources for somatic-cell genetic analysis of stem-cell pluripotency.     

Assessment of pluripotency and multilineage differentiation potential of NTERA-2 cells as a model for studying human embryonic stem cells

Embryonal carcinoma cells are pluripotent stem cells derived from teratocarcinomas and are considered to be the malignant counterparts of human embryonic stem cells. As there are few reliable experimental systems available to study the molecular mechanisms governing normal embryogenesis, well-characterized human embryonal carcinoma stem cell lines may provide a robust and simple model to study certain aspects of pluripotency and cellular differentiation. Here, we have analysed NTERA-2 cL.D1 cells at molecular and cellular levels during expansion and differentiation, via formation of cell aggregates similar to embryoid bodies in embryonic stem cells. Thus, human embryonal carcinoma cells may provide a valuable insight into cell fate determination, into the embryonic ectoderm, mesoderm and endoderm and their downstream derivatives.     

Gene expression in visceral endoderm: a comparison of mutant and wild- type F9 embryonal carcinoma cell differentiation

We have examined the abundance and cell specificity of several mRNAs that are regulated during the retinoic acid (RA)-induced differentiation of F9 embryonal carcinoma cells to visceral endoderm. The experiments confirmed the multistep nature of this process by demonstrating the expression of the ERA-1/Hox 1.6 message within 6 h after RA addition; the expression of messages specific for the extracellular matrix proteins laminin B1 and B2, and collagen IV(alpha 1) between days 4 and 12; and the expression of two visceral endoderm markers, alpha-fetoprotein (AFP) and H19, by days 8-15. In situ hybridization experiments revealed that the collagen IV(alpha 1) mRNA is restricted to the outer cell layer of F9 cell aggregates regardless of the presence or absence of RA. Laminin B1 and B2 mRNAs are concentrated in the outer cell layer of RA-treated aggregates although significant levels of message are also observed within the interior cells of the aggregates. Unexpectedly, AFP mRNA is detectable in only a subset of the outer cells of F9 cell aggregates grown 15 d in the presence of RA. The results obtained from wild-type F9 cells were compared with those from a mutant F9 cell line, RA-5-1, which was previously shown to synthesize collagen IV containing six- to ninefold less 4-hydroxyproline than that in wild-type F9 cells. RA-5-1 cells exhibit four- to sixfold less of the mRNAs encoding two visceral endoderm proteins, AFP and H19, than wild-type F9 cells after RA treatment of RA-5-1 aggregates. RA-5-1 cells, however, do exhibit an RA-associated increase in the level of ERA-1/Hox 1.6 mRNA within 6 h after adding RA. Although the collagen IV protein level is similar in wild-type F9 and RA-5-1 aggregates, the collagen IV(alpha 1) message level is 6-20-fold greater in aggregates of mutant cells than in aggregates of wild-type cells. Moreover, in situ hybridizations showed that this message is evenly distributed throughout the RA-5-1 aggregates rather than restricted to the outer cell layers as it is in wild-type F9 aggregates. These results suggest that abnormal collagen IV expression and localization are associated with decreased expression of the visceral endoderm markers, AFP and H19, in RA-5-1 cell aggregates.     

The response of several murine embryonal carcinoma cell lines to stimulation of differentiation by alpha-difluoromethylornithine

In an attempt to better establish the relationship between polyamine levels and the differentiation of embryonal carcinoma cells, we have examined the ability of alpha-difluoromethylornithine (DFMO), a known inducer of differentiation in one embryonal carcinoma cell line, to stimulate the differentiation of embryonal carcinoma cells from a variety of cell lines. Differentiation was monitored using a variety of criteria including morphological alterations and changes in biochemical and antigenic parameters. Depending on their response to difluoromethylornithine, three classes of cell lines could be identified, those which 1) differentiate extensively, 2) differentiate poorly, and 3) fail to differentiate. Three different classes of embryonal carcinoma cell lines reflect differential changes in polyamine levels resulting from inhibition of ornithine decarboxylase enzyme activity by DFMO. The specific cell lines which exhibit large decreases in both ornithine decarboxylase activity and polyamine levels also show extensive differentiation. The cell lines which show only moderate decreases in enzyme activity and polyamines differentiate poorly while the cell lines which fail to respond to DFMO in that polyamines do not drop below the threshold level necessary to induce differentiation fail to differentiate. These studies suggest that decreases in intracellular polyamines induce EC cell differentiation in vitro.     

Visceral-endoderm-like cell lines induce differentiation of murine P19 embryonal carcinoma cells

When P19 embryonal carcinoma (EC) cells were cocultured with cells from one of several established visceral-endoderm-like cell lines, the EC cells were rapidly induced to aggregate and differentiate, into cell types including mesoderm-derived cardiac and skeletal muscle. Neither parietal-endoderm- nor mesoderm-like cell lines induced aggregation or differentiation of EC cells in coculture, although a cell line with both parietal and visceral endoderm characteristics induced aggregation but not differentiation. Also, without the feeder cells aggregates of P19 failed to differentiate, provided that serum in the culture medium had been previously passed over dextran-coated charcoal to remove lipophilic substances, which may include endogenous retinoids. All experiments were carried out using serum treated in this way. Taken together, the results demonstrated that aggregation was necessary, but not sufficient, to make P19 EC cells differentiate. Direct contact between the two cell types was not necessary, since even when separated by an agar layer in cocultures, aggregates of P19 still differentiated. Medium conditioned by cells of the END-2 line, a visceral-endoderm-like derivative of P19, was particularly potent in inducing endodermal and mesodermal differentiation of single P19 aggregates, confirming the involvement of a diffusible factor secreted specifically by visceral-endoderm-like cells in this process.     

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.     

Biochemical markers of the progress of differentiation in cloned teratocarcinoma cell lines.

The progeny of single teratocarcinoma cells will give rise to several different cell types in vitro, and the latter were shown to be functionally differentiated by biochemical criteria. In all these studies, cloned lines of mouse teratocarcinoma cells were assayed during the course of differentiation for some biochemical products characteristic of the tissues formed. The carcinoembryonic protein, alpha-foetoprotein, was not synthesized by undifferentiated embryonal carcinoma (EC) cells, but was synthesized in increasing amounts during their differentiation to endoderm-type cells in suspension culture. alpha-Foetoprotein was shown to be a product of endoderm cells, but not all endoderm cells synthesized this protein. During the course of further differentiation when EC cells or aggregates were grown in tissue-culture dishes, other biochemical products appeared. In cultures containing predominantly nerve-type cells, there was a 30-fold increase in the specific activity of acetylcholinesterase, with concomitant appearance of the aldolase isoenzyme characteristic of mouse brain. In some cultures, a small amount of muscle-type cell formation was marked by the appearance of the MB isoenzyme of creatine phosphokinase. Generally, biochemical differentiation was immature.     

Effects of differentiation of embryonal carcinoma cells (P19) on mitochondrial DNA content in vitro

Summary The embryonal carcinoma cell line P19 is derived from mouse teratocarcinomas. These pluripotent cells can be induced to differentiate into a variety of cell types by exposure to various drugs. We used retinoic acid to induce embryonal carcinoma cells to differentiate into neuronlike cells. In this study, we show that changes occur in mitochondria during differentiation of embryonal carcinoma cells to neuronlike cells. We found that various morphologic parameters such as mitochondrial fractional area and mitochondrial size decrease as embryonal carcinoma cells differentiate into neuronlike cells. Similar changes were also observed in mitochondrial DNA content. Stereologic analysis of cell preparations provided a measure of mitochondrial fractional area per cell and mtDNA content was assessed by radiolabeled mtDNA probe. This study establishes that mitochondria are regulated as cells differentiate. This study was financially supported by the Medical Research Council of Canada.     

Characterization of a new human cell line derived from a xenografted embryonal carcinoma

Summary A human cell line has been established from a transplantable xenografted human testicular tumor, which, both in the original tumor and in the xenograft, exhibited the histological characteristics of an undifferentiated malignant teratoma (embryonal cell carcinoma). The cells in culture were undifferentiated by biochemical, morphological, and ultrastructural criteria, growing as small islands of cells that tended to form aggregates at high density. The cells showed some variation in chromosome number with 30 to 40% of the cells having a normal human karyotype. The cells expressed high levels of alkaline phosphatase, which by heat inactivation and inhibition studies was 40 to 50% placental type alkaline phosphatase. None of the cultures produced human chorionic gonadotrophin, alphafetoprotein, carcinoembryonic antigen, or fibronectin, although at high cell densities plasminogen activator could be detected at low levels. Cell surface studies showed that the cells shared antigens with the murine embryonal carcinoma cell line F9, expressedβ ₂-microglobulin at very low and variable levels, and bound the lectin peanut agglutinin. These studies suggest that this cell line has some of the characteristics described for murine embryonal carcinoma cell lines.