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.     

Identification of murine extra-embryonic endodermal cells by reaction with teratocarcinoma basement membrane antiserum

Embryonal carcinoma cells from the PSA1 cell line will differentiate in vitro to form structures called embryoid bodies composed of an inner core of embryonal carcinoma cells surrounded by a basement membrane matrix and an outer layer of extra-embryonic endodermal cells. Immunization of rabbits with basement membranes isolated from embryoid bodies resulted in an antiserum, which binds to fixed extra-embryonic endodermal cells of either embryonic or teratocarcinoma origin but does not bind substantially to mouse embryonal carcinoma cells, fibroblasts, myoblasts or erythroleukemic cells. The F9-22 embryonal carcinoma cell line normally differentiates only to a very limited extent in vitro or in vivo. However, incubation of these cells in medium containing retinoic acid results in the appearance of cells resembling extra-embryonic endoderm. The embryoid body basement membrane antibodies were used to measure, by flow microfluorometry, the appearance of reactive cells in F9-22 cultures treated with retinoic acid. The kinetics of appearance of cells reactive with the basement membrane antibodies are similar to the kinetics of appearance of cells secreting plasminogen activator, a known marker of extraembryonic endoderm.     

Flow Cytoenzymology of the Early Differentiation of Mouse Embryonal Carcinoma Cells

Dual parameter flow cytoenzymology was used to detect biochemical differentiation of embryonal carcinoma cells, the undifferentiated, multipotent stem cells of teratocarcinomas. With the use of fluorogenic substrates, two enzyme systems, alkaline phosphatase (EC 3.1.3.1.) and carboxyl esterase (EC 3.1.1.), were studied. Embryonal carcinoma cells passaged in vitro for several years retained high alkaline phosphatase activities similar to those of embryonal carcinoma cells in embryoid bodies grown in vivo. Similar to the embryonal carcinoma cells in vivo, the in vitro embryonal carcinoma cells were capable of giving rise to progeny with greatly decreased levels of alkaline phosphatase. The embryonal carcinoma cell alkaline phosphatase was inhibited by l-p-bromotetramisole, suggesting a relationship between this enzyme and somatic, nonintestinal alkaline phosphatase isoenzymes. Determinations of esterase activities in viable teratocarcinoma cells showed that prior to any evidence of morphologic differentiation, the embryonal carcinoma cells are quite heterogeneous with regard to esterase activities.     

Nucleolus-like bodies in embryonal carcinoma cells of the embryoid bodies isolated from mouse teratocarcinoma

Undifferentiated embryonal carcinoma cells (EC cells) in the embryoid bodies isolated from mouse teratocarcinoma contained nucleolus-like bodies (NLBs) of smaller sizes in their cytoplasm (their sectional area averaged about 0.036 μm²). At the onset of EC cell differentiation, the average sectional area of NLBs significantly increased (about 0.107 μm²). When EC cells had differentiated into mesenchymal cells and endothelial cells of primitive blood vessels, NLBs decreased dramatically both in size and number. The possible role of NLBs in the differentiation process of EC cells is discussed.     

Cell surface antigens of totipotent mouse teratocarcinoma cells grown in vivo: their relation to embryo, adult, and tumor antigens.

Cell surface antigens on mouse embryonal carcinoma (or teratocarcinoma) cells were investigated by means of a syngeneic antiserum prepared against small-size embryoid bodies from the ascites form of the OTT 6050 transplantable teratoma. These embryoid bodies consist of embryonal carcinoma cells which are usually covered by a yolk-sac-like epithelium. The choice of immunogen was based on the previous demonstration [Mintz, B., and Illmensee, K. (1975) Proc. Nat. Acad. Sci. USA72, 3585–3589] that embryonal carcinoma cells from this specific source are euploid, developmentally totipotent, and completely reversible to normalcy. In indirect immunofluorescence tests, anti-embryoid-body serum reacted with both cell types of the immunogen and with two in vitro lines of embryonal carcinoma cells. Absorption of antiserum with a pure yolk sac carcinoma derived from the epithelial component of the embryoid bodies enabled assessment of reactivity with the embryonal carcinoma component of the immunogen: The absorption revealed that some antigens recognized on the embryonal carcinoma cells were shared by the yolk sac epithelial cells but that some antigens were present only on the embryonal carcinoma cells. The antigens were not shared by sperm, which failed to fluoresce with unabsorbed antiserum and were ineffective when tested as absorbents of antiserum reactivity against embryoid body target cells. Unfertilized eggs also failed to fluoresce. Preimplantation embryos gave immunofluorescence evidence of some antigens shared with embryonal carcinoma cells (and some with yolk sac cells) during cleavage, and in the blastocyst on both inner cell mass and trophoblast. Postimplantation embryos were also antigen-positive (at least through Day 6) in immunofluorescence tests on endoderm as well as ectoderm cells. Absorption of the antiserum with various normal adult tissues showed substantial cross-reactivity, especially with ovary and testis. Other tumors were tested, but only hepatoma cells grown in vitro were reactive, thereby indicating lack of any general tumor recognition in the antiserum. The above results with syngeneic immunizations demonstrate that known totipotent teratocarcinoma cells possess surface molecules which, while not universal on normal cells or tumors, are shared with many other tissues, including developmentally plastic cells of early embryos, developmentally restricted cells of later embryos, and various adult tissues. Immunofluorescence tests of cleavage-stage (Day 2) embryos from matings of $\text{+}\text{t}{}^{12}\text{×}\text{+}\text{t}{}^{12}$ heterozygotes, yielding 40% mutant $\text{t}{}^{12}\text{t}{}^{12}$ homozygotes lethal on Day 3, were uniformly positive on all the embryos, including mutants and normals. Therefore, under these conditions, no evidence was adduced to support the hypothesis that surface components required for normal early development might be coded by the wild-type allele of t¹².     

Protein synthesis and differentiation in a clonal line of teratocarcinoma and in preimplantation mouse embryo.

Undifferentiated cells of a clonal line of teratocarcinoma can differentiate in vitro into embryoid bodies with morphological and biochemical features of early mouse embryo. During the first step of differentiation protein synthesis has been analysed by 2 dimensional gel electrophoresis. While new proteins are synthesized, the synthesis of others turned off with the appearance of endodermal cells in embryoid bodies. We have compared protein synthesis during teratocarcinoma differentiation and during early mouse embryogenesis at three stages of mouse preimplantation embryo. The results demonstrate that only the late blastocyst protein synthesis pattern shows most of the polypeptides identified in the differentiated protein synthesis pattern of teratocarcinoma. In contrast, protein synthesis during the early stages of mouse embryonic development is very different from protein synthesis in undifferentiated teratocarcinoma.     

PCC4azal teratocarcinoma stem cell differentiation in culture: I. Biochemical studies

PCC4azal embryonal carcinoma cells were observed to spontaneously differentiate under defined culture conditions to endoderm-like cells and subsequently to giant cells. This differentiation was examined by determining the specific activities of several enzymes in the stem and endoderm-like cell populations. With differentiation, the level of alkaline and acid phosphatase activities remained unchanged, plasminogen activator specific activity increased fivefold, and lactate dehydrogenase (LDH) specific activity decreased to 40% of its original level. Isozyme analysis revealed a shift of the LDH isozymes toward LDH₁ with the appearance of LDH₂ for the first time in the endoderm-like cells. The surface antigen SSEA-1 was detected by indirect immunofluorescence on virtually all of the stem cells. However, the SSEA-1 antigen was not present on many of the endoderm-like cells, and it was completely undetectable on giant cells as assayed by immunofluorescence. The expression of H-2 antigen was examined in a similar manner using anti-H-2^b antiserum; this antigen was not detected on the stem, endoderm-like, or giant cells. Thus, there are defined biochemical changes that accompany the differentiation of PCC4azal stem cells in culture.     

THE PROCESS OF DIFFERENTIATION OF EMBRYOID BODIES IN THE LUNG OF SYNGENEIC MICE

The process of differentiation of embryoid bodies of mouse teratocarcinoma OTT6050 transplanted into the lung of syngeneic mice (129/Sv) is described. Embryoid bodies took more than 2 weeks to differentiate, and several kinds of differentiated tissues appeared often in the colonies derived from a single embryoid body. All the colonies with differentiated tissues were larger than 100μm in diameter.
Three steps on the differentiation of embryoid bodies can be distinguished by microscopic observations on histological preparations of tumors at different periods after injection. The first step is the deformation of the embryoid bodies and the disappearance of the outer endodermal cells, which occurs within a few days after injection. In the second step, which begins 5–7 days after injection, clusters of embryonal carcinoma cells in the colony are identified by the PAS reaction. The third step starts about 10 days after injection, and is characterized by the formation of tubular structures in some clusters.     

Lipid composition and lateral diffusion in plasma membranes of teratocarcinoma-derived cell lines

We measured lipid lateral diffusion rates for a series of teratocarcinoma-derived and embryo-derived cell lines, using the technique of fluorescence photo-bleaching recovery with a fluorescent lipid probe, C₁₆dil. The probe diffuses more rapidly in plasma membranes of embryonal carcinoma cells than in plasma membranes of teratocarcinoma-derived endodermal cell lines. When embryonal carcinoma cells are induced to differentiate by treatment with retinoic acid, diffusion constants of C₁₆dil are reduced to levels typical of endoderm. These changes are paralleled by differences in membrane cholesterol content; membrane free cholesterol levels in embyronal carcinoma lines are approximately half those found in endodermal lines, and are markedly increased upon retinoic-acid-induced differentiation.     

PCC4azal teratocarcinoma stem cell differentiation in culture: II. Morphological characterization

The ultrastructural morphology of the PCC4azal embryonal carcinoma cells and their differentiated counterparts, endoderm-like cells and giant cells, was characterized and compared with that of the cells of embryoid bodies. The ultrastructure of the PCC4azal embryonal carcinoma cells is similar to that of the embryonal carcinoma cells of the embryoid body. These cells are small, with a large nucleus and relatively few cytoplasmic organelles. Gap junctions and modified adherens junctions are formed at some areas of intercellular contact between the embryonal carcinoma cells. The differentiated PCC4azal endoderm-like cells have a more developed cytoplasm, containing an extensive endoplasmic reticulum with large Golgi regions. Most striking is the de novo appearance of epithelial-like junctional complexes which join the apical borders between the endoderm-like cells, thus polarizing the cell monolayer. The zonula occludens junctions of the junctional complex are extensive, consisting of six or more strands of tight junctional ridges. Terminal webs are present in the apical regions that are inserted into the zonula adherens region of the junctional complex. Gap junctions continue to join neighboring cells, and some gap junctions are intercalated within tight junctional ridges. The ultrastructure of the differentiated endodermal cells of the embryoid bodies is very similar to that of the PCC4azal endoderm-like cells. The embryoid body endodermal cells form similar junctional complexes which also contain continuous belts of tight junctions that are intercalated with gap junctions. As the PCC4azal endoderm-like cells are transformed to giant cells, a massive cytoskeleton is formed, consisting of a large complex system of 10-nm filaments, microtubules, and 7-nm microfilaments. The junctional complexes that were present during the endodermal stage are partially disassembled as the giant cells migrate apart. Thus, the differentiation process in this system is characterized by significant and distinctive morphological changes.     

Lipid patterns of embryonal carcinoma cell lines and their derivatives: Changes with differentiation

The lipid composition of several teratocarcinoma cell lines has been examined by biochemical and immunological methods in order to identify properties that might be correlated with the state of cell differentiation. The data indicate qualitative and quantitative changes in the phospholipid, cholesterol, and glycolipid composition. In particular, the ratios of cholesterol/phospholipid and of sphingomyelin/phosphatidylcholine are higher in differentiated cells. Gangliosides with short glycosidic chains (GM₃ and GD₃) are characteristic of undifferentiated, multipotent, embryonal carcinoma cell lines. More complex gangliosides (GM₁ and GD_1a) appear early during the course of differentiation. Each differentiated cell line presents a unique ganglioside map. Results are tentatively correlated with a stabilization of the membrane bilayer in differentiated cell lines, whereas a more fluid state of the membrane in embryonal carcinoma cell lines would allow maximal flexibility. Subtle differences in ganglioside composition among embryonal carcinoma cell lines are discussed in relation with their potentialities, and their developmental age.     

Enolase isoenzymes as markers of differentiation in teratocarcinoma cells and normal tissues of mouse

The changing profile of enolase (EC 4.2.1.11) isoenzymes in differentiating mouse cells has been traced by the use of specific antisera to the three subunits α, β, and γ. The amounts of the isoenzymes were measured in a variety of tissues during normal mouse development and during the differentiation of mouse teratocarcinoma cells in culture and as tumors. One isoenzyme is predominant in the early cells of the developing mouse embryo, namely, the homodimer made up of α subunits. The same isoenzyme is also the sole form detected in undifferentiated teratocarcinoma (embryonal carcinoma) cells. The isoenzyme form remains unchanged in developing primitive and definitive endoderm of the embryo. Similarly, endoderm cells formed by differentiation of embryonal carcinoma cells contained only αα enolase. In contrast, during the development of striated muscle and of brain, increasing proportions of β and γ subunits, respectively, were detected. Thus enolase was found to be a marker of the differentiation of these tissues. This conclusion was substantiated by finding significant amounts of the β subunit in teratocarcinoma cell cultures which had formed beating striated muscle in vitro.     

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.     

The unique killing of embryo-derived teratocarcinoma cells by nonactivated murine macrophages is not due to a lack of H-2 antigen expression.

It is well documented that activated macrophages, but not nonactivated ones, kill tumor cells in vitro without damaging normal cells. We, however, have previously shown that embryo-derived teratocarcinoma cells (F9, P19, PCC4) are efficiently killed by nonactivated macrophages as well as by activated ones. Whereas other tumor cells are killed extracellularly by macrophages, we found that F9 teratocarcinoma cells are phagocytosed alive by macrophages and subsequently killed intracellularly by a process dependent on intact lysosomal function. Neither the H-2 antigens nor the mRNAs for the alpha-chain and beta 2-microglobulin are detectable in embryo-derived teratocarcinoma cells. An obvious explanation for this unique killing is that the nonactivated macrophages recognize and kill these cells due to their lack of class I MHC antigen expression, assuming that class I MHC gene products on the target cells switch off the cytolytic machinery of nonactivated macrophages. Our present findings demonstrate that there is no correlation between H-2 antigen expression on tumor cells and their susceptibility to killing by macrophages. Retinoic acid-differentiated F9 cells and P19 cells expressing H-2 antigen after exposure to MAF (IFN-gamma) were sensitive to the killing by nonactivated macrophages. Hybrids that arose from fusion of P19 teratocarcinoma cells with embryonal normal fibroblasts (C57BL/6), which displayed the morphology of embryonal carcinoma stem cells and expressed H-2 antigens, were also sensitive to the killing by nonactivated macrophages. On the other hand, the H-2-negative testicular 402AX teratocarcinoma cells and K1735P melanoma cells were both resistant to the killing by nonactivated macrophages. We concluded that the unique killing of embryo-derived teratocarcinoma cells by nonactivated murine macrophages is not related to a lack of H-2 antigen expression.     

The regulation of embryonic stem cell differentiation by leukaemia inhibitory factor (LIF)

LIF (leukaemia inhibitory factor) is commonly used to maintain mouse embryonic stem cells in an undifferentiated state. These cells spontaneously differentiate when allowed to aggregate in the absence of LIF, forming embryoid bodies in which early embryonic cell lineages develop. Using embryoid bodies cultured in the presence and absence of LIF, we show that although LIF inhibited the development of visceral and parietal endodermal cells, it did not affect the differentiation of the primitive endodermal cell precursors of these extraembryonic cell lineages. Furthermore, deposition of the basement membrane produced by the primitive endodermal cells, which separates them from the remaining cells of the embryoid body, still occurred. The differentiation of primitive ectodermal cells and their progeny was inhibited by LIF, as evidenced by the lack of expression of FGF-5, muscle, and neuronal markers. However, cavitation of the embryoid body and maintenance of the cells in contact with the primitive endodermal basement membrane as an epiblast epithelium still occurred normally in the presence of LIF. These results indicate that cavitation and formation of the epiblast epithelium are regulated by mechanisms distinct from those controlling the differentiation of epiblast cell lineages. Furthermore, although epithelium formation and cavitation do not require the differentiation of visceral endodermal cells, the results are consistent with the hypothesis that the primitive endodermal basement membrane is sufficient to induce the epithelialization of undifferentiated embryonic stem cells necessary for cavitation.     

Analysis of T-T lymphocytes and T-accessory cell interactions using cloned T cells: MHC I restricted cloned T cells activate MHC II restricted T helper cells

We evaluated the role of molecules of the major histocompatibility complex (MHC) involved in the cellular interactions of two T-cell clones by testing the effect of monoclonal antibodies on the responses of the clones in vitro. The two T-cell clones used in the study are specific for minor histocompatibility antigens and restricted to the H-2K^k. In the absence of exogenous IL-2 the clones require the presence of Ia⁺, Thy-1⁻ accessory cells and of Thy-1⁺, Lyt-1⁺2⁻ cells in the irradiated spleen cell suspension used as stimulator. It is also necessary that both the accessory cells and the T cells in the stimulator cell populations are recognized specifically by the clones. Monoclonal antibodies specific for the H-2K product inhibited the lytic effector function of the cytolytic clone. These antibodies when added to cultures of stimulator cells and clones inhibited also the proliferation of this clone and of a nonlytic clone. When antigen recognition was measured by the increase in sensitivity of the clones to IL-2 while confronted with uv-irradiated stimulator cells, both clones were blocked efficiently by anti-H-2K antibodies. Thus, these results suggest that the interaction of monoclonal antibodies with the restricting H-2K molecule is sufficient to block the recognition signal, a prerequisite for proliferation. In contrast, monoclonal antibodies specific for A_αA_β and/or E_αE_β had no effect on cytolysis or on restricted recognition. However, they inhibited the proliferative responses as efficiently as the H-2K specific antibodies. Inhibition by class II-specific antibodies was not abolished when stimulator cell populations were depleted of Lyt-2⁺ cells. The blocking effect, however, was reversed by the addition of IL-2. No inhibition was obtained with antibody specific for E_αE_β when B10.A(4R) spleen cells, which do not express E_αE_β, or when B10.A(4R) accessory cells, which were reconstituted with (BALB/c X B10.A(4R)) F1 T cells, were used as stimulators. Stimulator cells heterozygous for H-2 could be inhibited by antibodies to the parental haplotype not encoded in the clones (H-2K^d). These and previous results suggest that H-2K-restricted minor histocompatibility antigen-specific recognition transmits an activating signal to the clones and to the stimulator cells. The clones probably are induced to express more IL-2 receptors. The stimulator T cells seem to interact through A_αA_β and E_αE_β molecules with syngeneic accessory cells. This interaction results in IL-2 production by the stimulator T cells and thus in the proliferation of the clones.     

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.     

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.