Biochemical criteria for the in vitro differentiation of embryoid bodies produced by a transplantable teratoma of mice. The production of acetylcholine esterase and creatine phosphokinase by teratoma cells

When embryoid bodies are grown in suspension culture in vitro, they undergo only a limited amount of morphological development. When these same embryoid bodies are permitted to attach to the surface of a culture dish, a wide variety of new morphological cell types appear. Suspension cultures of embryoid bodies do not contain significant detectable levels of acetylcholine esterase or creatine phosphokinase. These same enzymes however are produced in cell cultures derived from embryoid bodies attached to the culture dish surface. Polyacrylamide gel electrophoresis has been employed to demonstrate that the electrophoretic form of creatine phosphokinase produced by teratoma cells in culture is the brain form of the enzyme. Solid transplantable tumors containing only embryonal carcinoma cells (stem cells) do not contain either of these enzymatic activities. Well differentiated transplantable teratomas contain both enzymes.     

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)     

Introduction and recovery of a selectable bacterial gene from the genome of mammalian cells.

The simian virus 40 (SV40)-pBR322 recombinant, pSV2, carrying the origin of SV40 replication and the gpt gene of Escherichia coli, has been stably introduced into Chinese hamster ovary hprt- cells. All gpt-transformed cell lines were found to contain one or more insertions of pSV2 sequences exclusively associated with high-molecular-weight DNA. Additional analyses showed that at least one integrated copy in each cell line retained an intact gpt gene and flanking SV40 sequences required for expression of xanthine-guanine phosphoribosyltransferase. Most cell lines contained pSV2 sequences which had integrated with partial sequence duplication. Upon fusion with COS-1 cells, a simian cell line permissive for autonomous pSV2 replication, most gpt-transformed cell lines produced low-molecular-weight DNA molecules related to pSV2. The majority of these replicating DNAs were indistinguishable from the original transfecting plasmid in both size and restriction enzyme cleavage pattern. In addition, the recovered DNA molecules were able to confer ampicillin resistance to E. coli and to transform mouse L cells and Gpt- E. coli to a Gpt+ phenotype. These studies indicate that all of the genetic information carried by this SV40-plasmid recombinant can be introduced into and retrieved from the genome of mammalian cells.     

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.     

Transformation of isolated rat hepatocytes with simian virus 40

Rat hepatocytes were transformed by simian virus 40 (SV40). Hepatocytes from two different strains of rats and a temperature-sensitive mutant (SV40tsA 1609), as well as wild-type virus were used. In all cases, transformed cells arose from approximately 50% of the cultures containing hepatocytes on collagen gels or a collagen gel-nylon mesh substratum. Cells did not proliferate in mock-infected cultures. SV40-transformed hepatocytes were epithelial in morphology, retained large numbers of mitochondria, acquired an increased nucleus to cytoplasm ratio, and contained cytoplasmic vacuoles. Evidence that these cells were transformed by SV40 came from the findings that transformants were 100% positive for SV40 tumor antigen expression, and that SV40 was rescued when transformed hepatocytes were fused with monkey cells. All SV40-transformed cell lines tested formed clones in soft agarose. Several cell lines transformed by SV40tsA 1609 were temperature dependent for colony formation on plastic dishes. Transformants were diverse in the expression of characteristic liver gene functions. Of eight cell lines tested, one secreted 24% of total protein as albumin, which was comparable to albumin production by freshly plated hepatocytes; two other cell lines produced 4.2 and 5.7%, respectively. Tyrosine aminotransferase activity was present in five cell lines tested but was inducible by dexamethasone treatment in only two. We conclude from these studies that adult, nonproliferating rat hepatocytes are competent for virus transformation.     

Comparison of buffers and detection systems for high-pressure liquid chromatography of peptide mixtures.

Cell culture lines were established from the transplantable mouse hepatomas H6 and H129. Both cell lines had a doubling time about 30 h when maintained in medium containing 5% foetal bovine serum. H6 cells contained about 3-4 times more DNA-dependent RNA polymerase I (Pol I; ribonucleoside triphosphate--RNA nucleotidyltransferase, EC 2.7.7.6) than did H129 cells. Moreover, the H6-cell enzyme was more heat-labile than that from H129 cells. Steady-state contents of 28S rRNA were measured in both cell lines. Exponentially growing cultures of H6 cells contained about 6.5pg of 28S rRNA/cell, and similar cultures of H129 cells contained about 5.8pg/cell. Stationary cultures of both cell lines contained about 2pg of 28S rRNA/cell. By two different techniques, the half-time for turnover of 28S rRNA was estimated to be 16-17h for both H6 and H129 cells. Knowing the turnover rate and the steady-state concentration, one may calculate that both H6 and H129 cells synthesize 28S rRNA at a rate of about 0.25 pg/h per cell. The amount of template-bound Pol I activity was similar in nuclei isolated from H6 and H129 cell cultures. These data indicate that, although H6 cells contained 3-4 times more Pol I than did H129 cells, both cell lines synthesized rRNA at about the same rate.     

Isolation of U1-snRNP(s) from mouse teratocarcinoma cells using immunochemical and biochemical procedures: high proportion of U1a-snRNP to U1b-snRNP

An attempt was made to immunochemically and biochemically purify and characterize the U1-snRNP(s) of mouse embryonal carcinoma cells. The results obtained by RNA analysis of U1-snRNP(s) purified immunochemically from embryoid bodies, F9 cells and PYS-2 cells indicated that the U1-snRNP(s) in these cells consisted of U1a-snRNP and U1b-snRNP. The proportion of U1a-snRNP to U1b-snRNP was also found to be high in the embryoid bodies and F9 cells. The U1a-snRNP predominance in U1-snRNP population was also detected in PYS-2 cells. The immunochemically purified U1-snRNP population from liver nuclei of 129 syngeneic male mouse (129/sv), a host mouse for transplantable tetratocarcinoma OTT6050, and ICR male mouse, contained approximately equal levels of the two U1-snRNP species (U1a- and U1b-snRNP). Partially purified U1-snRNP from embryoid bodies was also obtained by elution from a DEAE-Sepharose column at around 0.18 M NH4Cl or by fractionation by 5-20% linear sucrose gradient centrifugation. The electrophoretic RNA profiles of the partially purified U1-snRNP of embryoid bodies were similar to those obtained immunochemically.     

Hyaluronate degradation in 3T3 and simian virus-transformed 3T3 cells

The cellular control of hyaluronate levels was examined in cultures of simian virus 40-transformed 3T3 (SV3T3) and 3T3 cells which are known to differ in their metabolism of hyaluronate. When [3H]hyaluronate was added to cultures of the two cell lines, four times more ligand was bound per mg of protein by the SV3T3 cells than by the 3T3 cells. Of the bound [3H] hyaluronate, 40% was degraded by the SV3T3 cells to oligosaccharides characteristic of the breakdown of hyaluronate, but only 2% was degraded by 3T3 cells. Hyaluronidase activity was found in the cell layer and medium of the SV3T3 cultures, but was not detectable in 3T3 cells. The SV3T3 enzyme was active only at acidic pH, but at neutral pH the secreted SV3T3 hyaluronidase was thermally more stable then the cell-associated enzyme. In contrast, both cell lines were found to contain similar amounts of beta-glucuronidase and beta-N-acetylglucosaminidase activity. We conclude that the elevated capacity of SV3T3 cells to degrade hyaluronate may be partially responsible for their lack of the hyaluronate-containing pericellular coat which is prominent around 3T3 cells.     

Ultrastructural and biological characterization of human choroid cell cultures transformed by Simian Virus 40

A human diploid cell line of choroid origin was isolated from the retrouveal portion of an enucleated eye and designated HC. After 10 passages, when the proliferative capacity of HC cells decreased, they were infected and transformed by Simian Virus 40 (SV40). A proliferating long-term cultured cell line designated HC/SV40 was established and it has been maintained as monolayer for more than 100 passages so far. The two cell lines, HC and HC/SV40, were compared for growth characteristics, capacity to form colonies in soft agar, presence of nuclear T-antigen, and ultrastructure. Cytogenetic analysis was also performed to determine the presence of chromosomal aberrations due to the permanent viral transformation of the cell line. The results indicate that HC/SV40 should be considered the transformed counterparts of HC cells because they are morphologically similar to the latter but can grow in soft agar, possess T-antigen, and show a pattern of karyotypic changes similar to that induced by SV40 in human fibroblasts. The choroid origin of HC and HC/SV40 cell lines was confirmed by the presence, in their cytoplasm, of typical electron dense granules. Their neural origin will make these cell lines very useful for neuropharmacological and differentiation studies.     

Thymidine Kinase from Normal, Simian Virus 40-Transformed and Simian Virus 40-Lytically Infected Cells

Simian virus 40 (SV40) infection of human diploid cells failed to cause an enhanced production of thymidine kinase during the first 10 days after infection. Thymidine kinase activities from extracts of SV40-transformed cultures (human or simian) were considerably higher than the activity levels in extracts from the normal cells of origin. In addition, whereas the kinase activities obtained for human diploid cultures decreased as the cell sheet became confluent, the kinase activities for SV40-transformed human cells remained high after confluence was reached. Antisera obtained from hamsters bearing SV40 or adeno-7-SV40 hybrid virus tumors selectively inhibited enzyme from transformed sources (human or simian). Also, the antisera selectively inhibited enzyme extracted from SV40-lytically infected monkey cells. Sera from normal animals or from hamsters bearing polyoma tumors failed to inhibit enzymes from normal, SV40-transformed, or SV40-lytically infected cells. The Michaelis constant of partially purified enzyme from SV40-transformed cells was two to five times as high as that obtained for partially purified enzyme from human diploid cell cultures.     

Ultrastructural and biological characterization of human choroid cell cultures transformed by Simian Virus 40

Summary A human diploid cell line of choroid origin was isolated from the retrouveal portion of an enucleated eye and designated HC. After 10 passages, when the proliferative capacity of HC cells decreased, they were infected and transformed by Simian Virus 40 (SV40). A proliferating long-term cultured cell line designated HC/SV40 was established and it has been maintained as monolayer for more than 100 passages so far. The two cell lines, HC and HC/SV40, were compared for growth characteristics, capacity to form colonies in soft agar, presence of nuclear T-antigen, and ultrastructure. Cytogenetic analysis was also performed to determine the presence of chromosomal aberrations due to the permanent viral transformation of the cell line. The results indicate that HC/SV40 should be considered the transformed counterparts of HC cells because they are morphologically similar to the latter but can grow in soft agar, possess T-antigen, and show a pattern of karyotypic changes similar to that induced by SV40 in human fibroblasts. The choroid origin of HC and HC/SV40 cell lines was confirmed by the presence, in their cytoplasm, of typical electron dense granules. Their neural origin will make these cell lines very useful for neuropharmacological and differentiation studies. This work was supported by Grant 82.00346.96 from the C.N.R. finalized project “Control of Neoplastic Growth”.     

Influence of regulatory genes of type 1 human immunodeficiency virus on proliferation and differentiation of murine embryonic stem cells

Spontaneous formation of embryoid bodies and subsequent differentiation of some cells into cardiomyocytes were demonstrated on murine embryonic stem cells of R1 line. The lines of embryonic stem cells were obtained that had been transfected with genetic constructs carrying expressing regulatory genes of the human immunodeficiency virus tat and nef and "green protein" gene (GFP). The transfection of embryonic stem cells with the gene tat stimulated their proliferative activity, while this activity decreased in the cells transfected with the gene nef. The time necessary for the formation of embryoid bodies by all lines of transfected cells was similar to that in the control cells. In the cultures of cells transfected with nef and tat, the number of embryoid bodies and the percentage of embryoid bodies with contracting cardiomyocytes were higher and lower than in the control, respectively. Thus, an inverse correlation was observed between the effects of regulatory genes of the human immunodeficiency virus on proliferation and differentiation embryonic stem cells.     

Transformation of Simian Virus 40-resistant Hamster Cells with an Adenovirus 7-Simian Virus 40 Hybrid

When the hamster cell lines BHK21 and Nil-2 were infected at a multiplicity of 100 with the adenovirus 7-simian virus 40 (SV40) hybrid (strain LLE46), SV40 T antigen was induced in 0.1 to 6% of the cells during the first 96 hr postinfection, morphological changes occurred 3 to 7 weeks later, and eventually all the cells contained SV40 T antigen, but no adeno 7 T antigen. Results were similar when primary and secondary monolayer cultures of hamster embryo (HE) cells were infected with the adeno 7-SV40 hybrid, and when primary HE cells were infected with SV40. However, infection of BHK21, Nil-2, and secondary HE cells with the same multiplicity of SV40 did not induce SV40 T antigen or morphological transformation. This suggests that the target cells required for infection with SV40 virions, but not those required for infection with the hybrid, are lost or altered in secondary HE cultures and in the two cell lines. In most of the virus-host cell systems in which SV40 T antigen and transformation were induced, there was a decrease in the number of T antigen-positive cells after the initial infection. This was followed by a lag period of up to 2 months before the onset of a progressive increase in the number of positive cells. The beginning of the rise in T antigen production coincided with the first morphological changes.     

Umbilical cord endothelial cells expressing large T antigen: Comparison with primary cultures and effect of cell age

Summary A number of human endothelial cell lines from umbilical cord cells (HUVECs) have been generated by transfection with SV40 large T and small t antigen sequences. Comparison of these lines with primary cultures of HUVECs has been carried out by monitoring the expression of a number of endothelial cell markers with specific regard to cell age. The secreted levels of the protein plasminogen activator inhibitor (PAI) was found to be significantly reduced in SV40-transfected cells when compared to untransfected controls. Tissue plasminogen activator (tPA) and urokinase (uPA) levels were unchanged. As cells entered crisis, there was a rapid and significant increase in the levels of tPA, uPA, and PAI and this was observed for all clones screened. The endothelial cell marker von Willebrand Factor (vWF) was found intracellularly and was also secreted into the medium. The levels were not altered between transfected and untransfected cells. Angiotensin converting enzyme (ACE) activity was maintained in cell lines at levels found in nonimmortalized HUVECs. Both isoforms (α and β) of IL-1 (interleukin-1) increased as cells approached crisis, and the presence of these cytokines may be responsible for the increased levels of tPA, PAI, and uPA. With one exception, the ability of the transfected cells to produce prostacyclin (PGI₂) was lost by all clones.     

Virogenic Properties of Bromodeoxyuridine-sensitive and Bromodeoxyuridine-resistant Simian Virus 40-transformed Mouse Kidney Cells

When simian virus 40 (SV40)-transformed mouse kidney cells (mKS) were grown in the presence of susceptible indicator cells, SV40 was readily recovered from: (i) 15 transformed cell lines, (ii) transformed cells subcultured 45 times over a 7-month period in medium containing antiviral serum and bromodeoxyuridine (dBU), (iii) 45 of 46 clonal lines isolated in the presence of antiviral serum, (iv) 19 of 19 secondary clones isolated from two clonal lines, and (v) dBU-resistant transformed cell lines. dBU-resistant SV40-transformed mouse kidney cell lines were selected and shown to contain the T antigen and to have normal levels of thymidylate kinase and deoxyribonucleic acid (DNA) polymerase, but to be deficient in thymidine (dT) kinase. Radioautographic and biochemical experiments demonstrated that very little (3)H-dT was incorporated into DNA of dBU-resistant cells during a 6-hr labeling period. After infection of dT kinase-deficient mKS cells with vaccinia virus, high levels of dT kinase were induced. The properties of SV40 recovered from dBU-sensitive and dBU-resistant cells were studied. SV40 recovered from transformed cells was shown to express in CV-1 cells at least six functions characteristic of parental virus: synthesis of capsid antigen, synthesis of T antigen, synthesis of viral DNA, induction of dT kinase, induction of DNA polymerase, and induction of host cell DNA synthesis. In addition, SV40 recovered from the transformed cells induced T antigen, dT kinase, deoxycytidylate deaminase, thymidylate kinase, and DNA polymerase in abortively infected mouse kidney cultures, and the virus was also capable of transforming primary cultures of mouse kidney cells.     

Establishment and differentiation of murine EG cell lines derived from primordial germ cells]

Primordial germ cells (PGC) were isolated from 8.5, 10.5, 12.5 days post coitum (dpc) embryos of F1 (Balb/c x ICR), C57BL/6J, 129/svJ, 129/sv-ter mice, and cultured on mitotically inactive MEF or STO feeder layer cells with addition of leukemia inhibitory factor, stem cell factor and basic fibroblast growth factor in cultures. PGCs formed densely packed and AKP positive colonies with pluripotential marker gene (oct-4) expression resembling undifferentiated ES cells in morphology and growth pattern. Five EG cell lines derived from PGCs were established: EG1(8.5 dpc, F1), EG2 and EG3 (8.5 dpc, C57BL/6J), EG4 (10.5 dpc, 129/svJ), EG5 (10.5 dpc, 129/sv-ter). No long term culture was obtained from 12.5 dpc PGCs of 29 embryos. All five EG cell lines cultured on feeder layer cells or in LIF containing medium still remain undifferentiated state at 15 th passage. Under appropriate conditions, EG cells formed embryoid bodies in suspension culture and multiple types of differentiated cells in monolayer culture. When these EG cells were injected in nude mice, they formed teratocacinomas containing differentiated cells such as cartilage, neural tissue and epithelium. These results show that EG1-5 cell lines derived from 8.5, 10.5 dpc embryos are pluripotential.