Analysis of the cell culture fluid from two new human hepatoma-derived cell lines reveals that 17 of the major human plasma proteins are synthesized and secreted by these cells. One of these cell lines, Hep 3B, also produces the two major polypeptides of the hepatitis B virus surface antigen. When Hep 3B in injected into athymic mice, metastatic hepatocellular carcinomas appear. These cell lines provide experimental models for investigation of plasma protein biosynthesis and the relation of the hepatitis B viru genome to tumorigenicity.

A comprehensive analysis of transposable element (TE) expression in mammalian full-grown oocytes reveals that LTR class III retrotransposons make an unexpectedly high contribution to the maternal mRNA pool, which persists in cleavage stage embryos. The most abundant transcripts in the mouse oocyte are from the mouse transcript (MT) retrotransposon family, and expression of this and other TE families is developmentally regulated. Furthermore, TEs act as alternative promoters and first exons for a subset of host genes, regulating their expression in full-grown oocytes and cleavage stage embryos. To our knowledge, this is the first example of TEs initiating synchronous, developmentally regulated expression of multiple genes in mammals. We propose that differential TE expression triggers sequential reprogramming of the embryonic genome during the oocyte to embryo transition and in preimplantation embryos.

A significant aspect of primary hepatic carcinoma in man is the high positive correlation of hepatocellular carcinoma with infection with hepatitis B virus (HBV)1. Analysis of the relationship between HBV infection and oncogenesis is difficult because natural infection with HBV is limited to man and experimental infection has been achieved only in chimpanzees and gibbons. Furthermore, because HBV has not been successfully propagated in cell culture, basic study of virus-cell interaction of the aetiological agent of one of the most widespread infections of man has been impossible. Recently, however, a cell line (PLC/PRF/5) derived from a human hepatoma biopsy was described which produces the HRV surface antigen (HBsAg) and so provides a tool for the experimental investigation of HBV in viro. We now report the derivation and characterisation of two additional cell lines primary liver carcinomas. In contrast to the PLC/PRF/5 cell line, these cell lines retain the capacity to synthesise many human plasma proteins, including both albumin and alpha-fetoprotein (AFP). One of these lines also produces BHsAg. We also present evidence that HBsAg synthesis and secretion in this cell line are correlated with the growth state of the culture. This finding is in contrast to the continuous HBsAg production found in the PLC/PRF/5 cell line.

The importance of embryonic stage-specific molecules in the regulation of cell interactions and cell sorting during development and differentiation has been postulated; however, experimental confirmation of this postulate is scarce. Several candidates for stage-specific molecules have been found by using antisera, raised by both syngeneic (1, 2) and xenogeneic (3) immunization with embryonal carcinoma cells (ECG) or by xenogeneic immunization (4) with mouse embryos. Indeed, inhibition of development of preimplantation mouse embryos by Fab fragments isolated from antisera to the murine teratocarcinoma cell F9 has recently been reported (5). However, such sera contain antibodies to multiple antigenic determinants, which precludes precise definition of embryo stage-specific antigens. In order to circumvent this difficulty and to study the stage-specific molecules in a methodical fashion, we are producing monoclonal antibodies (6) reactive with teratocarcinoma cells and embryos. Production and characterization of one such antibody is described here. MATERIALS AND METHODSPreparation of Monoclonal Reagents. BALB/c mice were immunized by weekly intraperitoneal injection of 107 irradiated F9 cells. The mice were tail-bled 7 days after each injection, and sera were tested for reactivity on F9 cells. Three days after the seventh immunization, the spleen was removed from one mouse, which showed a high titer of antibody reactivity. Splenic lymphocytes were isolated and fused with the P3-X63-Ag8 mouse myeloma cell line as described (6-8). Hybrid cell lines were isolated by growth of the fusion mixtures in Dulbecco's modification of Eagle's minimal essential medium containing 10% fetal bovine serum, 2 mM glutamine, and the HAT components (hypoxanthine/aminopterin/thymidine) (9). Supernatants from Linbro wells containing growing colonies were tested for reactivity on F9 cells by an indirect antibody binding radioimmunoassay (RIA). One positive colony was transferred to mass culture and cloned. Supernatants from clones were also tested in RIA and the positive clones were maintained in tissue culture. Supernatants were collected from dense cultures of specific antibody-producing hybrids, clarified by centrifugaThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S.

Methylation of DNA is an essential epigenetic control mechanism in mammals. During embryonic development, cells are directed toward their future lineages, and DNA methylation poses a fundamental epigenetic barrier that guides and restricts differentiation and prevents regression into an undifferentiated state. DNA methylation also plays an important role in sex chromosome dosage compensation, the repression of retrotransposons that threaten genome integrity, the maintenance of genome stability, and the coordinated expression of imprinted genes. However, DNA methylation marks must be globally removed to allow for sexual reproduction and the adoption of the specialized, hypomethylated epigenome of the primordial germ cell and the preimplantation embryo. Recent technological advances in genome-wide DNA methylation analysis and the functional description of novel enzymatic DNA demethylation pathways have provided significant insights into the molecular processes that prepare the mammalian embryo for normal development.

Two monoclonal antibodies (MC631 and MC813‐70) raised against 4‐ to 8‐cell stage mouse embryos and a human teratocarcinoma cell line, respectively, detect the stage‐specific embryonic antigens, the previously defined SSEA‐3 and SSEA‐4, described herein. These antibodies were both reactive with a unique globo‐series ganglioside with the structure shown below: (formula; see text) The antibodies were found to recognize sequential regions of this ganglioside, i.e., MC813‐70 recognizes the terminal ‘a’ structure whereas antibody MC631 recognizes the internal ‘b’ structure. Thus, a set of two antibodies defines this unique embryonic antigen. During differentiation of human teratocarcinoma 2102Ep cells, the globo‐series glycolipids defined by these antibodies decrease and the lacto‐series glycolipids, reacting with the SSEA‐1 antibody appear. This antigenic conversion suggests that a shift of glycolipid synthesis from globo‐series to lacto‐series glycolipids occurs during differentiation of human teratocarcinoma and perhaps of pre‐implantation mouse embryos.

The International Stem Cell Initiative analyzed 125 human embryonic stem (ES) cell lines and 11 induced pluripotent stem (iPS) cell lines, from 38 laboratories worldwide, for genetic changes occurring during culture. Most lines were analyzed at an early and late passage. Single-nucleotide polymorphism (SNP) analysis revealed that they included representatives of most major ethnic groups. Most lines remained karyotypically normal, but there was a progressive tendency to acquire changes on prolonged culture, commonly affecting chromosomes 1, 12, 17 and 20. DNA methylation patterns changed haphazardly with no link to time in culture. Structural variants, determined from the SNP arrays, also appeared sporadically. No common variants related to culture were observed on chromosomes 1, 12 and 17, but a minimal amplicon in chromosome 20q11.21, including three genes, ID1, BCL2L1 and HM13, expressed in human ES cells, occurred in >20% of the lines. Of these genes, BCL2L1 is a strong candidate for driving culture adaptation of ES cells.

The International Stem Cell Initiative characterized 59 human embryonic stem cell lines from 17 laboratories worldwide. Despite diverse genotypes and different techniques used for derivation and maintenance, all lines exhibited similar expression patterns for several markers of human embryonic stem cells. They expressed the glycolipid antigens SSEA3 and SSEA4, the keratan sulfate antigens TRA-1-60, TRA-1-81, GCTM2 and GCT343, and the protein antigens CD9, Thy1 (also known as CD90), tissue-nonspecific alkaline phosphatase and class 1 HLA, as well as the strongly developmentally regulated genes NANOG, POU5F1 (formerly known as OCT4), TDGF1, DNMT3B, GABRB3 and GDF3. Nevertheless, the lines were not identical: differences in expression of several lineage markers were evident, and several imprinted genes showed generally similar allele-specific expression patterns, but some gene-dependent variation was observed. Also, some female lines expressed readily detectable levels of XIST whereas others did not. No significant contamination of the lines with mycoplasma, bacteria or cytopathic viruses was detected.