Human blastocyst-derived, pluripotent cell lines are described that have normal karyotypes, express high levels of telomerase activity, and express cell surface markers that characterize primate embryonic stem cells but do not characterize other early lineages. After undifferentiated proliferation in vitro for 4 to 5 months, these cells still maintained the developmental potential to form trophoblast and derivatives of all three embryonic germ layers, including gut epithelium (endoderm); cartilage, bone, smooth muscle, and striated muscle (mesoderm); and neural epithelium, embryonic ganglia, and stratified squamous epithelium (ectoderm). These cell lines should be useful in human developmental biology, drug discovery, and transplantation medicine.

Embryonic Stem Cell Lines Derived from Human Blastocysts

Pluripotential cells are present in a mouse embryo until at least an early post-implantation stage, as shown by their ability to take part hi the formation of chimaeric animals1 and to form teratocarcinomas2. Until now it has not been possible to establish progressively growing cultures of these cells in vitro, and cell lines have only been obtained after teratocarcinoma formation in vivo. We report here the establishment in tissue culture of pluripotent cell lines which have been isolated directly from in vitro cultures of mouse blastocysts. These cells are able to differentiate either in vitro or after innoculation into a mouse as a tumour in vivo. They have a normal karyotype.

Establishment in culture of pluripotential cells from mouse embryos

This report describes the establishment directly from normal preimplantation mouse embryos of a cell line that forms teratocarcinomas when injected into mice. The pluripotency of these embryonic stem cells was demonstrated conclusively by the observation that subclonal cultures, derived from isolated single cells, can differentiate into a wide variety of cell types. Such embryonic stem cells were isolated from inner cell masses of late blastocysts cultured in medium conditioned by an established teratocarcinoma stem cell line. This suggests that such conditioned medium might contain a growth factor that stimulates the proliferation or inhibits the differentiation of normal pluripotent embryonic cells, or both. This method of obtaining embryonic stem cells makes feasible the isolation of pluripotent cells lines from various types of noninbred embryo, including those carrying mutant genes. The availability of such cell lines should made possible new approaches to the study of early mammalian development.

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Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells

Fertilization of mammalian eggs is followed by successive cell divisions and progressive differentiation, first into the early embryo and subsequently into all of the cell types that make up the adult animal. Transfer of a single nucleus at a specific stage of development, to an enucleated unfertilized egg, provided an opportunity to investigate whether cellular differentiation to that stage involved irreversible genetic modification. The first offspring to develop from a differentiated cell were born after nuclear transfer from an embryo-derived cell line that had been induced to become quiescent. Using the same procedure, we now report the birth of live lambs from three new cell populations established from adult mammary gland, fetus and embryo. The fact that a lamb was derived from an adult cell confirms that differentiation of that cell did not involve the irreversible modification of genetic material required for development to term. The birth of lambs from differentiated fetal and adult cells also reinforces previous speculation that by inducing donor cells to become quiescent it will be possible to obtain normal development from a wide variety of differentiated cells.

Viable offspring derived from fetal and adult mammalian cells

http://web.mit.edu/7.31/restricted/pdfs/F05_and_earlier/Nichols.pdf

Formation of Pluripotent Stem Cells in the Mammalian Embryo Depends on the POU Transcription Factor Oct4

Completion of mouse embryogenesis requires both the maternal and paternal genomes

A monoclonal antibody derived by fusion of mouse myeloma cells with spleen cells from a mouse immunized with F9 teratocarcinoma cells is described. This antibody, which reacts with embryonal carcinoma cells of mouse and human origin and with some preimplantation stage mouse embryos, defines an embryonic stage-specific antigen. This stage-specific antigen (SSEA-1) is first detected on blastomeres of 8-cell stage embryos. Trophectodermal cells are transitorily positive; however, each cell in the inner cell mass eventually expresses this antigen.

https://www.pnas.org/content/pnas/75/11/5565.full.pdf

Monoclonal antibody defining a stage-specific mouse embryonic antigen (SSEA-1).

Mouse blastocysts with and without zonae pellucidae are susceptible to complement-dependent antibody cytotoxicity. Exposure of blastocysts to rabbit anti-mouse serum together with complement results in the death of all cells; however, when blastocysts are exposed to antiserum alone and then transferred to guinea pig complement, only the trophoblastic cells are killed. These results suggest that the mouse blastocyst is not permeable for certain antibodies. The inner cell masses can easily be separated from the remnants of trophoblastic cells and are then able to grow and differentiate in vitro. This method of immunosurgery can be used to obtain large quantities of pure inner cell masses in a relatively short period of time.

https://www.pnas.org/content/pnas/72/12/5099.full.pdf

Immunosurgery of mouse blastocyst.

There is much interest in developmentally regulated molecules which may have function in cell interactions and sorting during embryogenesis and differentiation. Numerous antisera have been raised which detect antigens that are expressed in early embryonic cells and become restricted during differentiation, being expressed in only a minority of adult cells (reviewed in refs 1–3). The precise antigenic determinants recognized by such antisera have not been defined. However, studies using a hybri-doma antibody against mouse spleen cells4 and monoclonal autoantibodies of patients with cold agglutinin disease5 have shown that two defined carbohydrate antigen systems, the Forssman and the li antigens, have stage-specific expression in early mouse embryos. We now describe evidence that the stage-specific embryonic antigen SSEA-1 (ref. 6) involves the carbohydrate sequence Galβ1 → 4GlcNAc ↑1,3 Fucα This determinant is formed by α 1 → 3 fucosylation of blood group I or i antigens which are branched or linear oligosaccharides, respectively7–9, built of Galβ1 →4GlcNAc units and known as type 2 precursor chains10 of the major blood group antigens. Thus, we introduce the concept of simple glycosylation changes as a basis for stage-specific expression of embryonic antigens.

Davor Solter

Papers

Completion of mouse embryogenesis requires both the maternal and paternal genomes

Monoclonal antibody defining a stage-specific mouse embryonic antigen (SSEA-1).

Immunosurgery of mouse blastocyst.

Stage-specific embryonic antigen involves alpha 1 goes to 3 fucosylated type 2 blood group chains.

Retrotransposons regulate host genes in mouse oocytes and preimplantation embryos.