We describe the derivation of pluripotent embryonic stem (ES) cells from human blastocysts. Two diploid ES cell lines have been cultivated in vitro for extended periods while maintaining expression of markers characteristic of pluripotent primate cells. Human ES cells express the transcription factor Oct-4, essential for development of pluripotential cells in the mouse. When grafted into SCID mice, both lines give rise to teratomas containing derivatives of all three embryonic germ layers. Both cell lines differentiate in vitro into extraembryonic and somatic cell lineages. Neural progenitor cells may be isolated from differentiating ES cell cultures and induced to form mature neurons. Embryonic stem cells provide a model to study early human embryology, an investigational tool for discovery of novel growth factors and medicines, and a potential source of cells for use in transplantation therapy.

Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro.

The earliest stages of development in most animals, including the few mammalian species that have been investigated, are regulated by maternally inherited information. Dependence on expression of the embryonic genome cannot be detected until the mid two-cell stage in the mouse, the four-cell stage in the pig (J. Osborn & C. Polge, personal communication), and the eight-cell stage in the sheep. Information about the timing of activation of the embryonic genome in the human is of relevance not only to the therapeutic practice of in vitro fertilization and embryo transfer (IVF), but more importantly for the successful development of techniques for the preimplantation diagnosis of certain inherited genetic diseases. We describe here changes in the pattern of polypeptides synthesized during the pre-implantation stages of human development, and demonstrate that some of the major qualitative changes which occur between the four- and eight-cell stages are dependent on transcription. In addition, it appears that cleavage is not sensitive to transcriptional inhibition until after the four-cell stage.

Human gene expression first occurs between the four- and eight-cell stages of preimplantation development

The derivation of neural progenitor cells from human embryonic stem (ES) cells is of value both in the study of early human neurogenesis and in the creation of an unlimited source of donor cells for neural transplantation therapy. Here we report the generation of enriched and expandable preparations of proliferating neural progenitors from human ES cells. The neural progenitors could differentiate in vitro into the three neural lineages--astrocytes, oligodendrocytes, and mature neurons. When human neural progenitors were transplanted into the ventricles of newborn mouse brains, they incorporated in large numbers into the host brain parenchyma, demonstrated widespread distribution, and differentiated into progeny of the three neural lineages. The transplanted cells migrated along established brain migratory tracks in the host brain and differentiated in a region-specific manner, indicating that they could respond to local cues and participate in the processes of host brain development. Our observations set the stage for future developments that may allow the use of human ES cells for the treatment of neurological disorders.

Neural progenitors from human embryonic stem cells.

Oxidative stress is involved in the aetiology of defective embryo development. Reactive oxygen species (ROS) may originate from embryo metabolism and/or embryo surroundings. Embryo metabolism generates ROS via several enzymatic mechanisms. The relative contribution of each source seems different depending on the species, the stage of development, and the culture conditions. Several exogenous factors and culture conditions can enhance the production of ROS by embryos. ROS can alter most types of cellular molecules, and also induce development block and retardation. Multiple mechanisms of embryo protection against ROS exist, and these have complementary actions. External protection, present in follicular and tubal fluids, mainly comprises non-enzymatic antioxidants such as hypotaurine, taurine and ascorbic acid. Internal protection mainly comprises antioxidant enzymes: superoxide dismutase, glutathione peroxidase and gamma-glutamylcysteine synthetase. Transcripts encoding for these enzymes are present in the oocyte, embryo and oviduct. It may be important that these transcripts are stored during oocyte maturation in order to allow the embryo to acquire the aptitude to develop. It is now common to add antioxidant compounds to culture media. Nevertheless, maintaining the pro-oxidant-antioxidant equilibrium in embryos through such supplementation is a complex problem. Further studies are necessary to limit oxidative stress during embryo culture.

Oxidative stress and protection against reactive oxygen species in the pre-implantation embryo and its surroundings

Pluripotent stem cells provide a potential solution to current epidemic rates of heart failure by providing human cardiomyocytes to support heart regeneration. Studies of human embryonic-stem-cell-derived cardiomyocytes (hESC-CMs) in small-animal models have shown favourable effects of this treatment. However, it remains unknown whether clinical-scale hESC-CM transplantation is feasible, safe or can provide sufficient myocardial regeneration. Here we show that hESC-CMs can be produced at a clinical scale (more than one billion cells per batch) and cryopreserved with good viability. Using a non-human primate model of myocardial ischaemia followed by reperfusion, we show that cryopreservation and intra-myocardial delivery of one billion hESC-CMs generates extensive remuscularization of the infarcted heart. The hESC-CMs showed progressive but incomplete maturation over a 3-month period. Grafts were perfused by host vasculature, and electromechanical junctions between graft and host myocytes were present within 2 weeks of engraftment. Importantly, grafts showed regular calcium transients that were synchronized to the host electrocardiogram, indicating electromechanical coupling. In contrast to small-animal models, non-fatal ventricular arrhythmias were observed in hESC-CM-engrafted primates. Thus, hESC-CMs can remuscularize substantial amounts of the infarcted monkey heart. Comparable remuscularization of a human heart should be possible, but potential arrhythmic complications need to be overcome.

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Human embryonic-stem-cell-derived cardiomyocytes regenerate non-human primate hearts

The idea that sperm cells could be used as an effective tool for introducing exogenous DNA into an oocyte at fertilization is generally regarded with scepticism. However, in recent years, several investigators have been working on different aspects of this intriguing research topic. In the present review, their results are summarised and discussed. Sections have been dedicated to the way DNA molecules bind to spermatozoa of different species, to the events regulating such binding, to the fate of the DNA within sperm cells, and to the attempts made to produce transgenic animals with this method. The data available on the interaction between DNA and spermatozoa begin to explain how this event takes place and how it is regulated. However, the stable integration of exogenous genes into the genome of adult animals mediated by sperm cells is a very rare event, although several reports describe forms of partial success. Available evidence suggests that changes to the DNA molecules, oc curring mostly within the oocyte, represents the limiting step in the production of transgenic animals using spermatozoa as vectors of exogenous genes. At present there are not enough data to understand what happens to sperm-associated DNA upon its entrance into the oocyte at fertilization. Therefore, it has not yet been resolved whether sperm-mediated gene transfer is a possible way to manipulate the genome or if evolution has imposed some unsurpassable barriers to its use

Spermatozoa, DNA binding and transgenic animals.

Chronic mastitis is associated with altered ovarian follicle development in dairy cattle.

Alkylphenolic compounds are a widespread family of xenoestrogens. High concentrations of these substances are present in sewage sludge that is spread on arable land and pasture as fertilizer. Because of their known endocrine system-disrupting activity, alkylphenols represent a potential risk for the reproductive health of farm animals. In this study, the impact of p-tert-octylphenol (OP) on the developmental competence of bovine oocytes was evaluated. Endocrine activity of OP was investigated for its effect on estrogen receptors α and β (ERα and ERβ) and progesterone receptor (PR) mRNA levels. Cumulus-oocyte complexes (COCs) were exposed during in vitro maturation to serial concentrations of OP (1–0.0001 μg/ml) and were compared with vehicle-treated controls and a group of COCs treated with 17β-estradiol (E2). A dose-related decrease in the percentage of oocytes that completed maturation after 24 h and in oocyte fertilization competence was observed at doses of OP as low as 0.01 μg/ml. Groups tre...

Toxic Effects of In Vitro Exposure to p-tert-Octylphenol on Bovine Oocyte Maturation and Developmental Competence

It is evident that members of several growth factor families are actively involved in embryogenesis from its earliest phases. Several reports also indicate the oviduct as a possible source of growth factors, suggesting an active role of this organ in mammalian embryonic development. The aim of this study was to investigate the presence of activin/inhibin subunits in bovine oviduct since activin is a well-characterised morphogen in amphibian development. The presence of transcripts for α. βA, and βB subunits was investigated by analysing oviduct epithelial cells mRNA with reverse transcription-polymerase chain reaction (RT-PCR). Moreover, antisera specific for the three subunits were used for the Western blot analysis of the proteins secreted by oviduct epithelial cells in vitro and for their immunohistochemical localisation in different oviductal regions. Oviduct epithelial cells expressed only the βA-subunit gene. Immunoreactive material was present among in vitro secreted proteins, indicating that the transcript is translated into a polypeptide that has been localised in the epithelium of both the ampullary and isthmic tract of the organ. Consistent with these results, the antisera for the α and βB subunits did not recognise any specific antigen either among secreted proteins or in the sections. These results indicate that βA subunit gene is expressed in bovine oviduct epithelial cells, and the protein is secreted in vitro and can be found along the whole extension of the organ. In the absence of α or βB subunits, this suggests that activin A is present in bovine oviduct. Such a finding would be consistent with an embry-otrophic activity of this organ, but definitive conclusions on the target tissue and the specific functions of oviductal activin require further studies. © 1995 Wiley-Liss, Inc.

Fulvio Gandolfi

Papers

Spermatozoa, DNA binding and transgenic animals.

Chronic mastitis is associated with altered ovarian follicle development in dairy cattle.

Toxic Effects of In Vitro Exposure to p-tert-Octylphenol on Bovine Oocyte Maturation and Developmental Competence

Activin βA subunit is expressed in bovine oviduct

Early embryonic signals: embryo-maternal interactions before implantation