Weak acids may act as teratogens by accumulating in the basic milieu of the early mammalian embryo

Among the eleven drugs or chemicals which are well-documented human teratogens, eight (or their main metabolites) are weak acids whereas none is a weak base. Moreover, 23 out of 32 acids tested have been found to be teratogenic in at least one animal species. The acidic property of drugs may therefore be an important determinant of teratogenicity. We demonstrate here that the intracellular pH (pHi) of the mouse and rat embryo is higher than that of maternal plasma, as determined by the relative accumulation of dimethadione. The antiepileptic drug valproic acid and its pharmacologically active unsaturated metabolite accumulate in embryonic tissue to higher concentrations than in maternal plasma, whereas the essentially neutral amide of valproic acid (valpromide) or ethosuximide do not accumulate in the embryo; we further demonstrate in the rat that the pHi of the embryo decreases with advancing gestation; in general agreement with the pH partition hypothesis, the exposure of the embryo to valproic acid also decreases significantly during that period. Furthermore, the amides of two weak acid teratogens, valpromide and methoxyacetamide, and the imide ethosuximide, are much less teratogenic than their acid counterparts. Our results suggest that weakly acidic drugs, by virtue of their physico-chemical nature, accumulate in the early embryo with its relatively high pHi.     

Schwann cells induce morphological transformation of sensory neurones in vitro

Cell-cell interactions are thought to play a crucial part in determining the developmental fate of vertebrate cells and regulating their subsequent differentiation. In the peripheral nervous system, for example, signals from neuronal axons determine whether or not some Schwann cells wrap their plasma membrane concentricially around the axon to form a myelin sheath. Moreover, there is some evidence that the interactions between Schwann cells and neurones are not all one way: for example, Schwann cells are thought to provide signals for neuronal sprouting and regeneration. However, there are no clear examples in which Schwann cells have been shown to influence the normal development of neurones. Here I have used purified populations of embryonic sensory neurones and Schwann cells to demonstrate that Schwann cells have a dramatic influence on the development of these neurones. In the presence of Schwann cells, but not other cell types, the sensory neurones undergo a morphological transformation from an immature bipolar form to a mature pseudo-unipolar form. This provides a striking example of the importance of glial cells for neuronal development.     

Evidence from reversal of handedness in C. elegans embryos for early cell interactions determining cell fates

Many animals with overall bilateral symmetry also exhibit some left-right asymmetries with generally invariant handedness. Therefore, the left-right embryonic axis must have a consistent polarity, whose origins and subsequent effects on development are not understood. Caenorhabditis elegans exhibits such left-right asymmetries at all developmental stages. The embryonic cell lineage is asymmetric as well: although the animal is generally bilaterally symmetric, many of its contralaterally analogous cells arise from different lineages on the two sides of the embryo. I accomplished reversal of embryonic handedness by micromanipulation at the 6-cell stage, which resulted in mirror-image but otherwise normal development into healthy, fertile animals with all the usual left-right asymmetries reversed. This result demonstrates that in the 6-cell embryo the pair of anterior (AB) blastomeres on the right is equivalent to the pair on the left, and that the extensive differences in fates between lineally homologous derivatives of these cells on the two sides of the animal must be dictated by cell interactions, most of which are likely to occur early in embryogenesis.     

Properties and applications of embryonic stem cells

Mouse embryonic stem (ES) cells are pluripotent cells derived from the early embryo and can be propagated stably in undifferentiated state in vitro. They retain the ability to differentiate into all cell types found in the embryonic and adult body in vivo, and can be induced to differentiate into many cell types under appropriate culture conditions in vitro. Using these properties, people have set up various differentiated systems of many cell types and tissues in vitro. Through analysis of these systems, one can identify novel bioactive factors and reveal mechanisms of cell differentiation and organogenesis. ES cell-derived differentiated cells can also be applied to cell transplantation therapy. In addition, we summarized the features and potential applications of human ES cells.     

Pluripotency of spermatogonial stem cells from adult mouse testis

Embryonic germ cells as well as germline stem cells from neonatal mouse testis are pluripotent and have differentiation potential similar to embryonic stem cells, suggesting that the germline lineage may retain the ability to generate pluripotent cells. However, until now there has been no evidence for the pluripotency and plasticity of adult spermatogonial stem cells (SSCs), which are responsible for maintaining spermatogenesis throughout life in the male. Here we show the isolation of SSCs from adult mouse testis using genetic selection, with a success rate of 27%. These isolated SSCs respond to culture conditions and acquire embryonic stem cell properties. We name these cells multipotent adult germline stem cells (maGSCs). They are able to spontaneously differentiate into derivatives of the three embryonic germ layers in vitro and generate teratomas in immunodeficient mice. When injected into an early blastocyst, SSCs contribute to the development of various organs and show germline transmission. Thus, the capacity to form multipotent cells persists in adult mouse testis. Establishment of human maGSCs from testicular biopsies may allow individual cell-based therapy without the ethical and immunological problems associated with human embryonic stem cells. Furthermore, these cells may provide new opportunities to study genetic diseases in various cell lineages.     

Inhibition of pluripotential embryonic stem cell differentiation by purified polypeptides

Murine embryonic stem (ES) cells are pluripotent cell lines established directly from the early embryo which can contribute differentiated progeny to all adult tissues, including the germ-cell lineage, after re-incorporation into the normal embryo. They provide both a cellular vector for the generation of transgenic animals and a useful system for the identification of polypeptide factors controlling differentiation processes in early development. In particular, medium conditioned by Buffalo rat liver cells contains a polypeptide factor, ES cell differentiation inhibitory activity (DIA), which specifically suppresses the spontaneous differentiation of ES cells in vitro, thereby permitting their growth as homogeneous stem cell populations in the absence of heterologous feeder cells. ES cell pluripotentiality, including the ability to give rise to functional gametes, is preserved after prolonged culture in Buffalo rat liver media as a source of DIA. Here, we report that purified DIA is related in structure and function to the recently identified hematopoietic regulatory factors human interleukin for DA cells and leukaemia inhibitory factor. DIA and human interleukin DA/leukaemia inhibitory factor have thus been identified as related multifunctional regulatory factors with distinct biological activities in both early embryonic and hematopoietic stem cell systems.     

Scatter factor is a fibroblast-derived modulator of epithelial cell mobility

Various factors are known to regulate cell growth and differentiation, but less is known of agents which affect movement and positioning, particularly in epithelial-mesenchymal interactions. Cultured human embryo fibroblasts release a protein with a relative molecular mass (Mr) of approximately 50,000 (50K) that affects epithelial cells by causing a disruption of junctions, an increase in local motility and a scattering of contiguous sheets of cells. To investigate specificity, a range of cells has been examined for the ability to produce the factor and for sensitivity to its action. Most freshly isolated normal epithelia and epithelia from cell lines of normal tissue, but not epithelia from tumour cell lines or fibroblasts, were sensitive to scatter factor. In contrast, production of the factor, as identified by activity and by chromatography, was restricted to embryonic fibroblasts and certain variants of 3T3 and BHK21 cells and their transformed derivatives. We conclude that the scatter factor is a paracrine effector of epithelial-mesenchymal interaction, which affects the intercellular connections and mobility of normal epithelial cells. The factor might be involved in epithelial migration, such as occurs in embryogenesis or wound healing.     

Myeloid leukaemia inhibitory factor maintains the developmental potential of embryonic stem cells

Embryonic stem (ES) cells, the totipotent outgrowths of blastocysts, can be cultured and manipulated in vitro and then returned to the embryonic environment where they develop normally and can contribute to all cell lineages. Maintenance of the stem-cell phenotype in vitro requires the presence of a feeder layer of fibroblasts or of a soluble factor, differentiation inhibitory activity (DIA) produced by a number of sources; in the absence of DIA the ES cells differentiate into a wide variety of cell types. We recently noted several similarities between partially purified DIA and a haemopoietic regulator, myeloid leukaemia inhibitory factor (LIF), a molecule which induces differentiation in M1 myeloid leukaemic cells and which we have recently purified, cloned and characterized. We demonstrate here that purified, recombinant LIF can substitute for DIA in the maintenance of totipotent ES cell lines that retain the potential to form chimaeric mice.     

Early motor activity drives spindle bursts in the developing somatosensory cortex

Sensorimotor coordination emerges early in development. The maturation period is characterized by the establishment of somatotopic cortical maps, the emergence of long-range cortical connections, heightened experience-dependent plasticity and spontaneous uncoordinated skeletal movement. How these various processes cooperate to allow the somatosensory system to form a three-dimensional representation of the body is not known. In the visual system, interactions between spontaneous network patterns and afferent activity have been suggested to be vital for normal development. Although several intrinsic cortical patterns of correlated neuronal activity have been described in developing somatosensory cortex in vitro, the in vivo patterns in the critical developmental period and the influence of physiological sensory inputs on these patterns remain unknown. We report here that in the intact somatosensory cortex of the newborn rat in vivo, spatially confined spindle bursts represent the first and only organized network pattern. The localized spindles are selectively triggered in a somatotopic manner by spontaneous muscle twitches, motor patterns analogous to human fetal movements. We suggest that the interaction between movement-triggered sensory feedback signals and self-organized spindle oscillations shapes the formation of cortical connections required for sensorimotor coordination.     

Inhibition of suppressor T-cell development following deoxyguanosine administration

The expression of immunodeficiency in patients with specific purine enzyme defects indicates a crucial role of the purine salvage pathway in the acquisition and expression of normal immune function. One current hypothesis links the failure of normal lymphocyte development in these diseases to the accumulation of deoxynucleotide triphosphates. In our studies of human in vitro IgM responses, we observed that antigen-induced T-suppressor cell activity was abrogated in the presence of micromolar concentrations of deoxyguanosine (dGuo). In contrast, more than 1,000-fold higher resistance to dGuo was found for both noin-proliferative T-helper cell activity and the differentiation and proliferation of the precursor B lymphocytes for direct haemolytic plaque forming cells (PFC). To determine whether these observations could have in vivo relevance, we monitored the generation of murine T-suppressor cells, capable of abrogating a primary IgM response. It was found that dGuo (but not guanosine) selectively inhibited the in vivo development of T-suppressor cells.     

Capillary endothelial cells express basic fibroblast growth factor, a mitogen that promotes their own growth

Angiogenesis, the formation of new capillaries, which is observed in embryonic and injured tissue and is particularly prominent in the vicinity of solid tumours, involves the migration and proliferation of capillary endothelial cells. It is probably triggered by agents, such as basic fibroblast growth factor (bFGF), thought to be released from tissues adjacent to proliferating capillaries. As well as being a potent inducer of cell division in capillary endothelial cells in vitro, bFGF can act as an angiogenic agent in vivo. It is present in a wide variety of richly vascularized tissues including brain, pituitary, retina, adrenal gland, kidney, corpus luteum, placenta and various tumours. So far, however, the normal bFGF-producing cell species in these tissues have not been identified. We report here that capillary endothelial cells express the bFGF gene, that they produce and release bFGF and that bFGF derived from them can stimulate the proliferation of capillary endothelial cells. We conclude that bFGF can act as a self-stimulating growth factor for capillary endothelial cells, and that it is possible that the formation of new capillaries is induced by capillary endothelial cells themselves.     

Human cardiovascular progenitor cells develop from a KDR+ embryonic-stem-cell-derived population

The functional heart is comprised of distinct mesoderm-derived lineages including cardiomyocytes, endothelial cells and vascular smooth muscle cells. Studies in the mouse embryo and the mouse embryonic stem cell differentiation model have provided evidence indicating that these three lineages develop from a common Flk-1(+) (kinase insert domain protein receptor, also known as Kdr) cardiovascular progenitor that represents one of the earliest stages in mesoderm specification to the cardiovascular lineages. To determine whether a comparable progenitor is present during human cardiogenesis, we analysed the development of the cardiovascular lineages in human embryonic stem cell differentiation cultures. Here we show that after induction with combinations of activin A, bone morphogenetic protein 4 (BMP4), basic fibroblast growth factor (bFGF, also known as FGF2), vascular endothelial growth factor (VEGF, also known as VEGFA) and dickkopf homolog 1 (DKK1) in serum-free media, human embryonic-stem-cell-derived embryoid bodies generate a KDR(low)/C-KIT(CD117)(neg) population that displays cardiac, endothelial and vascular smooth muscle potential in vitro and, after transplantation, in vivo. When plated in monolayer cultures, these KDR(low)/C-KIT(neg) cells differentiate to generate populations consisting of greater than 50% contracting cardiomyocytes. Populations derived from the KDR(low)/C-KIT(neg) fraction give rise to colonies that contain all three lineages when plated in methylcellulose cultures. Results from limiting dilution studies and cell-mixing experiments support the interpretation that these colonies are clones, indicating that they develop from a cardiovascular colony-forming cell. Together, these findings identify a human cardiovascular progenitor that defines one of the earliest stages of human cardiac development.     

The end of the beginning for pluripotent stem cells.

Pluripotent stem cells can be expanded seemingly indefinitely in culture, maintain a normal karyotype and have the potential to generate any cell type in the body. As such they represent an incredible resource for the repair of diseased or damaged tissues in our bodies. These cells also promise to open a new window into the embryonic development of our species.     

Platelet-derived growth factor from astrocytes drives the clock that times oligodendrocyte development in culture

The various cell types in a multicellular animal differentiate on a predictable schedule but the mechanisms responsible for timing cell differentiation are largely unknown. We have studied a population of bipotential glial (O-2A) progenitor cells in the developing rat optic nerve that gives rise to oligodendrocytes beginning at birth and to type-2 astrocytes beginning in the second postnatal week. Whereas, in vivo, these O-2A progenitor cells proliferate and give rise to postimitotic oligodendrocytes over several weeks, in serum-free (or low-serum) culture they stop dividing prematurely and differentiate into oligodendrocytes within two or three days. The normal timing of oligodendrocyte development can be restored if embryonic optic-nerve cells are cultured in medium conditioned by type-1 astrocytes, the first glial cells to differentiate in the nerve: in this case the progenitor cells continue to proliferate, the first oligodendrocytes appear on the equivalent of the day of birth, and new oligodendrocytes continue to develop over several weeks, just as in vivo. Here we show that platelet-derived growth factor (PDGF) can replace type-1-astrocyte-conditioned medium in restoring the normal timing of oligodendrocyte differentiation in vitro and that anti-PDGF antibodies inhibit this property of the appropriately conditioned medium. We also show that PDGF is present in the developing optic nerve. These findings suggest that type-1-astrocyte-derived PDGF drives the clock that times oligodendrocyte development.     

Caenorhabditis elegans gene ced-9 protects cells from programmed cell death.

The gene ced-9 of the nematode Caenorhabditis elegans acts to protect cells from programmed cell death. A mutation that abnormally activates ced-9 prevents the cell deaths that occur during normal C. elegans development. Conversely, mutations that inactivate ced-9 cause cells that normally live to undergo programmed cell death; these mutations result in embryonic lethality, indicating that ced-9 function is essential for development. The ced-9 gene functions by negatively regulating the activities of other genes that are required for the process of programmed cell death.     

Embryonic and extraembryonic stem cell lines derived from single mouse blastomeres

The most basic objection to human embryonic stem (ES) cell research is rooted in the fact that ES cell derivation deprives embryos of any further potential to develop into a complete human being. ES cell lines are conventionally isolated from the inner cell mass of blastocysts and, in a few instances, from cleavage stage embryos. So far, there have been no reports in the literature of stem cell lines derived using an approach that does not require embryo destruction. Here we report an alternative method of establishing ES cell lines-using a technique of single-cell embryo biopsy similar to that used in pre-implantation genetic diagnosis of genetic defects-that does not interfere with the developmental potential of embryos. Five putative ES and seven trophoblast stem (TS) cell lines were produced from single blastomeres, which maintained normal karyotype and markers of pluripotency or TS cells for up to more than 50 passages. The ES cells differentiated into derivatives of all three germ layers in vitro and in teratomas, and showed germ line transmission. Single-blastomere-biopsied embryos developed to term without a reduction in their developmental capacity. The ability to generate human ES cells without the destruction of ex utero embryos would reduce or eliminate the ethical concerns of many.     

Monoclonal antibodies identify a cell-surface antigen associated with an activated cellular oncogene

A variety of antigens have been identified on the surface of the malignant cell. However, identical antigens are often found on non-malignant cells of the same or different histological origin, or of a different stage of embryonic development. Many of these tumour-associated antigens appear to be only incidentally expressed on neoplastic cells. Clearly, it would be of great interest to identify cell-surface antigens whose expression is associated specifically with the transformed state and linked directly with the mechanisms responsible for transformation. The detection of activated cellular oncogenes in human and animal cancer cells by the technique of DNA transfection has allowed the isolation of genetic elements which are thought to have a critical role in malignancy. Here, in an effort to identify cell-surface antigens associated with the neoplastic process, we have generated hybridomas which secrete monoclonal antibodies that react specifically with cell-surface determinants found on NIH 3T3 cells transformed by transfection with a group of rat neuroblastoma oncogenes. These antibodies bind to and immunoprecipitate a phosphoprotein of relative molecular mass 185,000 (185 K) from a DNA donor rat neuroblastoma and 13 independent rat neuroblastoma DNA transfectants. There was no antibody reactivity with normal NIH 3T3 cells or with NIH 3T3 cells transformed by various other agents.