Forced expression of the homeobox-containing gene Pem blocks differentiation of embryonic stem cells.

Similarities in the differentiation of mouse embryos and ES cell embryoid bodies suggest that aspects of early mammalian embryogenesis can be studied in ES cell embryoid bodies. In an effort to understand the regulation of cellular differentiation during early mouse embryogenesis, we altered the expression of the Pem homeobox-containing gene in ES cells. Pem is normally expressed in the preimplantation embryo and expressed in a lineage-restricted fashion following implantation, suggesting a role for Pem in regulating cellular differentiation in the early embryo. Here, we show that the forced expression of Pem from the mouse Pgk-1 promoter in ES cells blocks the in vitro and in vivo differentiation of the cells. In particular, embryoid bodies produced from these Pgk-Pem ES cells do not differentiate into primitive endoderm or embryonic ectoderm, which are prominent features of early embryoid bodies from normal ES cells. This Pgk-Pem phenotype is also different from the null phenotype, as embryoid bodies derived from ES cells in which endogenous Pem gene expression has been blocked show a pattern of differentiation similar to that of normal ES cells. When the Pgk-Pem ES cells were introduced into subcutaneous sites of nude mice, only undifferentiated EC-like cells were found in the teratomas derived from the injected cells. The Pem-dependent block of ES cell differentiation appears to be cell autonomous; Pgk-Pem ES cells did not differentiate when mixed with normal, differentiating ES cells. A block to ES cell differentiation, resulting from the forced expression of Pem, can also be produced by the forced expression of the nonhomeodomain region of Pem. These studies are consistent with a role for Pem in regulating the transition between undifferentiated and differentiated cells of the early mouse embryo.     

The responsiveness of embryonic stem cells to alpha and beta interferons provides the basis of an inducible expression system for analysis of developmental control genes.

Embryonic stem (ES) cells, derived from the inner cell mass of the preimplantation mouse embryo, are used increasingly as an experimental tool for the investigation of early mammalian development. The differentiation of these cells in vitro can be used as an assay for factors that regulate early developmental decisions in the embryo, while the effects of altered gene expression during early embryogenesis can be analyzed in chimeric mice generated from modified ES cells. The experimental versatility of ES cells would be significantly increased by the development of systems which allow precise control of heterologous gene expression. In this paper, we report that ES cells are responsive to alpha and beta interferons (IFNs). This property has been exploited for the development of inducible ES cell expression vectors, using the promoter of the human IFN-inducible gene, 6-16. The properties of these vectors have been analyzed in both transiently and stably transfected ES cells. Expression was minimal or absent in unstimulated ES cells, could be stimulated up to 100-fold by treatment of the cells with IFN, and increased in linear fashion with increasing levels of IFN. High levels of induced expression were maintained for extended periods of time in the continuous presence of the inducing signal or following a 12-h pulse with IFN. Treatment of ES cells with IFN did not affect their growth or differentiation in vitro or compromise their developmental potential. This combination of features makes the 6-16-based expression vectors suitable for the functional analysis of developmental control control genes in ES cells.     

Mouse and human embryonic stem cell models of hematopoiesis: past, present, and future

Human embryonic stem (ES) cells provide a unique model and an important resource to analyze early hematopoietic development. Other systems to study mammalian hematopoiesis include mouse ES cells, dissection of timed mouse embryos, or use of human postnatal hematopoietic tissue typically isolated from bone marrow or umbilical cord blood. All these models have particular strengths and weaknesses. The extensive studies on murine hematopoiesis provide a basis for work on the human developmental system. Since there are likely some important species differences, use of human ES cells now provides an optimal means to evaluate basic cellular and molecular mechanisms that regulate the beginning stages of human blood development, prior to derivation of hematopoietic stem cells (HSCs). Eventually, research on human ES cells may provide an alternative source of HSCs and other blood products for hematopoietic cell transplantation or other cellular therapies.     

Forced Expression of the Homeobox-Containing Gene Pem Blocks Differentiation of Embryonic Stem Cells

Similarities in the differentiation of mouse embryos and ES cell embryoid bodies suggest that aspects of early mammalian embryogenesis can be studied in ES cell embryoid bodies. In an effort to understand the regulation of cellular differentiation during early mouse embryogenesis, we altered the expression of the Pem homeobox-containing gene in ES cells. Pem is normally expressed in the preimplantation embryo and expressed in a lineage-restricted fashion following implantation, suggesting a role for Pem in regulating cellular differentiation in the early embryo. Here, we show that the forced expression of Pem from the mouse Pgk-1 promoter in ES cells blocks the in vitro and in vivo differentiation of the cells. In particular, embryoid bodies produced from these Pgk-Pem ES cells do not differentiate into primitive endoderm or embryonic ectoderm, which are prominent features of early embryoid bodies from normal ES cells. This Pgk-Pem phenotype is also different from the null phenotype, as embryoid bodies derived from ES cells in which endogenous Pem gene expression has been blocked show a pattern of differentiation similar to that of normal ES cells. When the Pgk-Pem ES cells were introduced into subcutaneous sites of nude mice, only undifferentiated EC-like cells were found in the teratomas derived from the injected cells. The Pem-dependent block of ES cell differentiation appears to be cell autonomous;Pgk-Pem ES cells did not differentiate when mixed with normal, differentiating ES cells. A block to ES cell differentiation, resulting from the forced expression of Pem, can also be produced by the forced expression of the nonhomeodomain region of Pem. These studies are consistent with a role for Pem in regulating the transition between undifferentiated and differentiated cells of the early mouse embryo.     

Lineage choice and differentiation in mouse embryos and embryonic stem cells

The use of embryonic stem (ES) cells for generating healthy tissues has the potential to revolutionize therapies for human disease or injury, for which there are currently no effective treatments. Strategies for manipulating stem cell differentiation should be based on knowledge of the mechanisms by which lineage decisions are made during early embryogenesis. Here, we review current research into the factors influencing lineage differentiation in the mouse embryo and the application of this knowledge to in vitro differentiation of ES cells. In the mouse embryo, specification of tissue lineages requires cell-cell interactions that are influenced by coordinated cell migration and cellular neighborhood mediated by the key WNT, FGF, and TGFbeta signaling pathways. Mimicking the cellular interactions of the embryo by providing appropriate signaling molecules in culture has enabled the differentiation of ES cells to be directed predominately toward particular lineages. Multistep strategies incorporating the provision of soluble factors known to influence lineage choices in the embryo, coculture with other cells or tissues, genetic modification, and selection for desirable cell types have allowed the production of ES cell derivatives that produce beneficial effects in animal models. Increasing the efficiency of this process can only result from a better understanding of the molecular control of cell lineage determination in the embryo.     

Self-renewal vs. differentiation of mouse embryonic stem cells

Embryonic stem (ES) cells are typically derived from the inner cell mass of the preimplantation blastocyst and can both self-renew and differentiate into all the cells and tissues of the embryo. Because they are pluripotent, ES cells have been used extensively to analyze gene function in development via gene targeting. The embryonic stem cell is also an unsurpassed starting material to begin to understand a critical, largely inaccessible period of development. If their differentiation could be controlled, they would also be an important source of cells for transplantation to replace cells lost through disease or injury or to replace missing hormones or genes. Traditionally, ES cells have been differentiated in suspension culture as embryoid bodies, named because of their similarity to the early postimplantation-staged embryo. Unlike the pristine organization of the early embryo, differentiation in embryoid bodies appears to be largely unpatterned, although multiple cell types form. It has recently been possible to separate the desired cell types from differentiating ES cells in embryoid bodies by using cell-type-restricted promoters driving expression of either antibiotic resistance genes or fluorophores such as EGFP. In combination with growth factor exposure, highly differentiated cell types have successfully been derived from ES cells. Recent technological advances such as RNA interference to knock down gene expression in ES cells are also producing enriched populations of cells and elucidating gene function in early development.     

Potential of embryonic stem cells

Embryonic stem (ES) cells are pluripotent cell lines established from undifferentiated embryonic cells characterized by nearly unlimited self-renewal and differentiation capacity. During differentiation in vitro, ES cells were found to be able to develop into specialized somatic cells types and to recapitulate processes of early embryonic development. These properties allow to use ES cells as model system for studying early embryonic development by gain- or loss-of-function approaches, or to investigate the effects of drugs and environmental factors on differentiation and cell function in embryotoxicity and pharmacology. Now, ES cells derived of human blastocysts may be used for the generation of somatic precursor or differentiated cells in cell and tissue therapy. The review presents data of mouse ES cell differentiation and gives an outlook on future perspectives and problems of using human ES cells in regenerative medicine.     

Murine embryonic stem cell in vitro differentiation: applications to the study of vascular development

The present review summarizes knowledge accumulated during the last decade concerning in vitro endothelial differentiation from embryonic stem (ES) cells. There is now growing evidence that ES cells may provide a powerful model system to determine the cellular and molecular mechanisms of vascular development. ES cells differentiate into the endothelial lineage by successive maturation steps recapitulating in vivo events observed in the embryo. Further maturation of ES-derived embryoid bodies either in three dimensional gels or in confrontation cultures with tumor spheroids can also provide a model of physiological or tumoral angiogenesis. The data obtained from experimental in vitro differentiation of genetically modified mouse ES cells highlight the potential and the complementarity of this model system to in vivo gene knock out studies. We also consider and discuss some of the potential applications of ES cell technology in vascular biology for future directions in basic research and medicine, by manipulation of differentiation and the generation of cell populations for analysis and transplantation for therapeutic use.     

Derivation, propagation and differentiation of human embryonic stem cells

Embryonic stem (ES) cells are in vitro cultivated pluripotent cells derived from the inner cell mass (ICM) of the embryonic blastocyst. Attesting to their pluripotency, ES cells can be differentiated into representative derivatives of all three embryonic germ layers (endoderm, ectoderm and mesoderm) both in vitro and in vivo. Although mouse ES cells have been studied for many years, human ES cells have only more recently been derived and successfully propagated. Many biochemical differences and culture requirements between mouse and human ES cells have been described, yet despite these differences the study of murine ES cells has provided important insights into methodologies aimed at generating a greater and more in depth understanding of human ES cell biology. One common feature of both mouse and human ES cells is their capacity to undergo controlled differentiation into spheroid structures termed embryoid bodies (EBs). EBs recapitulate several aspects of early development, displaying regional-specific differentiation programs into derivatives of all three embryonic germ layers. For this reason, EB formation has been utilised as an initial step in a wide range of studies aimed at differentiating both mouse and human ES cells into a specific and desired cell type. Recent reports utilising specific growth factor combinations and cell-cell induction systems have provided alternative strategies for the directed differentiation of cells into a desired lineage. According to each one of these strategies, however, a relatively high cell lineage heterogeneity remains, necessitating subsequent purification steps including mechanical dissection, selective media or fluorescent or magnetic activated cell sorting (FACS and MACS, respectively). In the future, the ability to specifically direct differentiation of human ES cells at 100% efficiency into a desired lineage will allow us to fully explore the potential of these cells in the analysis of early human development, drug discovery, drug testing and repair of damaged or diseased tissues via transplantation.     

Pluripotency in the light of the developmental hourglass

The hourglass model of development postulates divergence in early and late embryo development bridged by a period of developmental constraint at mid‐embryogenesis. Recently, molecular support for the hourglass model of development has accumulated, with the emphasis on studies using zebrafish and Drosophila species. Across mammals, the hourglass model and specifically divergence in early development has thus far received little attention. Divergence in mammalian pre‐implantation development is particularly interesting because of its potential impact on derivation of pluripotent embryonic stem cells. Here, we review recent findings that support the hourglass model of development. We provide striking examples of variation in key events in mammalian peri‐implantation development and their potential consequences for pluripotency of embryonic stem cell lines, including mechanisms of cell signalling and differentiation, gene regulatory networks, X‐chromosome inactivation, and epigenetic regulation. The variation in these processes indicates divergence in early mammalian development as was postulated by the hourglass model of development. We discuss the naive and primed states of pluripotency in light of this developmental divergence and their implications for human pluripotent stem cell states.     

The circadian clock starts ticking at a developmentally early stage

Although overt diurnal rhythms of behavior do not begin until well after birth, molecular studies suggest that the circadian clock may begin much earlier at a cellular level: mouse embryonic fibroblasts, for example, already possess robust clocks. By multiple criteria, we found no circadian clock present in mouse embryonic stem cells. Nevertheless, upon their differentiation into neurons, circadian gene expression was observed. In the first steps along the pathway from ES cells to neurons, a neural precursor cell (NPC) line already showed robust circadian oscillations. Therefore, at a cellular level, the circadian clock likely begins at the very earliest stages of mammalian development.     

Teratocarcinoma stem cells as a model for differentiation in the mouse embryo

Embryonal carcinoma (EC) cells, which are the malignant stem cells of teratocarcinomas, are considered similar to early embryo cells. The EC cells can be grown in vitro, and many of them can be experimentally induced to differentiate; upon differentiation, the cells become benign. Here we review some of the changes that take place in the cellular and molecular characteristics of murine F9 EC cells as they differentiate into endodermal cells. Upon differentiation of F9 cells, distinct changes occur in their cell surface molecules, cytoskeleton-associated proteins and cell adhesion properties. Simultaneously, the rate of cell proliferation decreases due to a dramatic increase in duration of G1 and S phases of the cell cycle. The changes in gene expression and cell behavior occurring during endodermal differentiation of EC cells closely resemble those occurring when the endoderm differentiates in the embryo. Teratocarcinoma stem cell lines may thus be exploited to enhance understanding of both teratoma-type neoplasms and embryonic development.     

An efficient system to establish multiple embryonic stem cell lines carrying an inducible expression unit

The growing use of mouse embryonic stem (ES) cells in research emphasizes their importance in studies of molecular mechanisms that maintain pluripotency and direct cellular differentiation. Although systems for regulatable transgene expression are essential for fine analysis of cellular processes at the molecular level, a strategy for the establishment of multiple ES cell lines carrying any of these systems has not yet been described. Here, we report our development of the ROSA-TET system, an effective system for the establishment of multiple ES cell lines carrying a tetracycline (Tc)-regulatable transgene at the Gt (ROSA)26asSor (ROSA26) locus. This system contains a knock-in step of a construct carrying both loxP and its mutant sequences into the ROSA26 locus, followed by a subsequent exchange step that introduces a cDNA to be Tc-regulated to the locus using the recombinase-mediated cassette exchange reaction. Both steps are demonstrated to give desired clones with high efficiency, suggesting that this system can be introduced readily into any ES cell lines, leading to the simultaneous establishment of multiple cell lines carrying different Tc-regulated cDNAs. We believe that use of this system will strongly accelerate molecular biological research using ES cells.     

The core endodermal gene network of vertebrates: combining developmental precision with evolutionary flexibility

Embryonic development combines paradoxical properties: it has great precision, it is usually conducted at breakneck speed and it is flexible on relatively short evolutionary time scales, particularly at early stages. While these features appear mutually exclusive, we consider how they may be reconciled by the properties of key early regulatory networks. We illustrate these ideas with the network that controls development of endoderm progenitors. We argue that this network enables precision because of its intrinsic stability, self propagation and dependence on signalling. The network enables high developmental speed because it is rapidly established by maternal inputs at multiple points. In turn these properties confer flexibility on an evolutionary time scale because they can be initiated in many ways, while buffering essential progenitor cell populations against changes in their embryonic environment on both evolutionary and developmental time scales. Although stable, these networks must be capable of rapid dissolution as cell differentiation progresses. While we focus on the core early endodermal network of vertebrates, we argue that these properties are likely to be general in early embryonic stem cell populations, such as mammalian ES cells.