Influence of clinorotation on embryoid body morphology

The compensatory effects of gravitation at early stages of embryonic development have been investigated using the slow clinorotation of embryoid bodies generated from R1 mouse embryonic stem cells. An analysis of semithin sections (1–2μm) and an electron microscopy study of embryoid bodies revealed cells at different stages of maturation. A significant decrease (compared to the control) in embryonic stem cells undergoing apoptosis, as well as in noticeably reduced hollow areas, were found in clinorotated embryonic bodies. We propose that the lack of large cysts may be caused by the delay in initial differentiation and morphogenesis stages associated with autophagy processes in embryonic bodies.     

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 regulation of embryonic stem cell differentiation by leukaemia inhibitory factor (LIF)

LIF (leukaemia inhibitory factor) is commonly used to maintain mouse embryonic stem cells in an undifferentiated state. These cells spontaneously differentiate when allowed to aggregate in the absence of LIF, forming embryoid bodies in which early embryonic cell lineages develop. Using embryoid bodies cultured in the presence and absence of LIF, we show that although LIF inhibited the development of visceral and parietal endodermal cells, it did not affect the differentiation of the primitive endodermal cell precursors of these extraembryonic cell lineages. Furthermore, deposition of the basement membrane produced by the primitive endodermal cells, which separates them from the remaining cells of the embryoid body, still occurred. The differentiation of primitive ectodermal cells and their progeny was inhibited by LIF, as evidenced by the lack of expression of FGF-5, muscle, and neuronal markers. However, cavitation of the embryoid body and maintenance of the cells in contact with the primitive endodermal basement membrane as an epiblast epithelium still occurred normally in the presence of LIF. These results indicate that cavitation and formation of the epiblast epithelium are regulated by mechanisms distinct from those controlling the differentiation of epiblast cell lineages. Furthermore, although epithelium formation and cavitation do not require the differentiation of visceral endodermal cells, the results are consistent with the hypothesis that the primitive endodermal basement membrane is sufficient to induce the epithelialization of undifferentiated embryonic stem cells necessary for cavitation.     

Antiproliferative and cytotoxic effects of different type cytostatics on mouse pluripotent stem and teratocarcinoma cells

Pluripotent stem cells are able to proliferate indefinitely and differentiate in vitro into various cell types. However, in most cases in vitro differentiation of the pluripotent stem cells is asynchronous and incomplete, and the residual undifferentiated cells can initiate teratoma development after transplantation into recipients. These features of the pluripotent stem cells are the major issue for development of safe cell therapy technologies based on pluripotent stem cells. Considering significant resemblance of growth rates of pluripotent stem and cancer cells we investigated antiproliferative and cytotoxic effects of different type cytostatics (mitomycin C, etoposide, vinblastine and cycloheximide) on the undifferentiated and differentiating mouse embryonic stem cells, embryonic germ cells, blastocyst and on mouse embryonal teratocarcinoma cells and mouse embryonic fibroblasts. The findings showed that all cytostatics used induced both antiproliferative effects and acute toxic processes in undifferentiated pluripotent stem cells and embryonal teratocarcinoma cells whereas these effects were less in differentiating embryonic stem cells and embryonic fibroblast. Moreover, the trophoblast cells of mouse blastocysts were less sensitive to damaging effects of cytostatics than inner cell mass cells. The examination of deferred effects of cytostatics revealed that the effects of mitomycin C, etoposide and vinblastine, but not cycloheximide, were irreversible because survived cells were not able to proliferate. Nevertheless, the numbers of embryonic fibroblasts exposed to etoposide or vinblastine remained unchanged while vast majority of undifferentiated pluripotent cells treated underwent apoptosis. Thus, diverse effects of etoposide and vinblastine on the undifferentiated pluripotent stem cells and differentiated embryonic cells allow us to consider these cytostatics and their analogs as drug-candidates for selective elimination of the residual undifferentiated pluripotent stem cells from population of differentiating cells. These findings demonstrate for the first time the possibility of selective elimination of undifferentiated pluripotent stem cells using cytostatic drugs approved for clinic practice. However, to improve effectiveness and safety of this approach and to prevent mutagenic, carcinogenic and teratogenic effects on undifferentiated pluripotent stem cells and their differentiated cell derivatives large-scale studies of cytostatic effects using different experimental design and active doses must be performed.     

Protein synthesis and differentiation in a clonal line of teratocarcinoma and in preimplantation mouse embryo.

Undifferentiated cells of a clonal line of teratocarcinoma can differentiate in vitro into embryoid bodies with morphological and biochemical features of early mouse embryo. During the first step of differentiation protein synthesis has been analysed by 2 dimensional gel electrophoresis. While new proteins are synthesized, the synthesis of others turned off with the appearance of endodermal cells in embryoid bodies. We have compared protein synthesis during teratocarcinoma differentiation and during early mouse embryogenesis at three stages of mouse preimplantation embryo. The results demonstrate that only the late blastocyst protein synthesis pattern shows most of the polypeptides identified in the differentiated protein synthesis pattern of teratocarcinoma. In contrast, protein synthesis during the early stages of mouse embryonic development is very different from protein synthesis in undifferentiated teratocarcinoma.     

Activation of retinoic acid receptor signaling coordinates lineage commitment of spontaneously differentiating mouse embryonic stem cells in embryoid bodies

Retinoid signaling has been implicated in embryonic stem cell differentiation. Here we present a systematic analysis of gene expression changes in mouse embryonic stem cells (mESCs), during their spontaneous differentiation into embryoid bodies and the effect of all-trans retinoic acid (ATRA) on this process. We show that retinoic acid is present in the serum and is sufficient to activate retinoid signaling at a basal level in undifferentiated mESCs. This signal disappears during embryoid body formation. However exogenously added ATRA resets the spontaneous differentiation programs in embryoid bodies and initiates a distinct genetic program. These data suggest that retinoid signaling not only promotes a particular pathway but also acts as a context dependent general coordinator of the differentiation states in embryonic stem cells.     

Use of developmental marker genes to define temporal and spatial patterns of differentiation during embryoid body formation.

Mouse embryonic stem cells are pluripotent cells that are derived from the inner cell mass of blastocysts. When induced to synchronously enter a program of differentiation in vitro, they form embryoid bodies that contain cells of the mesodermal, hematopoietic, endothelial, muscle, and neuronal lineages. Here, we used a panel of marker genes with early expression within the germ layers (oct-3, Brachyury T, Fgf-5, nodal, and GATA-4) or a variety of lineages (flk-1, Nkx-2.5, EKLF, and Msx3) to determine how progressive differentiation of embryoid bodies in culture correlated with early postimplantation development of mouse embryos. Using RNA in situ hybridization, we found that the temporal and spatial relationships existing between these marker genes in vivo were maintained also in vitro. Studying the onset of marker gene expression allowed us also to determine the time course of differentiation during the formation of embryoid bodies. Thus, stages equivalent to embryogenesis between implantation and the beginning of gastrulation (4.5-6.5 d.p.c.) occur within the first two days of embryoid body differentiation. Between days 3 and 5, embryoid bodies contain cell lineages found in embryos during gastrulation at 6.5 to 7.0 d.p.c., and after day 6 in culture, embryoid bodies are equivalent to early organogenesis-stage embryos (7.5 d.p.c.). In addition, we demonstrate that the panel of developmental markers can be applied in a screen for stage- or lineage-specific genes. Reporter gene expression from entrapment vector insertions can be co-localized with expression of specific markers within the same cell during embryoid body formation as well as during embryogenesis. Our results thus demonstrate the power of embryoid body formation as an in vitro model system to study early lineage determination and organogenesis in mammals, and indicate that they will prove to be useful tools for identifying developmental genes whose expression is restricted to particular lineages.     

Differentiation of Pluripotent Embryonic Stem Cells in the Peritoneal Cavity of Irradiated Mice

We studied the behavior and differentiation of pluripotent embryonic stem cells of R1 mice in vivo. Undifferentiated embryonic stem cells and differentiating embryoid bodies implanted in the abdominal cavity of irradiated mice were shown to form tumors containing the derivatives of all germ layers. Cells of the embryoid bodies form tumors two weeks after implantation, while undifferentiated embryonic stem cells form tumors only by week three.     

Cell autonomous sorting and surface positioning in the formation of primitive endoderm in embryoid bodies

The differentiation and formation of the primitive endoderm in early embryos can be mimicked in vitro by the aggregation of embryonic stem cells to form embryoid bodies. We present morphological evidence that primitive endoderm cells often first locate in the interior of embryoid bodies and subsequently migrate to the surface. Cell mixing experiments indicate that surface positioning is an intrinsic property of endoderm epithelial cells. Moreover, Disabled-2 (Dab2) is required for surface sorting and positioning of the endoderm cells: when Dab2 expression was eliminated, the differentiated endoderm epithelial cells distributed throughout the interior of the embryoid bodies. Surprisingly, E-cadherin is dispensable for primitive endoderm differentiation and surface sorting in embryoid bodies. These results support the model that primitive endoderm cells first emerge in the interior of the inner cell mass and are subsequently sorted to the surface to form the primitive endoderm.     

Autophagy in human embryonic stem cells

Autophagy (macroautophagy) is a degradative process that involves the sequestration of cytosolic material including organelles into double membrane vesicles termed autophagosomes for delivery to the lysosome. Autophagy is essential for preimplantation development of mouse embryos and cavitation of embryoid bodies. The precise roles of autophagy during early human embryonic development, remain however largely uncharacterized. Since human embryonic stem cells constitute a unique model system to study early human embryogenesis we investigated the occurrence of autophagy in human embryonic stem cells. We have, using lentiviral transduction, established multiple human embryonic stem cell lines that stably express GFP-LC3, a fluorescent marker for the autophagosome. Each cell line displays both a normal karyotype and pluripotency as indicated by the presence of cell types representative of the three germlayers in derived teratomas. GFP expression and labelling of autophagosomes is retained after differentiation. Baseline levels of autophagy detected in cultured undifferentiated hESC were increased or decreased in the presence of rapamycin and wortmannin, respectively. Interestingly, autophagy was upregulated in hESCs induced to undergo differentiation by treatment with type I TGF-beta receptor inhibitor SB431542 or removal of MEF secreted maintenance factors. In conclusion we have established hESCs capable of reporting macroautophagy and identify a novel link between autophagy and early differentiation events in hESC.     

Genome-wide gene expression analysis in mouse embryonic stem cells

Embryonic stem cell studies have generated great interest, due to their ability to form a wide variety of matured cells. However, there remains a poor understanding of mechanisms regulating the cell state of embryonic stem cells (ESCs) and of the genes they express during early differentiation. Gene expression analysis may be a valuable tool to elucidate either the molecular pathways involved in self-renewal and pluripotency, or early differentiation and to identify potential molecular therapy targets. The aim of this study was to characterize at the molecular level the undifferentiated mouse ESC state and the early development towards embryoid bodies. To attempt this issue, we performed CodeLink Mouse Uniset I 20K bioarrays in a well-characterized mouse ESC line, MES3, 3- and 7 day-old embryoid bodies and we compared our findings with those in adult tissue cells. Gene expression results were subsequently validated in a commercial stem cell line, CGR8 (ATCC). Significance Analysis of Microarrays (SAM) was used to identify statistically significant changes in microarray data. We identified 3664 genes expressed at significantly greater levels in MES3 stem cells than in adult tissue cells, which included 611 with 3-fold higher gene expression levels versus the adult cells. We also investigated the gene expression profile during early embryoid body formation, identifying 2040 and 2243 genes that were up-regulated in 3- and 7- day-old embryoid bodies, respectively. Our gene expression results in MES3 cells were partially confirmed in CGR8 cells, showing numerous genes that are expressed in both mouse stem cells. In conclusion, our results suggest that commonly expressed genes may be strong candidates for involvement in the maintenance of a pluripotent and undifferentiated phenotype and in early development.     

Differential expression of lectin receptors in germ layers of the mouse egg cylinder and teratocarcinomas

Receptors for three lectins with restricted specificities, namely fucose-binding protein of Lotus tetragonolobus (FBP), peanut agglutinin (PNA) and Dolichos biflorus agglutinin (DBA), were distinctively located in 6- and 7-day mouse embryos and in embryoid bodies of teratocarcinoraa OTT6050 grown in vivo. Thus, FBP reacted mainly with the inner cells (embryonic ectoderm and teratocarcinoma stem cells), DBA reacted with the outer cells (endoderm) and PNA reacted with all the germ layers including mesoderm. Upon in vitro culture of the embryoid bodies, the exposed stem cells express DBA receptors. Since the receptors for the three lectins in teratocarcinomas are known to be carried by the large carbohydrate chains characteristic of early embryonic cells, the present result suggests that terminal structure of the large carbohydrates is altered according to the direction of the differentiation or to the position of the cells in embryos and in teratocarcinomas.     

Altered patterns of differentiation in karyotypically abnormal human embryonic stem cells

Upon prolonged culture, human embryonic stem (hES) cells undergo adaptation, exhibiting decreased population doubling times and increased cloning efficiencies, often associated with karyotypic changes. To test whether culture adaptation influences the patterns of differentiation of hES cells, we compared the expression of genes indicative of distinct embryonic lineages in the embryoid bodies produced from two early passage, karyotypically normal hES cell lines, and two late passage, karyotypically abnormal hES cell lines. One of the abnormal lines was a subline of one of the normal early passage lines. The embryoid bodies from each of the lines showed evidence of extensive differentiation. However, there were differences in the expression of several genes, indicating that the culture adapted hES cells show altered patterns of differentiation compared to karyotypically normal hES cells. The loss of induction of alphafetoprotein in the culture-adapted cells was especially marked, suggesting that they had a reduced capacity to produce extra-embryonic endoderm. These changes may contribute to the growth advantages of genetically variant cells, not only by reflecting an increased tendency to self renewal rather than to differentiate, but also by reducing spontaneous differentiation to derivatives that themselves may produce factors that could induce further differentiation of undifferentiated stem cells.     

Influence of regulatory genes of type 1 human immunodeficiency virus on proliferation and differentiation of murine embryonic stem cells

Spontaneous formation of embryoid bodies and subsequent differentiation of some cells into cardiomyocytes were demonstrated on murine embryonic stem cells of R1 line. The lines of embryonic stem cells were obtained that had been transfected with genetic constructs carrying expressing regulatory genes of the human immunodeficiency virus tat and nef and "green protein" gene (GFP). The transfection of embryonic stem cells with the gene tat stimulated their proliferative activity, while this activity decreased in the cells transfected with the gene nef. The time necessary for the formation of embryoid bodies by all lines of transfected cells was similar to that in the control cells. In the cultures of cells transfected with nef and tat, the number of embryoid bodies and the percentage of embryoid bodies with contracting cardiomyocytes were higher and lower than in the control, respectively. Thus, an inverse correlation was observed between the effects of regulatory genes of the human immunodeficiency virus on proliferation and differentiation embryonic stem cells.     

Nodal inhibits differentiation of human embryonic stem cells along the neuroectodermal default pathway

Genetic studies in fish, amphibia, and mice have shown that deficiency of Nodal signaling blocks differentiation into mesoderm and endoderm. Thus, Nodal is considered as a major inducer of mesendoderm during gastrulation. On this basis, Nodal is a candidate for controlling differentiation of pluripotent human embryonic stem cells (hESCs) into tissue lineages with potential clinical value. We have investigated the effect of Nodal, both as a recombinant protein and as a constitutively expressed transgene, on differentiation of hESCs. When control hESCs were grown in chemically defined medium, their expression of markers of pluripotency progressively decreased, while expression of neuroectoderm markers was strongly upregulated, thus revealing a neuroectodermal default mechanism for differentiation in this system. hESCs cultured in recombinant Nodal, by contrast, showed prolonged expression of pluripotency marker genes and reduced induction of neuroectoderm markers. These Nodal effects were accentuated in hESCs expressing a Nodal transgene, with striking morphogenetic consequences. Nodal-expressing hESCs developing as embryoid bodies contained an outer layer of visceral endoderm-like cells surrounding an inner layer of epiblast-like cells, each layer having distinct gene expression patterns. Markers of neuroectoderm were not upregulated during development of Nodal-expressing embryoid bodies, nor was there induction of markers for definitive mesoderm or endoderm differentiation. Moreover, the inner layer expressed markers of pluripotency, characteristic of undifferentiated hESCs and of epiblast in mouse embryos. These results could be accounted for by an inhibitory effect of Nodal-induced visceral endoderm on pluripotent cell differentiation into mesoderm and endoderm, with a concomitant inhibition of neuroectoderm differentiation by Nodal itself. There could also be a direct effect of Nodal in the maintenance of pluripotency. In summary, analysis of the Nodal-expressing phenotype suggests a function for the transforming growth factor-beta (TGF-beta) growth factor superfamily in pluripotency and in early cell fate decisions leading to primary tissue layers during in vitro development of pluripotent human stem cells. The effects of Nodal on early differentiation illustrate how hESCs can augment mouse embryos as a model for analyzing mechanisms of early mammalian development.     

Differentiation-promoting effects of mammary-derived growth inhibitor (MDGI) on pluripotent mouse embryonic stem cells

Pluripotent embryonic stem cells (line BLC6), when cultivated in vitro as embryoid bodies and injected subcutaneously into syngeneic mice, form teratocarcinomas consisting of embryonal carcinoma cells and differentiated tissues of all three primary germ layers. In order to study the possible effects of the mammary-derived growth inhibitor (MDGI) on the differentiation pattern of the tumors developing in the mice, BLC6 cell-derived embryoid bodies were treated in vitro for 4 days with either MDGI or a synthetic peptide composed of the C-terminal 11 amino acids of MDGI. In those tumors, significantly more differentiated neural tissue and lesser proportions of undifferentiated embryonic carcinoma cells (ECC) were found in the MDGI-and peptide-treated groups, compared with controls. The results are discussed with respect to a possible differentiation-promoting capacity of MDGI.     

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.