Induced neuronal differentiation of human embryonic stem cells.

Human embryonic stem (ES) cells are pluripotent cells capable of forming differentiated embryoid bodies (EBs) in culture. We examined the ability of growth factors under controlled conditions to increase the number of human ES cell-derived neurons. Retinoic acid (RA) and nerve growth factor (betaNGF) were found to be potent enhancers of neuronal differentiation, eliciting extensive outgrowth of processes and the expression of neuron-specific molecules. Our findings show that human ES cells have great potential to become an unlimited cell source for neurons in culture. These cells may then be used in transplantation therapies for neural pathologies.

Establishment of human embryonic stem cell-transfected clones carrying a marker for undifferentiated cells.

Human embryonic stem (ES) cells are pluripotent cell lines that have been derived from the inner cell mass (ICM) of blastocyst stage embryos [1--3]. They are characterized by their ability to be propagated indefinitely in culture as undifferentiated cells with a normal karyotype and can be induced to differentiate in vitro into various cell types [1, 2, 4-- 6]. Thus, human ES cells promise to serve as an unlimited cell source for transplantation. However, these unique cell lines tend to spontaneously differentiate in culture and therefore are difficult to maintain. Furthermore, colonies may contain several cell types and may be composed of cells other than pluripotent cells [1, 2, 6]. In order to overcome these difficulties and establish lines of cells with an undifferentiated phenotype, we have introduced a reporter gene that is regulated by a promoter of an ES cell-enriched gene into the cells. For the introduction of DNA into human ES cells, we have established a specific transfection protocol that is different from the one used for murine ES cells. Human ES cells were transfected with enhanced green fluorescence protein (EGFP), under the control of murine Rex1 promoter. The transfected cells show high levels of GFP expression when in an undifferentiated state. As the cells differentiate, this expression is dramatically reduced in monolayer cultures as well as in the primitive endoderm of early stage (simple) embryoid bodies (EBs) and in mature EBs. The undifferentiated cells expressing GFP can be analyzed and sorted by using a Fluorescence Activated Cell Sorter (FACS). Thus, we have established lines of human ES cells in which only undifferentiated cells are fluorescent, and these cells can be followed and selected for in culture. We also propose that the pluripotent nature of the culture is made evident by the ability of the homogeneous cell population to form EBs. The ability to efficiently transfect human ES cells will provide the means to study and manipulate these cells for the purpose of basic and applied research.

Effects of eight growth factors on the differentiation of cells derived from human embryonic stem cells.

Human embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of in vitro fertilized human blastocysts. We examined the potential of eight growth factors [basic fibroblast growth factor (bFGF), transforming growth factor beta1 (TGF-beta1), activin-A, bone morphogenic protein 4 (BMP-4), hepatocyte growth factor (HGF), epidermal growth factor (EGF), beta nerve growth factor (betaNGF), and retinoic acid] to direct the differentiation of human ES-derived cells in vitro. We show that human ES cells that have initiated development as aggregates (embryoid bodies) express a receptor for each of these factors, and that their effects are evident by differentiation into cells with different epithelial or mesenchymal morphologies. Differentiation of the cells was assayed by expression of 24 cell-specific molecular markers that cover all embryonic germ layers and 11 different tissues. Each growth factor has a unique effect that may result from directed differentiation and/or cell selection, and we can divide the overall effects of the factors into three categories: growth factors (Activin-A and TGFbeta1) that mainly induce mesodermal cells; factors (retinoic acid, EGF, BMP-4, and bFGF) that activate ectodermal and mesodermal markers; and factors (NGF and HGF) that allow differentiation into the three embryonic germ layers, including endoderm. None of the growth factors directs differentiation exclusively to one cell type. This analysis sets the stage for directing differentiation of human ES cells in culture and indicates that multiple human cell types may be enriched in vitro by specific factors.

Differentiation of human embryonic stem cells into embryoid bodies compromising the three embryonic germ layers.

BACKGROUND: Embryonic stem (ES) cells are lines of cells that are isolated from blastocysts. The murine ES cells were demonstrated to be true pluripotent cells as they differentiate into all embryonic lineages. Yet, in vitro differentiation of rhesus ES cells was somewhat inconsistent and disorganized. The recent isolation of human ES cells calls for exploring their pluripotential nature. MATERIALS AND METHODS: Human ES cells were grown in suspension to induce their differentiation into embryoid bodies (EBs). The differentiation status of the human ES cells and EBs was analyzed by following the expression pattern of several lineage-specific molecular markers using reverse transcription polymerase chain reaction (RT-PCR) and in situ hybridization. RESULTS: Here we report the induction in vitro of cystic embryoid bodies from human ES cells. Our findings demonstrate induction of expression of cell-specific genes during differentiation of the human ES cells into EBs. In the human EBs, we could show a characteristic regional expression of embryonic markers specific to different cellular lineages, namely, zeta-globin (mesoderm), neurofilament 68Kd (ectoderm), and alpha-fetoprotein (endoderm). Moreover, we present a synchronously pulsing embryoid body that expresses the myocardium marker alpha-cardiac actin. In addition, dissociating the embryoid bodies and plating the cells as monolayers results in multiple morphologies, among them cells with neuronal appearance that express neurofilament 68Kd chain. CONCLUSION: Human ES cells can reproducibly differentiate in vitro into EBs comprising the three embryonic germ layers. The ability to induce formation of human embryoid bodies that contain cells of neuronal, hematopoietic and cardiac origins will be useful in studying early human embryonic development as well as in transplantation medicine.

The tmp gene, encoding a membrane protein, is a c-Myc target with a tumorigenic activity.

The c-Myc oncoprotein induces cell proliferation and transformation through its activity as a transcription factor. Uncovering the genes regulated by c-Myc is an essential step for understanding these processes. We recently isolated the tumor-associated membrane protein gene, Tmp, from a c-myc-induced mouse brain tumor. Here we show that Tmp is specifically highly expressed in mammary tumors and T-cell lymphomas which develop in c-myc transgenic mice, suggesting that Tmp expression is a general characteristic of c-Myc-induced tumors. In addition, Tmp expression is induced upon serum stimulation of fibroblasts as shown in a time course closely correlated with c-myc expression. We have isolated the Tmp promoter region and identified a putative c-Myc binding element, CACGTG, located in the first intron of the gene. We show here that constructs containing the Tmp regulatory region fused to a reporter gene are activated by c-Myc through this CACGTG element and that the c-Myc-Max protein complex can bind to this element. Moreover, an inducible form of c-Myc, the MycER fusion protein, can activate the endogenous Tmp gene. We also show that Tmp-overexpressing fibroblasts induce rapidly growing tumors when injected into nude mice, suggesting that Tmp may possess a tumorigenic activity. Thus, TMP, a member of a novel family of membrane glycoproteins with a suggested role in cellular contact, is a c-Myc target and is possibly involved in c-Myc-induced transformation.

Involvement of Myc targets in c-myc and N-myc induced human tumors.

The myc proto-oncogenes are transcription factors that directly regulate the expression of other genes, by binding to the specific DNA sequence, CACGTG. Among the target genes for c-Myc regulation are ECA39, p53, ornithine decarboxylase (ODC), alpha-prothymosin and Cdc25A. In this study we examined the involvement of c-Myc target genes in human oncogenesis induced by c-myc or N-myc. In MCF-7 breast cancer cells, the induction of c-myc expression by estrogen was followed by the induction of all the Myc targets that we examined, indicating that those genes can serve as c-Myc targets in human oncogenesis. Moreover, in breast tumors exhibiting c-myc overexpression, several Myc targets were also overexpressed. A clear correlation between the expression of c-myc and its targets was also detected in Burkitt's lymphomas, which involve a specific translocation of c-myc gene, but not in other lymphoma cells. Yet, in cells derived from a neuronal origin the pattern of expression of Myc targets was more complex. In a neuroepithelioma cell line that overexpresses c-myc, only some targets were expressed. In addition in neuroblastomas, in which N-myc is amplified and overexpressed, only ODC was overexpressed in all cell lines, while all other target genes were expressed in only some of the cell lines. The more complex expression pattern found for the Myc targets in neuroblastomas suggests that genes that were identified originally as targets for c-Myc regulation may be regulated by N-Myc, but other cell specific factors are also needed for transcription of the target genes.

ECA39 is regulated by c-Myc in human and by a Jun/Fos homolog, Gcn4, in yeast.

myc oncogenes are transcription factors regulating the level of expression of other genes. Using a subtraction/coexpression strategy, a murine genetic target for Myc regulation was isolated. To further characterize this target gene, named ECA39, we have recently isolated the human, nematode and budding yeast homologs of the mouse gene. The recognition site for Myc binding, located 3' to the start site of transcription in the mouse gene, is conserved in the human homolog. Transfection experiments demonstrated that the Myc binding site of the human gene, mediates activation of a reporter gene in response to over-expression of c-myc. The activation was better executed when the c-Myc binding element was positioned downstream to the promoter, which is the usual position of the c-Myc DNA binding element in its genetic targets. The tissue specific expression of human ECA39 during embryogenesis is similar to that of the mouse homolog. Moreover, ECA39 is expressed in c-myc induced human tumors. It is expressed in Burkitt's lymphoma (where c-myc is translocated and activated) but not in non Burkitt's B-cell lymphoma or in T-cell lymphoma. Thus, it seems that ECA39 is a target for c-myc oncogenesis in humans. In yeast, where c-myc is absent, the ECA39 sequences lack the c-Myc binding element. However, the promoter region of the yeast ECA39 harbors several Gcn4 binding elements. Moreover, ECA39 is markedly down regulated in cells deleted for gcn4, and deletion of Gcn4 binding elements down regulated the transcription from ECA39 promoter. We thus suggest that ECA39 is a target for c-Myc regulation in mammals, while in yeast the regulator is not c-Myc but the c-Jun/c-Fos homolog - Gcn4.

ECA39, a conserved gene regulated by c-Myc in mice, is involved in G1/S cell cycle regulation in yeast.

The c-myc oncogene has been shown to play a role in cell proliferation and apoptosis. The realization that myc oncogenes may control the level of expression of other genes has opened the field to search for genetic targets for Myc regulation. Recently, using a subtraction/coexpression strategy, a murine genetic target for Myc regulation, called EC439, was isolated. To further characterize the ECA39 gene, we set out to determine the evolutionary conservation of its regulatory and coding sequences. We describe the human, nematode, and budding yeast homologs of the mouse ECA39 gene. Identities between the mouse ECA39 protein and the human, nematode, or yeast proteins are 79%, 52%, and 49%, respectively. Interestingly, the recognition site for Myc binding, located 3' to the start site of transcription in the mouse gene, is also conserved in the human homolog. This regulatory element is missing in the ECA39 homologs from nematode or yeast, which also lack the regulator c-myc. To understand the function of ECA39, we deleted the gene from the yeast genome. Disruption of ECA39 which is a recessive mutation that leads to a marked alteration in the cell cycle. Mutant haploids and homozygous diploids have a faster growth rate than isogenic wild-type strains. Fluorescence-activated cell sorter analyses indicate that the mutation shortens the G1 stage in the cell cycle. Moreover, mutant strains show higher rates of UV-induced mutations. The results suggest that the product of ECA39 is involved in the regulation of G1 to S transition.