PGD-derived human embryonic stem cell lines as a powerful tool for the study of human genetic disorders.

Human embryonic stem (ES) cells are derived from the inner cell mass (ICM) of blastocyst embryos. They are established from spare embryos that have been obtained by in vitro fertilization (IVF) and donated for research purposes. The ICM-derived cell lines have two unique properties, they can be propagated indefinitely in culture and have the potential to develop into practically any cell type in vitro and in vivo. Human embryonic stem (hES) cells carrying specific mutations can be used as a valuable tool for studying genetic disorders in human. One favorable approach to obtain such mutant ES cell lines is their derivation from affected preimplantation genetic diagnosed (PGD) embryos. This review focuses on the importance of deriving human ES cell lines from genetically abnormal embryos, especially in cases where no good cellular and/or animal models exist.

Activation and transposition of endogenous retroviral elements in hypomethylation induced tumors in mice.

Genomewide DNA hypomethylation is a consistent finding in human tumors, but the importance of this change for human tumorigenesis remains an open question. We have previously reported that mice carrying a hypomorphic allele for the maintenance DNA methyltransferase (Dnmt1(chip/-)) are hypomethylated and develop thymic lymphomas, demonstrating that genomewide DNA hypomethylation can induce tumors. Hypomethylated cells exhibit inherent chromosomal instability, which is revealed in the lymphomas as a consistent trisomy of chromosome 15. We now report another aspect of the molecular basis for tumor development upon DNA hypomethylation. Seven out of 16 hypomethylation-induced lymphomas were found to contain an intracisternal A particle (IAP) somatic insertion in the middle of the Notch1 genomic locus, leading to generation of an oncogenic form of Notch1 in the tumors. This finding suggests that the molecular basis for hypomethylation-induced tumors in this model involves chromosomal instability events accompanied by activation of endogenous retroviral elements. Our findings validate the proposed role of DNA methylation in suppression of transposable elements in mammalian cells and demonstrate the importance of DNA methylation for normal cell function as well as the potential consequences of spontaneously occurring or chemically induced DNA hypomethylation.

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.