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 d erived from the inner cell mass (ICM) of blastocyst stage embryos [1-3], Th ey are characterized by their ability to be propagated indefinitely in cult ure 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. Ho wever, these unique cell lines tend to spontaneously differentiate in cultu re and therefore are difficult to maintain. Furthermore, colonies may conta in several cell types and may be composed of cells other than pluripotent c ells II, 2, 6]. In order to overcome these difficulties and establish lines of: cells with an undifferentiated phenotype, we have introduced a reporte r 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 establishe d a specific transfection protocol that is different from the one used for murine ES cells. Human ES cells were transfected with enhanced green fluore scence protein (EGFP), under the control of murine Rex1 promoter. The trans fected 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 cell s expressing GFP can be analyzed and sorted by using a Fluorescence Activat ed 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 cel l population to form EBs, The ability to efficiently transfect human ES cel ls will provide the means to study and manipulate these cells for the purpo se of basic and applied research.