Do IPS Colonies Derive From One Cell Or Many Cells?

Like ES (embryonic stem) cells, IPS (induced pluripotent stem) cells can differentiate into all cell types of the body, facilitating potential cell therapy strategies. IPS cells are produced by introducing four important regulatory genes into fibroblasts or other fully differentiated cells, reverting the cells to a primitive state similar to ES cells. In addition to their research and clinical values, IPS cells are important because they by pass the ethical issues associated with ES cells. It is not clear if IPS colonies derive from a single cell or a mixture of cells, which has implications regarding the propagation and therapeutic value of the cells. A mixed population of IPS cells may involve multiple gene expression profiles and synergistic support of growth, which makes propagation and genetic manipulation more complicated. Preliminary examinations of this issue using mixtures of GFP⁺ and GFP^- cells strongly suggested that IPS colonies are heterogeneous. However, this result may have been confounded by silencing and down regulation of the GFP reporter. I propose to repeat the experiment in a more controlled fashion. First, two fibroblast cultures will be transfected with GFP and M-Cherry reporters. A line of reporter-positive cells will be maintained for a number of generations to be sure that the reporters express consistently. Cells will be combined in one dish and transfected with the regulatory genes (Oct3/4, Sox2, Klf4, c-Myc) and IPS formation observed. Clonal versus mixed colonies will be determined on the basis of reporter expression.