Stem cells, pluripotency and nuclear reprogramming

The recent demonstration of in vitro reprogramming using transduction of 4 transcription factors by Yamanaka and colleagues represents a major advance in the field. However, major questions regarding the mechanism of in vitro reprogramming need to be understood and will be one focus of the talk. During cellular reprogramming only a small fraction of cells become induced pluripotent stem cells (iPSCs). Previous analyses of gene expression during reprogramming were based on populations of cells and impeded identification of events at the single‐cell level. We utilized two different gene expression technologies to profile 48 genes in single cells at various stages during the reprogramming process. Analysis of early stages revealed considerable variation in gene expression between cells in contrast to late stages. We show that Esrrb, Utf1, Lin28, and Dppa2 have a better potential to predict cells that will become iPSCs, compared to Fbxo15, Fgf4, and Oct4, which were previously suggested to be markers of reprogramming. Our data suggest that stochastic gene expression early in reprogramming is followed by a late sequential phase with Sox2 activation upstream in a gene expression hierarchy. Finally, we demonstrate that subsets of downstream factors derived from the sequential phase can activate the pluripotency circuitry. A major impediment in realizing the potential of ES and iPS cells to study human diseases is the inefficiency of gene targeting. Using Zn finger or TALEN mediated genome editing we have established efficient protocols to target expressed and silent genes in human ES and iPS cells. Finally, our progress in using iPS cells for therapy and for the study of complex human diseases will be summarized.