NOVOSTEM · Deprogramming of the mammalian genome for stem cell production

Final Activity Report Summary - NOVOSTEM (Deprogramming of the mammalian genome for stem cell production)

Cell based therapies potentially allow for alternative treatment of several human diseases. The search for an autologous cell source merits the investigation of novel means to de-program patient-derived differentiated cells into pluripotent stem cells with therapeutic potentials. The technique of cloning by somatic cell nuclear transfer (SCNT), where a differentiated somatic cell is transferred into an enucleated oocyte, has in several mammalian species proven that the oocyte possesses factors that can entirely de- and re-program the somatic cell genome. In order to further develop and evaluate our nucleolus-associated markers for genomic de- and re-programming, we have focused on genomic de- and re-programming in bovine in vitro produced (IVP) and SCNT embryos, respectively. It was shown, that RNA polymerase I (RPI) transcription and de novo protein synthesis in bovine IVP embryos are required for development of functional nucleoli. Failure in embryonic RPI activation during embryonic genome activation leads to specific aberrations in nucleolar development and such aberrations may serve as markers for early embryo quality. In SCNT embryos, the genome was transcriptionally inactive already 0.5 h post activation (hpa), and around 4 hpa the nucleus seemed to be de-programmed and under ooplasmic influence. The proteins of the ribosomal RNA (rRNA) transcriptional and processing machinery were located to substructes of nucleolus precursor bodies (NPBs), and probably will be used in the following cell cycles during the embryonic rRNA gene transcriptional activation. An apparent concentration of somatic cell components around the somatic cell nucleus for the initial 0.5-3 hpa in SCNT embryos may hamper the import of ooplasmic factors to the somatic cell nucleus during this period, which may represent a particularly permissive period for de-programming. Genomic de-programming may allow for production of pluripotent stem cells from somatic cells, and the SCNT-based de-programming may serve as a model for studying genomic de-programming ex ovo. It has been demonstrated that purified ooplasmic extract from non-mammalian species such as Xenopus leavis may exert de-programming effects. We utilised such extracts to de-program human 293T lymphocytes and bovine fibroblasts. Pluripotency factors, as e.g. OCT4, were first detected at 8 h after extract treatment, however, with decreasing amounts from Day 1 and onwards. At Day 10, no pluripotency factors were detected signalling that only a partial and temporal genomic de-programming had occurred. Moreover, extract treatment led to nuclear envelope changes and nucleolar disassembly. Again, these were only temporary changes. We believe that the knowledge generated in this research training project will increase the number of checkpoints suitable for evaluation of genomic de-programming in biomedical research. This knowledge may potentially be paving the way for autologous cell therapy.

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