We will develop and provide technologies useful for other members of the Consortium and the scientific community. These include a heterokaryon system to elucidate the critical network of reprogramming factors;combined with new bioinformatics web-based tools for analysis. A focus of the proposal is non-viral derivation of induced pluripotent stem cells;using fusion proteins synthesized by cell-free protein synthesis technology in a Protein Chemistry Core available to the Consortium. In addition, we can distribute hESC lines, maintain an hESC/germ cell tissue bank, and provide critical services such as karyotypic analysis of stem cell lines and single cell analysis via microfluidic methods. Finally, the induced pluripotent stem cells (iPSCs) generated in this proposal will have many uses: studies of human developmental pathways;drug screening;creating patient- specific lines for studying disease;and for pre-clinical studies leading to their therapeutic use. We will deploy local expertise in translational vascular and stem cell biology in three related projects: 1) We will develop and refine a non-viral technology for iPSC induction. We will synthesize fusion proteins that each comprise one of the known reprogramming factors linked to a translocating factor for cytoplasmic translocation, nuclear localization and transactivation of genes involved in reprogramming. The concentration and duration of exposure to each of the reprogramming factors will be optimized. The iPSCs generated will be characterized for markers of pluripotency, global epigenetic and transcriptional patterns, and the capacity to form all three germ layers. 2) We will discover new approaches to derive and differentiate iPSCs to vascular lineages. . The heterokaryon model induces rapid and efficient nuclear reprogramming. Transcriptional and epigenetic profiling, combined with the innovative bioinformatics, will provide new insights into determinants of nuclear reprogramming. We anticipate mechanistic insights and the discovery of novel factors to enhance the efficiency of iPSC generation. We will improve methods to differentiate vascular cells, using reporter constructs and nanoarrays to assess media and matrix conditions. 3) We will assess the therapeutic potential of iPSC-derived vascular cells: We will assess angiogenic functions of iPSC-derived vascular cells (eg. proliferation, migration, NO synthesis) and their capacity to incorporate into vascular networks in vitro and in the murine ischemic hindlimb using Doppler perfusion studies, and confocal microscopy and molecular imaging to assess cell incorporation and survival (as well as to detect complications, such as muscle necrosis or teratoma). We will also investigate the importance of paracrine effects, as well as the need for other vascular progenitors.
Our diverse group of scientists will determine how to cause adult cells to turn back the clock and become young healthy stem cells. These insights will help us to make stem cells from skin cells, using a non-viral approach, that will allow us to use these cells to treat diseases of blood vessels.
| Yang, William C; Patel, Kedar G; Lee, Jieun et al. (2009) Cell-free production of transducible transcription factors for nuclear reprogramming. Biotechnol Bioeng 104:1047-58 |
