The overriding hypothesis of this application is that the generation and characterization of non Human primate (NHP) induced pluripotent stem cell lines (iPS) will hasten the development of replacement tissues and organs in Humans. Macaque monkeys share an extremely close phylogenetic relationship with Man, and develop natural or experimentally induced disease states which accurately mimic the Human condition. NHPs can provide a unique preclinical model to test the usefulness of iPS cells and regenerative medicine approaches to the treatment and cure of disease, where other models may fail. The ability to generate autologous, pluripotent stem cells from skin fibroblasts, by relatively simple reprogramming steps, is a quantum scientific advance which will have major ramifications in many areas of basic research and applied clinical medicine. It is highly unlikely, however, that the use of viral vectors in the reprogramming protocol (as it exists now) will be acceptable for use in patients. It is vital, therefore, that alternative ways of reprogramming, without virus, be investigated. The short 11 amino acid protein transduction domain (PTD) derived from the HIV transactivator, TAT has been used to deliver large (~110kD), biologically active proteins directly into cells in vitro and in vivo. TAT fusion proteins and peptides have also been used to treat mouse models of cancer, inflammation and other diseases. It is our hypothesis that recombinant TAT and be able to reprogram cells into a pluripotent state. Once well characterized, lines derived by viral or protein transduction methods can be used in NHP preclinical models for a number of different diseases. We therefore propose to: (1) generate induced pluripotent stem cell lines (iPS) from the non-human primate, Macaca fascicularis (cynomolgus macaque) (2) To determine if M. fascicularis iPS cells can be produced non-virally using recombinant proteins modified with an HIV derived, TAT cell penetrating motif to aid in protein translocation across the plasma membrane and (3) characterize and compare iPS lines generated by viral and protein transduction.
| Pan, Chuanying; Jia, Wenchao; Lu, Baisong et al. (2015) Expression of TAT recombinant Oct4, Sox2, Lin28, and Nanog proteins from baculovirus-infected Sf9 insect cells. Gene 556:245-8 |
| Pan, Chuanying; Lan, Xianyong; Chen, Hong et al. (2010) An economical single-sided antibody incubation method for Western blotting. J Virol Methods 169:409-11 |
| Pan, Chuanying; Hicks, Amy; Guan, Xuan et al. (2010) SNL fibroblast feeder layers support derivation and maintenance of human induced pluripotent stem cells. J Genet Genomics 37:241-8 |
| Pan, Chuanying; Lu, Baisong; Chen, Hong et al. (2010) Reprogramming human fibroblasts using HIV-1 TAT recombinant proteins OCT4, SOX2, KLF4 and c-MYC. Mol Biol Rep 37:2117-24 |
