Reprogramming of murine and human somatic cells using a single polycistronic vector

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 106; no. 1; pp. 157 - 162
• Main Authors: Carey, Bryce W, Markoulaki, Styliani, Hanna, Jacob, Saha, Kris, Gao, Qing, Mitalipova, Maisam, Jaenisch, Rudolf
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 06.01.2009; National Acad Sciences
• Subjects: adults; Animals; B lymphocytes; Biological Sciences; Cell lines; Cells; Cells, Cultured; Cellular Reprogramming - genetics; Cloning; Embryonic stem cells; Embryos; ethics; Fibroblasts; Fibroblasts - cytology; Genetic Vectors - genetics; Human subjects; humans; Induced pluripotent stem cells; insertional mutagenesis; keratinocytes; Keratinocytes - cytology; Kruppel-Like Transcription Factors - genetics; methodology; Mice; Neural stem cells; Octamer Transcription Factor-3 - genetics; Peptides; Pluripotent Stem Cells - cytology; Proto-Oncogene Proteins c-myc - genetics; Rodents; Somatic cells; SOXB1 Transcription Factors - genetics; Stem cells; Transfection - methods; Transgenes - genetics; Viruses
• ISSN: 0027-8424; 1091-6490; 1091-6490
• DOI: 10.1073/pnas.0811426106

Directed reprogramming of somatic cells by defined factors provides a novel method for the generation of patient-specific stem cells with the potential to bypass both the practical and ethical concerns associated with somatic cell nuclear transfer (SCNT) and human embryonic stem (hES) cells. Although the generation of induced pluripotent stem (iPS) cells has proven a robust technology in mouse and human, a major impediment to the use of iPS cells for therapeutic purposes has been the viral-based delivery of the reprogramming factors because multiple proviral integrations pose the danger of insertional mutagenesis. Here we report a novel approach to reduce the number of viruses necessary to reprogram somatic cells by delivering reprogramming factors in a single virus using 2A "self-cleaving" peptides, which support efficient polycistronic expression from a single promoter. We find that up to four reprogramming factors (Oct4, Sox2, Klf4, and c-Myc) can be expressed from a single virus to generate iPS cells in both embryonic and adult somatic mouse cells and we show that a single proviral copy is sufficient to generate iPS cells from mouse embryonic fibroblasts. In addition we have generated human induced pluripotent stem (hiPS) cell lines from human keratinocytes, demonstrating that a single polycistronic virus can reprogram human somatic cells.