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

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. Althoug...

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Vydané v:	Proceedings of the National Academy of Sciences - PNAS Ročník 106; číslo 1; s. 157
Hlavní autori:	Carey, Bryce W, Markoulaki, Styliani, Hanna, Jacob, Saha, Kris, Gao, Qing, Mitalipova, Maisam, Jaenisch, Rudolf
Médium:	Journal Article
Jazyk:	English
Vydavateľské údaje:	United States 06.01.2009
Predmet:	Animals Cells Cells, Cultured Cellular Reprogramming - genetics Fibroblasts - cytology Genetic Vectors - genetics Keratinocytes - cytology Kruppel-Like Transcription Factors - genetics Mice Octamer Transcription Factor-3 - genetics Pluripotent Stem Cells - cytology Proto-Oncogene Proteins c-myc - genetics SOXB1 Transcription Factors - genetics Transfection - methods Transgenes - genetics
ISSN:	1091-6490, 1091-6490
On-line prístup:	Zistit podrobnosti o prístupe
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Abstract	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.
AbstractList	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. 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.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.
Author	Mitalipova, Maisam Gao, Qing Carey, Bryce W Hanna, Jacob Jaenisch, Rudolf Saha, Kris Markoulaki, Styliani
Author_xml	– sequence: 1 givenname: Bryce W surname: Carey fullname: Carey, Bryce W organization: Whitehead Institute for Biomedical Research and Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02142, USA – sequence: 2 givenname: Styliani surname: Markoulaki fullname: Markoulaki, Styliani – sequence: 3 givenname: Jacob surname: Hanna fullname: Hanna, Jacob – sequence: 4 givenname: Kris surname: Saha fullname: Saha, Kris – sequence: 5 givenname: Qing surname: Gao fullname: Gao, Qing – sequence: 6 givenname: Maisam surname: Mitalipova fullname: Mitalipova, Maisam – sequence: 7 givenname: Rudolf surname: Jaenisch fullname: Jaenisch, Rudolf
BackLink	https://www.ncbi.nlm.nih.gov/pubmed/19109433$$D View this record in MEDLINE/PubMed
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Snippet	Directed reprogramming of somatic cells by defined factors provides a novel method for the generation of patient-specific stem cells with the potential to...
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SubjectTerms	Animals Cells Cells, Cultured Cellular Reprogramming - genetics Fibroblasts - cytology Genetic Vectors - genetics Keratinocytes - cytology Kruppel-Like Transcription Factors - genetics Mice Octamer Transcription Factor-3 - genetics Pluripotent Stem Cells - cytology Proto-Oncogene Proteins c-myc - genetics SOXB1 Transcription Factors - genetics Transfection - methods Transgenes - genetics
Title	Reprogramming of murine and human somatic cells using a single polycistronic vector
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