Polycistronic lentiviral vector for "hit and run" reprogramming of adult skin fibroblasts to induced pluripotent stem cells.
We report the derivation of induced pluripotent stem (iPS) cells from adult skin fibroblasts using a single, polycistronic lentiviral vector encoding the reprogramming factors Oct4, Sox2, and Klf4. Porcine teschovirus-1 2A sequences that trigger ribosome skipping were inserted between human cDNAs for these factors, and the polycistron was subcloned downstream of the elongation factor 1 alpha promoter in a self-inactivating (SIN) lentiviral vector containing a loxP site in the truncated 3' long terminal repeat (LTR). Adult skin fibroblasts from a humanized mouse model of sickle cell disease were transduced with this single lentiviral vector, and iPS cell colonies were picked within 30 days. These cells expressed endogenous Oct4, Sox2, Nanog, alkaline phosphatase, stage-specific embryonic antigen-1, and other markers of pluripotency. The iPS cells produced teratomas containing tissue derived from all three germ layers after injection into immunocompromised mice and formed high-level chimeras after injection into murine blastocysts. iPS cell lines with as few as three lentiviral insertions were obtained. Expression of Cre recombinase in these iPS cells resulted in deletion of the lentiviral vector, and sequencing of insertion sites demonstrated that remnant 291-bp SIN LTRs containing a single loxP site did not interrupt coding sequences, promoters, or known regulatory elements. These results suggest that a single, polycistronic "hit and run" vector can safely and effectively reprogram adult dermal fibroblasts into iPS cells.
Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL, USA.
Chia-Wei
CW
Chang
Yi-Shin
YS
Lai
Kevin M
KM
Pawlik
Kaimao
K
Liu
Chiao-Wang
CW
Sun
Chao
C
Li
Trenton R
TR
Schoeb
Tim M
TM
Townes
D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D015139:Blotting, Southern; D065150:Cellular Reprogramming / Q000235:genetics*; D002678:Chimera; D003114:Colony-Forming Units Assay; D005347:Fibroblasts / Q000166:cytology* / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors / Q000378:metabolism; D016086:Lentivirus / Q000235:genetics*; D051379:Mice; D050814:Octamer Transcription Factor-3 / Q000378:metabolism; D039904:Pluripotent Stem Cells / Q000166:cytology* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D055748:SOXB1 Transcription Factors / Q000378:metabolism; D017422:Sequence Analysis, DNA; D012867:Skin / Q000166:cytology*; D013724:Teratoma / Q000473:pathology
D015415:Biomarkers; C000715467:KLF4 protein, human; C000715468:Klf4 protein, mouse; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors; D050814:Octamer Transcription Factor-3; C494173:POU5F1 protein, human; C527782:SOX2 protein, human; D055748:SOXB1 Transcription Factors