Adenoviral gene delivery can reprogram human fibroblasts to induced pluripotent stem cells.
Mouse and human fibroblasts have been transformed into induced pluripotent stem (iPS) cells by retroviral transduction or plasmid transfection with four genes. Unfortunately, viral and plasmid DNA incorporation into chromosomes can lead to disruption of gene transcription and malignant transformation. Tumor formation has been found in offspring of mice generated from blastocysts made mosaic with iPS cells. To proceed with iPS cells for human therapy, reprogramming should be done with transient gene expression. Recently, adenoviral vectors have been used to produce mouse iPS cells without viral integration. Here, we report the successful creation of human iPS cells from embryonic fibroblasts using adenoviral vectors expressing c-Myc, Klf4, Oct4, and Sox2. After screening 12 colonies, three stable iPS cell lines were established. Each cell line showed human embryonic stem cell morphology and surface markers. Southern blots and polymerase chain reaction demonstrated that there was no viral DNA integration into iPS cells. Fingerprinting and karyotype analysis confirmed that these iPS cell lines are derived from the parent human fibroblasts. The three human iPS cell lines can differentiate to all three germ layers in vitro, including dopaminergic neurons. After s.c. injection into nonobese diabetic-severe combined immunodeficient mice, each human iPS line produced teratomas within 5 weeks postimplantation. We conclude that adenoviral vectors can reprogram human fibroblasts to pluripotent stem cells for use in individualized cell therapy without the risk for viral or oncogene incorporation.
Zhou
Wenbo
W
Department of Medicine, University of Colorado Denver, School of Medicine, Aurora, Colorado 80045, USA. wenbo.zhou@ucdenver.edu
Freed
Curt R
CR
D000256:Adenoviridae / Q000235:genetics* / Q000502:physiology; D000818:Animals; D015139:Blotting, Southern; D002454:Cell Differentiation / Q000235:genetics / Q000502:physiology; D002460:Cell Line; D016172:DNA Fingerprinting; D005347:Fibroblasts / Q000166:cytology* / Q000378:metabolism*; D005822:Genetic Vectors / Q000235:genetics / Q000502:physiology; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology* / Q000378:metabolism* / Q000637:transplantation; D007621:Karyotyping; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors / Q000235:genetics / Q000502:physiology; D051379:Mice; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D050814:Octamer Transcription Factor-3 / Q000235:genetics / Q000502:physiology; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics / Q000502:physiology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D055748:SOXB1 Transcription Factors / Q000235:genetics / Q000502:physiology; D013724:Teratoma / Q000473:pathology
C000715467:KLF4 protein, human; C000715468:Klf4 protein, mouse; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors; D050814:Octamer Transcription Factor-3; D016271:Proto-Oncogene Proteins c-myc; D055748:SOXB1 Transcription Factors