title: Deep sequencing reveals low incidence of endogenous LINE-1 retrotransposition in human induced pluripotent stem cells.
abstract: Long interspersed element-1 (LINE-1 or L1) retrotransposition induces insertional mutations that can result in diseases. It was recently shown that the copy number of L1 and other retroelements is stable in induced pluripotent stem cells (iPSCs). However, by using an engineered reporter construct over-expressing L1, another study suggests that reprogramming activates L1 mobility in iPSCs. Given the potential of human iPSCs in therapeutic applications, it is important to clarify whether these cells harbor somatic insertions resulting from endogenous L1 retrotransposition. Here, we verified L1 expression during and after reprogramming as well as potential somatic insertions driven by the most active human endogenous L1 subfamily (L1Hs). Our results indicate that L1 over-expression is initiated during the reprogramming process and is subsequently sustained in isolated clones. To detect potential somatic insertions in iPSCs caused by L1Hs retotransposition, we used a novel sequencing strategy. As opposed to conventional sequencing direction, we sequenced from the 3' end of L1Hs to the genomic DNA, thus enabling the direct detection of the polyA tail signature of retrotransposition for verification of true insertions. Deep coverage sequencing thus allowed us to detect seven potential somatic insertions with low read counts from two iPSC clones. Negative PCR amplification in parental cells, presence of a polyA tail and absence from seven L1 germline insertion databases highly suggested true somatic insertions in iPSCs. Furthermore, these insertions could not be detected in iPSCs by PCR, likely due to low abundance. We conclude that L1Hs retrotransposes at low levels in iPSCs and therefore warrants careful analyses for genotoxic effects.
authors.affiliation: Department of Microbiology, Immunology and Molecular Genetics, University of California Los Angeles, David Geffen School of Medicine, Los Angeles, California, United States of America.
authors.forename: Hubert
authors.initials: H
authors.lastname: Arokium
authors.affiliation: Department of Microbiology, Immunology and Molecular Genetics, University of California Los Angeles, David Geffen School of Medicine, Los Angeles, California, United States of America.
authors.forename: Masakazu
authors.initials: M
authors.lastname: Kamata
authors.affiliation: Department of Microbiology, Immunology and Molecular Genetics, University of California Los Angeles, David Geffen School of Medicine, Los Angeles, California, United States of America.
authors.forename: Sanggu
authors.initials: S
authors.lastname: Kim
authors.affiliation: Korean Bioinformation Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, South Korea.
authors.forename: Namshin
authors.initials: N
authors.lastname: Kim
authors.affiliation: Department of Microbiology, Immunology and Molecular Genetics, University of California Los Angeles, David Geffen School of Medicine, Los Angeles, California, United States of America.
authors.forename: Min
authors.initials: M
authors.lastname: Liang
authors.affiliation: Department of Biostatistics, University of California Los Angeles School of Public Health, University of California Los Angeles, Los Angeles, California, United States of America.
authors.forename: Angela P
authors.initials: AP
authors.lastname: Presson
authors.affiliation: Department of Microbiology, Immunology and Molecular Genetics, University of California Los Angeles, David Geffen School of Medicine, Los Angeles, California, United States of America.
authors.forename: Irvin S
authors.initials: IS
authors.lastname: Chen
mesh_terms: D002460:Cell Line; D065150:Cellular Reprogramming / Q000235:genetics; D005786:Gene Expression Regulation; D055106:Genome-Wide Association Study; D005854:Germ Cells / Q000378:metabolism; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000378:metabolism*; D020084:Long Interspersed Nucleotide Elements; D016254:Mutagenesis, Insertional; D018626:Retroelements
chemical_list: D018626:Retroelements