LINE-1-mediated retrotransposition in human pluripotent stem cells: Consequences for genomic stability of hES and hiPS cells and its derivatives
Final Report Abstract
Human induced pluripotent stem cells (hiPSCs) hold substantial promise for regenerative medicine. However, reprogramming and subsequent hiPSC cultivation can result in hiPSCs with genetic and epigenetic abnormalities, and genomic mutations that can undermine their use in regenerative medicine because they question biosafety of hiPSC-derived differentiated cells. Activation of endogenous mobile retrotransposons LINE-1 (Long Interspersed Element-1, L1), Alu and SVA can cause such mutations. In differentiated cells, L1 is suppressed by methylation of its CpG-rich promoter. The results of our funded project show that reprogramming triggers transcription of endogenous L1 elements via demethylation. To investigate if the observed L1 activation in hiPSCs leads to L1-mediated mobilization, we applied retrotransposon capture-sequencing (RC-seq) to eight hiPSC lines, their parental cells and human embryonic stem cell lines (hESCs). We identified, mapped and validated individual L1, Alu and SVA de novo retrotransposition events that occurred during reprogramming into hiPSCs and cultivation of hiPSCs and hESCs, and timed the period during hiPSC cultivation when individual de novo insertions occurred. Each hiPSC was estimated to carry approximately one L1 de novo insertion. As ~50% of all de novo retrotransposition events occurred in protein-coding genes that are actively transcribed in hiPSCs, including genes playing roles in oncogenesis, development or signal transduction, we investigated effects of these intronic insertions on host gene expression in hiPSCs. To exemplify the consequences of even short intronic L1 de novo insertions, we analyzed effects of the 390-bp L1 de novo insertion L1-dn13 in CADPS2 intron 7 on CADPS2 transcription in hiPSCs, and demonstrate significant interference of L1-dn13 with allelic CADPS2 gene expression. Our experiments demonstrate incidence and functional impact of reprogramming-activated endogenous retrotransposition in hiPSCs and imply consequences for the biological safety of hiPSC-derived cell therapies. RNA preparations from five hiPSC lines and one hESC line used to analyze expression of functional L1 elements at different passages after reprogramming were also used to quantify transcription of endogenous retroviruses in pluripotent stem cell lines. These analyses uncovered that HERV-H was the only endogenous retrovirus expressed in all six hPSC lines analyzed and led to the finding that naÏvelike stem cells are associated with elevated expression of the primate-specific endogenous retrovirus HERV-H. We generated six L1 integrome libraries from three different hiPSC lines and two integrome libraries from one hESC line facilitating the identification of 1778 and 501 tagged L1 de novo insertions that occurred in hiPSCs and hESCs, respectively. These are the first comprehensive L1 integrome libraries that allow for the identification of potential target site preferences in cultivated human pluripotent stem cells due to the so far unmatched number of tagged and independent L1 de novo insertions. We found that 43% (1.5%) and 76% (3%) of all marked L1 de novo insertions occurred into introns (exons) of host genes in hiPSCs and hESCs, respectively. This is the first time that exonic L1 de novo insertions are demonstrated to occur in cultured human cells. It is unexpected that the fraction of L1 de novo insertions that occur into genes is twice as high in hESCs (3%) than in hiPSCs (1.5%). ∼2.9% and ∼5% of the L1 de novo insertions in hiPSCs and hESCs integrated into cancer-related genes. Chromosomal abnormalities including deletions of 3-17 Mb and trisomies ocf chromosome 7 and 20 were found in transgenic hiPSC lines L1-ORFeusHs-C1 and C7 that are characterized by the highest numbers of L1 de novo insertions in their genomes, suggesting that L1 de novo insertions support genomic alterations. Bioinformatic analyses of the 1778 L1 de novo insertions in hiPSCs suggest that L1 elements have a preference for integrating into the proximity of transcription start sites (TSSs) of those genes that are transcribed in hiPSCs. In conjunction with establishing the most efficient method to introduce tagged L1 retrotransposition reporter cassettes into pluripotent stem cell lines, we participated in the development of novel lentiviral vectors targeted to the hiPSC surface receptor CD30 (CD30-LV) to transfer genes (and L1 reporter cassettes) into hiPSCs. CD30-LV may serve as novel tool for the selective gene transfer into PSCs with broad applications in basic and therapeutic research.
Publications
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(2014). Primate-specific endogenous retrovirus-driven transcription defines naive-like stem cells. Nature 516: 405-9
Wang J, Xie G, Singh M, Ghanbarian AT, Raskó T, Szvetnik A, Cai H, Besser D, Prigione A, Fuchs NV, Schumann GG, Chen W, Lorincz MC, Ivics Z, Hurst LD, Izsvák Z
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(2016). CD30 Receptor-Targeted Lentiviral Vectors for Human Induced Pluripotent Stem Cell-Specific Gene Modification. Stem Cells Dev. 25: 729-39
Friedel T, Jung-Klawitter S, Sebe A, Schenk F, Modlich U, Ivics Z, Schumann GG, Buchholz CJ, Schneider IC
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(2016). Reprogramming triggers endogenous L1 and Alu retrotransposition in human induced pluripotent stem cells. Nat Commun 7:10286
Klawitter, S., Fuchs, N. V., Upton, K. R., Munoz-Lopez, M., Shukla, R., Wang, J., Garcia-Canadas, M., Lopez- Ruiz, C., Gerhardt, D. J., Sebe, A., Grabundzija, I., Merkert, S., Gerdes, P., Pulgarin, J. A., Bock, A., Held, U., Witthuhn, A., Haase, A., Sarkadi, B., Lower, J., Wolvetang, E. J., Martin, U., Ivics, Z., Izsvak, Z., Garcia-Perez, J. L., Faulkner, G. J., and Schumann, G. G.
