Lower Genomic Stability of Induced Pluripotent Stem Cells Reflects

Zhang et al. Cancer Commun (2018) 38:49 https://doi.org/10.1186/s40880-018-0313-0 Cancer Communications ORIGINAL ARTICLE Open Access Lower genomic stability of induced pluripotent stem cells refects increased non‑homologous end joining Minjie Zhang1,2†, Liu Wang3†, Ke An1,2†, Jun Cai1, Guochao Li1,2, Caiyun Yang1, Huixian Liu1, Fengxia Du1, Xiao Han1,2, Zilong Zhang1,2, Zitong Zhao1,2, Duanqing Pei4, Yuan Long5, Xin Xie5, Qi Zhou3 and Yingli Sun1* Abstract Background: Induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs) share many common features, including similar morphology, gene expression and in vitro diferentiation profles. However, genomic stability is much lower in iPSCs than in ESCs. In the current study, we examined whether changes in DNA damage repair in iPSCs are responsible for their greater tendency towards mutagenesis. Methods: Mouse iPSCs, ESCs and embryonic fbroblasts were exposed to ionizing radiation (4 Gy) to introduce double-strand DNA breaks. At 4 h later, fdelity of DNA damage repair was assessed using whole-genome re-sequencing. We also analyzed genomic stability in mice derived from iPSCs versus ESCs. Results: In comparison to ESCs and embryonic fbroblasts, iPSCs had lower DNA damage repair capacity, more somatic mutations and short indels after irradiation. iPSCs showed greater non-homologous end joining DNA repair and less homologous recombination DNA repair. Mice derived from iPSCs had lower DNA damage repair capacity than ESC-derived mice as well as C57 control mice. Conclusions: The relatively low genomic stability of iPSCs and their high rate of tumorigenesis in vivo appear to be due, at least in part, to low fdelity of DNA damage repair. Keywords: Genomic stability, DNA damage repair, iPSCs, ESCs Background morphology, gene expression profle, epigenetic status Embryonic stem cells (ESCs) are pluripotent and could and in vitro diferentiation capacity. Te development of diferentiate into all types of somatic cells [1]. ESCc have iPSCs raises new hope for personalized clinical therapy enormous potential in the treatment of a variety of dis- [3–5]. eases, but their clinical application has been limited by Te four transcription factors (Oct4, Sox2, c-Myc and ethical controversy. In 2006, Yamanaka and colleagues Klf4) that are critical for the production of iPSCs are overexpressed four transcription factors (Oct4, Sox2, frequently overexpressed in various cancers, and mice c-Myc and Klf4) in mouse somatic cells and obtained derived from iPSCs are prone to develop tumors [6–9]. ESC-like pluripotent stem cells, termed induced pluri- Although only a small population of transformed cells potent stem cells (iPSCs) [2]. iPSCs resemble ESCs in with genetic mutations is likely to develop into tumors [10], the genomic instability of iPSCs is a major concern *Correspondence: [email protected]; [email protected] that could produce huge impact on their eventual clinical †Minjie Zhang, Liu Wang and Ke An are co-frst authors and contributed use [11–16]. equally to this work One possible explanation for the observed greater 1 Key Laboratory of Genomic and Precision Medicine, China Gastrointestinal Cancer Research Center, Beijing Institute of Genomics, genomic instability of iPSCs is alterations in the fdelity of Chinese Academy of Sciences, Beijing 100101, P. R. China DNA repair pathways. Double-stranded DNA breaks, for Full list of author information is available at the end of the article example, can be repaired via homologous recombination © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Zhang et al. Cancer Commun (2018) 38:49 Page 2 of 22 (HR) with high fdelity, or via non-homologous end join- 1500g for 10 min at 4 °C. Te pellet was washed with 1.5- ing (NHEJ) with lower fdelity [17–20]. In the current mL TEB, re-suspended in 0.2 mol/L HCl, and incubated study, we examined whether iPSCs difer from other at 4 °C overnight. Samples were centrifuged at 6500g types of pluripotent cells in their ability to perform these for 10 min, after which 200-µL supernatant was trans- types of DNA repair. Briefy, ionizing radiation was used ferred to a new tube, and neutralized with 20-µL 2 mol/L to induce double-stranded DNA breaks in the following NaOH. cells: mouse iPSCs induced using lentivirus (lv-iPSCs) Samples were separated using SDS-PAGE and trans- or chemically with CHR99021, Repsox and forskolin (ci- ferred to PVDF membranes (Millipore, Billerica, MA, iPSCs) [21]; mouse ESCs; and mouse embryonic fbro- USA). Blots were incubated with a primary antibody blasts (MEFs) [22–26]. against one of the following proteins: phospho-ATM Te experiments showed that lv-iPSCs are more likely (1:1000; R&D Systems, Minneapolis, MN, USA), β-actin than the other cell types to harbor genomic abnormali- (1:3000; Beyotime Biotech, Beijing, China), H3 (1:30,000; ties, likely due to lower genomic fdelity of DNA damage Abcam, Cambridge, MA, USA) and H3K9me3 (1:3000; repair. We also found greater genomic stability in ci- Abcam). Blots were washed three times with phosphate- iPSCs than lv-iPSCs. bufered saline (PBS), and then incubated with a horse- radish peroxidase-conjugated anti-mouse secondary Methods antibody (1:3000; Gene Tex, San Diego, CA, USA) or Cell lines and culture anti-rabbit secondary antibody (1:3000; Abcam). Protein Te lv- and ci-iPSCs were derived from female transgenic bands of interest were visualized using an Image Quant OG2 mice carrying an Oct4-GFP transgene. Both types ECL system (GE Healthcare, Piscataway, NJ, USA). of iPSCs and ESCs were cultured in Dulbecco’s Modifed Eagle Medium (DMEM; Gibco, Grand Island, NY, USA) Immunofuorescence labeling of γ‑H2AX foci supplemented with 15% fetal bovine serum (FBS; Gibco), Cells were passaged onto slides, exposed 24 h later to 1% MEM non-essential amino acids (Gibco), 1% penicil- 4 Gy of γ-irradiation, and incubated at 37 °C for 4 h. Cells lin/streptomycin (Gibco), 2 mmol/L l-glutamine (Gibco), were washed with PBS, fxed with 4% paraformaldehyde 3 1 × 10 units/mL of mouse leukemia inhibitory factor for 10 min at room temperature, washed again with PBS, (Millipore, Temecula, CA, USA) and 0.1 mmol/L 2-mer- permeabilized for 10 min using 0.05% Triton X-100 and captoethanol (Gibco) [27]. Te medium was changed 0.5% NP-40, and then washed three times (5 min each) in daily, and cells were passaged every 2 days using 0.25% PBS. Te cells were blocked for 1 h with 2% bovine serum trypsin (Termo Fisher Scientifc, Beijing, China) [28]. albumin (BSA), and then incubated for 1 h at room tem- MEFs were cultured in DMEM supplemented with 15% perature with a mouse anti-γH2AX antibody (1:500; Mil- FBS, 1% non-essential amino acids and 1% penicillin/ lipore, Temecula, CA, USA). Cells were washed three streptomycin [29]. times with PBS containing 0.05% Tween 20, and then incubated with a goat anti-mouse secondary antibody Irradiation (1:800; Abcam) for 1 h in the dark at room temperature. Cells were passaged 1 day before γ-irradiation (4 Gy) with Cells were counterstained with 0.2 mg/mL 4′,6-diamid- a cobalt irradiator (Termo Fisher Scientifc). After the ino-2-phenylindole (DAPI, 1:2000; Sigma, Shanghai, irradiation, cells were immediately returned to the incu- China). Confocal images were acquired and analyzed bator, and cultured for 4 h prior to analyses as described using a TCS SP5 (Leica) microscope equipped with an below. HCX PL 63 × 1.4 CS oil-immersion objective lens. Western blotting DNA extraction To test the phosphorylation level of ATM, cells were Tree types of cells (lv-iPSCs, ci-iPSCs, ESCs) were lysed in ATM lysis bufer [20 mmol/L HEPES (pH 7.4), digested with 0.25% trypsin and re-suspended in gela- 150 mmol/L NaCl, 0.2% Tween-20, 1.5 mmol/L MgCl2, tin-coated dishes. After incubation at 37 °C for 15 min, 1 mmol/L EGTA, 2 mmol/L dithiothreitol, 50 mmol/L supernatants were transferred to 15-mL centrifuge tubes, NaF, 500 μmol/L NaVO4, 1 mmol/L phenylmethylsulfo- and cells were collected by centrifugation at 500g for nyl fuoride, 0.1 μg/mL aprotinin and 0.1 µg/mL leupep- 5 min at room temperature. DNA was extracted using a tin], and centrifuged, as describe previously [30]. QIAamp DNA Mini Kit (Qiagen, Hilden, Germany). In assays of histone modifcation, cells were re-suspended in 1-mL triton extraction bufer (TEB) contain- Whole‑genome re‑sequencing ing 0.5% Triton X-100 and 2 mmol/L PMSF, and then Whole-genome DNA libraries suitable for sequencing lysed on ice for 10 min. Te lysates were centrifuged at using an Illumina sequencing platform were generated Zhang et al. Cancer Commun (2018) 38:49 Page 3 of 22 from 1-µg genomic DNA. Te DNA was sheared to Generation of iPSC‑ and ESC‑derived mice approximately 300–500 bp using a Covaris S220 instru- Two cell-stage ICR embryos were electrofused to pro- ment (Life Technologies, Carlsbad, CA, USA). A total duce tetraploid embryos, and 10–15 iPSCs and ESCs were of 2× 101-bp paired-end reads were produced using the subsequently injected into the reconstructed tetraploid HiSeq 2000 DNA Sequencer.

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