Myotonic dystrophy type 1 patient-derived iPSCs for the investigation of CTG repeat instability

www.nature.com/scientificreports OPEN received: 28 September 2016 accepted: 09 January 2017 Published: 13 February 2017 Myotonic dystrophy type 1 patientderived iPSCs for the investigation of CTG repeat instability Junko Ueki1,2, Masayuki Nakamori3,*, Masahiro Nakamura1, Misato Nishikawa1, Yoshinori Yoshida1, Azusa Tanaka1, Asuka Morizane1, Masayoshi Kamon4, Toshiyuki Araki4, Masanori P. Takahashi3,5, Akira Watanabe1, Nobuya Inagaki2 & Hidetoshi Sakurai1,* Myotonic dystrophy type 1 (DM1) is an autosomal-dominant multi-system disease caused by expanded CTG repeats in dystrophia myotonica protein kinase (DMPK). The expanded CTG repeats are unstable and can increase the length of the gene with age, which worsens the symptoms. In order to establish a human stem cell system suitable for the investigation of repeat instability, DM1 patient-derived iPSCs were generated and differentiated into three cell types commonly affected in DM1, namely cardiomyocytes, neurons and myocytes. Then we precisely analysed the CTG repeat lengths in these cells. Our DM1-iPSCs showed a gradual lengthening of CTG repeats with unchanged repeat distribution in all cell lines depending on the passage numbers of undifferentiated cells. However, the average CTG repeat length did not change significantly after differentiation into different somatic cell types. We also evaluated the chromatin accessibility in DM1-iPSCs using ATAC-seq. The chromatin status in DM1 cardiomyocytes was closed at the DMPK locus as well as at SIX5 and its promoter region, whereas it was open in control, suggesting that the epigenetic modifications may be related to the CTG repeat expansion in DM1. These findings may help clarify the role of repeat instability in the CTG repeat expansion in DM1. Myotonic dystrophy type 1 (DM1) is a chronic, slowly progressive, autosomal-dominant and multisystem disease1. DM1 is caused by expanded CTG repeats in dystrophia myotonica-protein kinase (DMPK). Its symptoms, such as myotonia, muscle wasting and cardiac conduction defects, have been thought to be the result of splicing defects caused by toxic mRNA that includes expanded CUG repeats2. The expanded CTG repeats in DM1 patients are unstable and can reach several thousand CTG repeats. In addition, the continuous growth of the expanded repeats can affect the progression of the symptoms3. Furthermore, the repeat size can vary between tissues (somatic instability), and the main affected tissues in DM1, such as neurons as well as skeletal and cardiac muscles, normally show longer CTG repeats than other tissues4–7. Previous studies have confirmed these phenotypes in patient-derived tissues and cell models, but have yet to explain the mechanism causing the expansion of the CTG repeats or the reason why the expansion is more apparent in specific tissues. This may be due to the difficulty in acquiring a sufficient number of DM1 patient cells, especially from neural or cardiac tissues. However, this problem can be remedied by using patient-derived induced pluripotent stem cells (iPSCs), which have made it possible to study several diseases that refer to the cell types commonly targeted by DM18–11. Additionally, protocols for differentiation into cardiomyocytes (CMs), neurons and myocytes from iPSCs with high efficiency and stability have also been established10–13. 1 Center for iPS Cell Research and Application (CiRA), Kyoto University, 53 Shogoin-Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan. 2Department of Diabetes, Endocrinology and Nutrition, Graduate School of Medicine, Kyoto University, 54 Shogoin-Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan. 3Department of Neurology, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan. 4Department of Peripheral Nervous System Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry, 4-1-1 Ogawa-Higashi, Kodaira, Tokyo 187-8502, Japan. 5Department of Functional Diagnostic Science, Osaka University Graduate School of Medicine, 1-7 Yamadaoka, Suita, Osaka 565-0871, Japan. *These authors contributed equally to this work. Correspondence and requests for materials should be addressed to M.N. (email: . osaka-u.ac.jp) or H.S. (email: ) Scientific Reports | 7:42522 | DOI: 10.1038/srep42522 1 www.nature.com/scientificreports/ There is only one previous study on DM1 using iPSCs, which showed that the peak length of the CTG repeats increased with the passaging of the cells, but not after their differentiation into neurospheres14. Although the results recapitulate the gradual increase of repeat length during propagation, as seen in DM1 fibroblasts15, the changes in repeat-size distribution during propagation or after differentiation were not clear. Another study using human embryonic stem cells (hESCs) showed that the peak length of the CTG repeats and repeat size distribution changed with passaging, but that it stabilized once the cells were differentiated into osteoprogenitor-like cells16. However, in the previous study, the repeat size changes varied among the clones, and the tendencies toward contraction or expansion were not consistent. Therefore, in this study, to establish a human stem cell system suitable for the investigation of repeat instability, we acquired iPSCs from DM1 patients. We then analysed repeat instability not only in the undifferentiated state but also in the differentiated cells which form the main affected tissues of DM1. We then analysed repeat instability by means of small-pool PCR (spPCR) not only in the undifferentiated state but also in the differentiated cells which form the main affected tissues of DM1. SpPCR is so far the best way to evaluate all the different CTG repeat lengths in a tissue with heterogeneity17. In spPCR, the DNA is diluted to the equivalent of a small number of genomes before amplification. This dilution allows the identification of PCR products derived from single-input molecules by agarose gel electrophoresis and Southern blot hybridization. This technique provides detailed information about the repeat-length variation in a sample, including rare large contracted or expanded alleles as well as the repeat size distribution, whereas conventional bulk PCR just gives the peak length of all the different repeats. Furthermore, several lines of evidence suggest that the epigenetic modifications may be related to the CTG/ CAG repeat instability in DM118. Epigenetic regulations, such as DNA methylation and chromatin structure, play a central role in gene expression. The open chromatin regions indicate the transcriptionally active regions. We next address the epigenetic regulation, which affects the gene expression profile. We performed Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq), which detects the transcriptionally active open chromatin regions. ATAC-seq requires very low numbers of cells (~1000 s cells)19. The number of cells of our iPSC-CMs was very limited. However, AT (...truncated)