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Here we describe a highly sensitive tumorigenicity assay for the detection of these cells in hiPSC-CMs. The soft agar colony formation assay and cell growth analysis were unable to detect malignantly transformed cells in hiPSC-CMs. There were no karyotypic abnormalities during hiPSCs subculture and differentiation. The hiPSC markers TRA1-60 and LIN28 showed the highest sensitivity for detecting undifferentiated hiPSCs among primary cardiomyocytes. Transplantation of hiPSC-CMs with a LIN28-positive fraction &gt; 0.33% resulted in tumor formation in nude rats, whereas no tumors were formed when the fraction was &lt; 0.1%. These findings suggested that combination of these <jats:italic>in vitro<\/jats:italic> and <jats:italic>in vivo<\/jats:italic> tumorigenecity assays can verify the safety of hiPSC-CMs for cell transplantation therapy.<\/jats:p>","DOI":"10.1038\/s41598-018-38325-5","type":"journal-article","created":{"date-parts":[[2019,2,13]],"date-time":"2019-02-13T11:05:13Z","timestamp":1550055913000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":47,"title":["Tumorigenicity assay essential for facilitating safety studies of hiPSC-derived cardiomyocytes for clinical application"],"prefix":"10.1038","volume":"9","author":[{"given":"Emiko","family":"Ito","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shigeru","family":"Miyagawa","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Maki","family":"Takeda","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ai","family":"Kawamura","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Akima","family":"Harada","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hiroko","family":"Iseoka","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shin","family":"Yajima","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nagako","family":"Sougawa","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Noriko","family":"Mochizuki-Oda","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1011-0815","authenticated-orcid":false,"given":"Satoshi","family":"Yasuda","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yoji","family":"Sato","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yoshiki","family":"Sawa","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2019,2,13]]},"reference":[{"key":"38325_CR1","doi-asserted-by":"publisher","first-page":"327","DOI":"10.1016\/j.yjmcc.2010.10.026","volume":"50","author":"Y Yoshida","year":"2011","unstructured":"Yoshida, Y. & Yamanaka, S. iPS cells: a source of cardiac regeneration. 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