1 Scientific RepoRts | 7: 5289 | DOI:10.1038/s41598-017-05616-2 www.nature.com/scientificreports Elimination of undiferentiated human embryonic stem cells by cardiac glycosides Yu-Tsen Lin 1,2 , Cheng-Kai Wang 2,3 , Shang-Chih Yang 2,3 , Shu-Ching Hsu 4 , Hsuan Lin 5 , Fang-Pei Chang 2 , Tzu-Chien Kuo 6 , Chia-Ning Shen 2 , Po-Ming Chiang 7 , Michael Hsiao 2 , Frank Leigh Lu 5 & Jean Lu 1,2,3,8,9,10 An important safety concern in the use of human pluripotent stem cells (hPSCs) is tumorigenic risk, because these cells can form teratomas after an in vivo injection at ectopic sites. Several thousands of undiferentiated hPSCs are sufcient to induce teratomas in a mouse model. Thus, it is critical to remove all residue-undiferentiated hPSCs that have teratoma potential before the clinical application of hPSC-derived cells. In this study, our data demonstrated the cytotoxic efects of cardiac glycosides, such as digoxin, lanatoside C, bufalin, and proscillaridin A, in human embryonic stem cells (hESCs). This phenomenon was not observed in human bone marrow mesenchymal stem cells (hBMMSCs). Most importantly, digoxin and lanatoside C did not afect the stem cells’ diferentiation ability. Consistently, the viability of the hESC-derived MSCs, neurons, and endothelium cells was not afected by the digoxin and lanatoside C treatment. Furthermore, the in vivo experiments demonstrated that digoxin and lanatoside C prevented teratoma formation. To the best of our knowledge, this study is the frst to describe the cytotoxicity and tumor prevention efects of cardiac glycosides in hESCs. Digoxin and lanatoside C are also the frst FDA-approved drugs that demonstrated cytotoxicity in undiferentiated hESCs. Human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) are human pluripotent stem cells (hPSCs) that have unique self-renewal (ability to replicate almost indefnitely) and pluripotency (ability to diferentiate into all cell types of the human body except for placental cells) properties. Tese abilities make hPSCs promising resources for regeneration therapy 1 . However, substantial challenges remain to be overcome before applying hPSCs to cell therapy. An important safety concern of hPSCs is their tumorigenic risk because these cells can form teratomas afer in vivo injections at ectopic sites 2, 3 . Tousands of undiferentiated hPSCs residing in millions of diferentiated cells are sufcient to induce teratomas in a mouse model 4 . Tus, it is critical to remove all or most of the residue-undiferentiated hPSCs that have teratoma potential before clinical applications using hPSC-derived cells. Tere are several strategies to selectively remove hPSCs. Tese methods include the use of cytotoxic antibod- ies 5, 6 , specifc antibody cell sorting 7–9 , genetic manipulations 10–12 , and pharmacological approaches 13–16 . However, each method has certain disadvantages, such as a high cost (cytotoxic antibodies and specifc antibody cell sort- ing), variation among diferent lots (cytotoxic antibodies and specifc antibody cell sorting) 17, 18 , non-specifc binding (cytotoxic antibodies) 18–20 , requirement of genetic manipulation and stable integration of toxic genes (genetic manipulation), and time-consuming procedures (genetic manipulation, specifc antibody cell sorting 1 Graduate institute of Life Sciences, national Defense Medical center, taipei, taiwan. 2 Genomics Research center, Academia Sinica, taipei, taiwan. 3 institute of Biochemistry and Molecular Biology, national Yang-Ming University, taipei, taiwan. 4 national institute of infectious Diseases and Vaccinology, national Health Research institute, Zhunan, taiwan. 5 Department of Pediatrics, national taiwan University Hospital and national taiwan University Medical college, taipei, taiwan. 6 Department of Biotechnology and Laboratory Science in Medicine, national Yang- Ming University, taipei, taiwan. 7 institute of clinical Medicine, national cheng Kung University, tainan, taiwan. 8 Genomics and System Biology Program, college of Life Science, national taiwan University, taipei, taiwan. 9 national core facility Program for Biotechnology, national RnAi Platform, taipei, taiwan. 10 Department of Life Science, tzu chi University, Hualien, taiwan. correspondence and requests for materials should be addressed to J.L. (email: jeanlu@gate.sinica.edu.tw) Received: 3 January 2017 Accepted: 5 June 2017 Published: xx xx xxxx OPEN