Elimination of undifferentiated human embryonic stem cells by cardiac glycosides

www.nature.com/scientificreports OPEN Received: 3 January 2017 Accepted: 5 June 2017 Published: xx xx xxxx Elimination of undifferentiated human embryonic stem cells by cardiac glycosides Yu-Tsen Lin1,2, Cheng-Kai Wang2,3, Shang-Chih Yang2,3, Shu-Ching Hsu4, Hsuan Lin5, Fang-Pei Chang2, Tzu-Chien Kuo6, Chia-Ning Shen 2, Po-Ming Chiang 7, Michael Hsiao2, Frank Leigh Lu5 & Jean Lu1,2,3,8,9,10 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 undifferentiated hPSCs are sufficient to induce teratomas in a mouse model. Thus, it is critical to remove all residue-undifferentiated hPSCs that have teratoma potential before the clinical application of hPSC-derived cells. In this study, our data demonstrated the cytotoxic effects 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 affect the stem cells’ differentiation ability. Consistently, the viability of the hESC-derived MSCs, neurons, and endothelium cells was not affected 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 first to describe the cytotoxicity and tumor prevention effects of cardiac glycosides in hESCs. Digoxin and lanatoside C are also the first FDA-approved drugs that demonstrated cytotoxicity in undifferentiated 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 indefinitely) and pluripotency (ability to differentiate into all cell types of the human body except for placental cells) properties. These abilities make hPSCs promising resources for regeneration therapy1. 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 after in vivo injections at ectopic sites2, 3. Thousands of undifferentiated hPSCs residing in millions of differentiated cells are sufficient to induce teratomas in a mouse model4. Thus, it is critical to remove all or most of the residue-undifferentiated hPSCs that have teratoma potential before clinical applications using hPSC-derived cells. There are several strategies to selectively remove hPSCs. These methods include the use of cytotoxic antibodies5, 6, specific antibody cell sorting7–9, genetic manipulations10–12, and pharmacological approaches13–16. However, each method has certain disadvantages, such as a high cost (cytotoxic antibodies and specific antibody cell sorting), variation among different lots (cytotoxic antibodies and specific antibody cell sorting)17, 18, non-specific binding (cytotoxic antibodies)18–20, requirement of genetic manipulation and stable integration of toxic genes (genetic manipulation), and time-consuming procedures (genetic manipulation, specific antibody cell sorting 1 Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan. 2Genomics Research Center, Academia Sinica, Taipei, Taiwan. 3Institute of Biochemistry and Molecular Biology, National Yang-Ming University, Taipei, Taiwan. 4National Institute of Infectious Diseases and Vaccinology, National Health Research Institute, Zhunan, Taiwan. 5Department of Pediatrics, National Taiwan University Hospital and National Taiwan University Medical College, Taipei, Taiwan. 6Department of Biotechnology and Laboratory Science in Medicine, National YangMing University, Taipei, Taiwan. 7Institute 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. 10Department of Life Science, Tzu Chi University, Hualien, Taiwan. Correspondence and requests for materials should be addressed to J.L. (email: ) Scientific Reports | 7: 5289 | DOI:10.1038/s41598-017-05616-2 1 www.nature.com/scientificreports/ and cytotoxic antibodies). Although many studies have attempted to prevent or block teratoma formation in residual hPSCs, a clinically applicable strategy to eliminate teratoma formation remains to be developed2, 21. In contrast, small molecule approaches have several advantages as follows: these approaches are robust, efficient, fast, simple, and inexpensive, and there is no need to insert genes into cells. Certain small molecules have been shown to inhibit teratoma formation in hPSCs. The inhibitor of stearoyl-CoA desaturase PluriSin #1 prevented teratoma formation15. Stearoyl-CoA desaturase is a key enzyme in the biosynthesis of mono-saturated fatty acids and is required for hPSC survival15. The N-benzylnonanamide JC011 induced ER stress through the PERK/AT4/DDIT3 pathway22. Chemical inhibitors of survivin, such as quercetin and YM155, induced selective cell death and efficiently inhibited teratoma formation14. However, neither of these drugs is well defined or approved by the FDA. In this study, we investigated the roles of cardiac glycosides in human PSCs. Cardiac glycosides (CGs) (also named cardiotonic steroids, CSs) belong to a large family of compounds that can be derived from nature products. Although these compounds have diverse structures, they share a common structural motif. These compounds are specific inhibitors of the transmembrane sodium pump (Na+/K+-ATPase). CGs inhibit the Na+/ K+-ATPase and then increase the intracellular concentrations of calcium ions23. These compounds act as positive inotropic agents, and members of this group have been used in the treatment of heart failure for more than 200 years. One member of this family, digoxin, is still in clinical use24. Furthermore, CGs are currently considered to have a potential therapeutic role in cancer therapy25. Several studies have reported that CGs play important roles in inducing cell death in several cancer cells23. Cancer cells show more susceptibility than cells in normal tissues. The molecular mechanism may be the overexpression of specific alpha subunits of Na+/K+-ATPase in cancerous cells26. These studies indicate that CGs are selective according to the cell type and distinguish between normal cells and transformed cells. Although cardiac glycosides act as multiple signal transducers, no studies have investigated whether these drugs can eliminate undifferentiated PSCs while sparing their progeny or differentiated cells. In this study, we used digoxin, lanatoside C, bufalin, and proscillaridin A to investigate whether CGs can targ (...truncated)