Standardization of the Teratoma Assay for Analysis of Pluripotency of Human ES Cells and Biosafety of Their Differentiated Progeny Michal Gropp1., Vitali Shilo2., Gilad Vainer3, Miri Gov2, Yaniv Gil1, Hanita Khaner1, Limor Matzrafi2, Maria Idelson1, Juri Kopolovic3, Naomi B. Zak2, Benjamin E. Reubinoff1* 1 The Hadassah Human Embryonic Stem Cell Research Center, The Goldyne Savad Institute of Gene Therapy and Department of Gynecology, Hadassah-Hebrew University Medical Center, Jerusalem, Israel, 2 CellCure NeuroSciences Ltd., Jerusalem, Israel, 3 The Department of Pathology, Hadassah-Hebrew University Medical Center, Jerusalem, Israel Abstract Teratoma tumor formation is an essential criterion in determining the pluripotency of human pluripotent stem cells. However, currently there is no consistent protocol for assessment of teratoma forming ability. Here we present detailed characterization of a teratoma assay that is based on subcutaneous co-transplantation of defined numbers of undifferentiated human embryonic stem cells (hESCs) with mitotically inactivated feeder cells and Matrigel into immunodeficient mice. The assay was highly reproducible and 100% efficient when 100,000 hESCs were transplanted. It was sensitive, promoting teratoma formation after transplantation of 100 hESCs, though larger numbers of animals and longer follow-up were required. The assay could detect residual teratoma forming cells within differentiated hESC populations however its sensitivity was decreased in the presence of differentiated cells. Our data lay the foundation, for standardization of a teratoma assay for pluripotency analysis. The assay can also be used for bio-safety analysis of pluripotent stem cell- derived differentiated progeny. Citation: Gropp M, Shilo V, Vainer G, Gov M, Gil Y, et al. (2012) Standardization of the Teratoma Assay for Analysis of Pluripotency of Human ES Cells and Biosafety of Their Differentiated Progeny. PLoS ONE 7(9): e45532. doi:10.1371/journal.pone.0045532 Editor: Dimas Tadeu Covas, University of Sao Paulo - USP, Brazil Received April 19, 2012; Accepted August 20, 2012; Published September 25, 2012 Copyright: 2012 Gropp et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by Cell Cure Neurosciences Ltd., and by a grant to the company from the Bereshith (Genesis) Magnet Consortium of the Israel Office of the Chief Scientist of the Minister of Industry, Trade, and Labor. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: BR is the CSO and holds shares in CellCure Neurosciences Ltd. VS, M. Gov, LM and NBZ were employees of CellCure Neurosciences Ltd. when the study was performed. M. Gropp was funded for this study by CellCure Neurosciences Ltd. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials. * E-mail: benjaminr@ekmd.hugi.ac.il . These authors contributed equally to this work. Introduction Human embryonic stem cells (hESCs) are pluripotent cells derived from preimplantation embryos. These cells can self-renew for long periods, and have the potential to differentiate into any cell type [1,2]. Hence they may be exploited for applications in regenerative medicine, drug discovery and basic research of early human development. When transplanted into immune-deficient mice at growth- permissive sites, hESCs form teratoma-like masses containing differentiated progeny representing the three germ layers [1–4]. Formation of cells from all three germ layers within a teratoma is considered as an essential criterion to define the pluripotent potential of a hESC line [5]. In the consensus guidance for the banking, testing and distribution of hESC lines, published by the International Stem Cell Banking Initiative (ISCBI), the teratoma formation assay is defined as the ‘‘gold standard’’ for pluripotency [6]. In accordance with these guidelines many publications on the derivation of new hESC lines include characterization of the line’s pluripotency by a teratoma formation assay. In addition, the teratoma assay is considered by many as a criterion that should be fulfilled in order to ascertain that a genuine induced pluripotent stem (iPS) cell line has been obtained [7]. However, the methods that different research groups use to induce teratomas differ in important parameters such as cell preparation, number of transplanted cells, mode of transplantation, site of transplantation, and the length of time that animals are monitored for tumor formation. Moreover, the pathological analysis of tumors and the report of experimental data are often incomplete and highly variable. If the teratoma assay is regarded as the ‘‘gold standard’’ for defining pluripotency, standardization of the assay is invalu- able. Indeed, recently Mueller and colleagues published a call for the standardization of the teratoma formation assay [8]. Standardization of the teratoma assay is not important only for assessing hESC pluripotency but also for evaluating the tumori- genic potential of hESC-derived progeny. The field of hESCs is moving rapidly towards clinical applications, with the first spinal injury patient being recently transplanted with hESC derived cells [9]. A key hazard in the implementation of hESC-based cell therapy is potential tumor formation caused by the presence of pluripotent hESCs within the transplanted cell preparations. A standardized sensitive teratoma assay to detect low numbers of PLOS ONE | www.plosone.org 1 September 2012 | Volume 7 | Issue 9 | e45532