Standardization of the teratoma assay for analysis of pluripotency of human ES cells and biosafety of their differentiated progeny

• Published in: PloS one Vol. 7; no. 9; p. e45532
• Main Authors: Gropp, Michal, Shilo, Vitali, Vainer, Gilad, Gov, Miri, Gil, Yaniv, Khaner, Hanita, Matzrafi, Limor, Idelson, Maria, Kopolovic, Juri, Zak, Naomi B, Reubinoff, Benjamin E
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 25.09.2012; Public Library of Science (PLoS)
• Subjects: Analysis; Animals; Assaying; Biological Assay - standards; Biology; Biomarkers - metabolism; Biosafety; Cell Count; Cell cycle; Cell Differentiation; Collagen - administration & dosage; Diagnosis; Drug Combinations; Embryo cells; Embryonic stem cells; Embryonic Stem Cells - pathology; Embryonic Stem Cells - transplantation; Embryos; Feeder Cells - cytology; Feeder Cells - transplantation; Fibroblasts - cytology; Fibroblasts - transplantation; Forming; Gene therapy; Gynecology; Health aspects; Humans; Immunodeficiency; Injections, Subcutaneous; Karyotyping; Laminin - administration & dosage; Medical research; Mice; Mice, Inbred NOD; Mice, SCID; Neurosciences; Pathology; Physiological aspects; Pluripotency; Pluripotent Stem Cells - pathology; Pluripotent Stem Cells - transplantation; Progeny; Proteoglycans - administration & dosage; Sensitivity and Specificity; Standardization; Stem cell transplantation; Stem cells; Survival Rate; Teratoma; Teratoma - mortality; Teratoma - pathology; Transplantation; Tumors; Jerusalem Israel; Israel
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0045532

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.