Human embryonic stem cell (hESC) and reprogrammed/induced pluripotent stem cell (iPSC) research is becoming the ‘‘flavor of the month’’for downstream applications such as drug screening, disease modeling, and future regenerative medicine and cell therapies [1–4]. Pluripotency (the ability to give rise to any cell type of the three germ layers: mesoderm, ectoderm, and endoderm) is the defining feature of hESCs and iPSCs [5]. In vivo teratoma formation in immune-compromised mice is the ‘‘gold-standard’’assay to define bona fide pluripotent stem cells capable of generating tumoral disorganized structures containing tissues representing the three germ layers [5, 6]. Despite the importance of teratoma assay as an extended screen for the pluripotency of hESCs and iPSCs and as in vivo assay to explore molecular and cellular mechanisms underlying the biology of human teratomas and their transition to teratocarcinomas, there are no standard procedures for performing this assay [5–7]. Different studies on hESCs have correlated the site of implantation with the efficiency of teratoma formation and histology tissue composition [6, 8]. However, limited data are available regarding the teratoma development latency. More importantly, no study so far has compared side-by-side the efficiency, latency, and histological tumor composition of hESCs-and iPSCs-derived teratomas. In addition, a new generation of immunodeficient mice has been developed: the NOD/SCID IL2Rc À/À mouse. This strain carries a IL2Rc-chain deficiency that blocks signaling through multiple cytokine receptors leading to many innate immune defects [9, 10]. The non obese diabetic/severe combined immune-deficient (NOD/SCID) IL2Rc À/À strain facilitates engraftment and tumor formation and does not develop thymic lymphoma, ensuring a longer lifespan of inoculated mice. Here, we followed the improved teratoma protocol previously developed by Prokhorova et al.[6, 11–13] to transplant side-by-side as few as 1 Â 106 of either fully characterized undifferentiated hESCs or iPSCs in 6-to 8-week-old non obese diabetic/severe combined immune-deficient (NOD/SCID) IL2Rc À/À mice [11, 13–15]. The following hESC lines were used: H9, H1, AND1, AND2, AND3, HS181, and ECAT. The following iPSC lines were used: MSHU-001, iAND4, CB-CD34þ iPSC1, and CB-CD34þ iPSC2. These lines have been fully characterized and deposited according to Spanish Legislation at The Spanish Stem Cell Bank (http://www. isciii. es/htdocs/terapia/terapia_lineas. jsp)[16]. Briefly, cells were resuspended in phosphate buffered saline (PBS) supplemented with 30% matrigel (Becton Dickinson, San Jose, CA, http://www. bd. com)[6] and transplanted subcutaneously (200 ll volume) or by intratesticular injection (60 ll volume). Figure 1A depicts the experimental strategy used. We then analyzed efficiency, latency, and histological tumor composition. In hESCs, the rate of teratoma formation was 81% subcutaneously versus 94% intratesticularly (n ¼ 30 mice; Fig. 1B). However, the intratesticular injection, despite showing higher efficiency of teratoma formation, displayed a slightly longer latency (66 vs. 59 days; p-value> 0.05). There were no site-specific differences in the teratoma composition at the histological level (Fig. 1C). Interestingly, when iPSCs were transplanted the rate of teratoma formation was 100%(n ¼ 16 mice), regardless the type of injection. More importantly, iPSCs seem more aggressive in vivo as the latency was shortened 52%(from 59 days to 31 days) upon subcutaneous injection and 26%(from 66 days to 49 days) upon intratesticular injection. As with hESCs, no differences in teratoma …