cells (16104, 56103, 26103, 16103, 56102, 16102, and 16101) using the transplantation method described above. Since we expected lower efficiencies of teratoma formation after trans- plantation of decreased numbers of hESCs, we increased the size of the transplanted animal groups (15–30 animals/group) per each specific number of hESCs. In addition, in each trans- plantation experiment, we included f a positive control of a mouse transplanted with 16105 hESCs. In accordance with our protocol, we first confirmed that the majority of transplanted hESCs expressed high levels of pluripotency associated cell surface markers (Fig. S1B) and had a normal karyotype. All the positive control animals (n = 6), transplanted with 16105 hESCs, rapidly developed teratomas, while we did not observe tumors in any of the negative control animals (n = 30), transplanted with 16106 feeders cells (in the presence; n = 15 and absence; n = 15 of ROCK inhibitor). As the number of transplanted hESCs decreased we observed lower efficiencies of teratoma formation (Fig. 1A). The smallest number of undifferentiated HES-1 cells that could still generate teratomas in our assay was 100 hESCs (Fig. 2 D–G). We did not observe tumor formation in animals transplanted with 10 hESCs. When we subjected the data in Fig. 1A to a regression analysis, a trendline with a high R squared value (R2 = 0.91) was obtained. This analysis revealed that the predicted detection limit of the assay is 28 hESCs (Fig. 1A). Decreased numbers of transplanted hESCs affected not only teratoma formation efficiency but also the kinetics of teratoma formation. When lower numbers of hESCs were transplanted, the time intervals for tumor detection were prolonged (Fig. 1C). A Kaplan-Meier analysis of the fraction of the animals that survived at different time points along the experiment (1 W–30 W) revealed a significant increase in the fraction of surviving animals (p, 0.0001) as the number of transplanted hESCs decreased (Fig. 1B). Moreover, when 5,000 or less hESCs were transplanted in many animals the tumors did not reach a volume of 1 cm3 by 30 weeks (Fig. 1D). In addition, histological analysis showed that with low numbers of trans- planted hESC (#5,000 cells) some small tumors (8 tumors with average volume of 0.360.12 cm3) did not include derivatives of all three germ layers, and therefore did not meet the histological criteria of a teratoma tumor. These non-teratoma tumors were excluded from the analysis. We confirmed with an additional line (HES-2) that the efficiency of teratoma formation decreases with the reduction of the number of transplanted cells, and that transplantation of 100 cells could give rise to teratoma formation (Table S1). These results demonstrate that the assay is highly sensitive, promoting and detecting teratoma formation by as few as 100 undifferentiated hESCs. Effect of ROCK Inhibitor on Teratoma Formation As mentioned above, for each experimental group with a specific number of hESC, cells were transplanted in the presence or absence of ROCK inhibitor. Given the improved survival of dissociated hESCs in the presence of ROCK inhibitor in vitro [16], we tested whether its supplementation would improve the efficiency of teratoma formation, in particular when a small number of hESCs are transplanted. We did not observe any beneficial effect of ROCK inhibitor supplementation, neither on tumor formation efficiency nor on tumor growth (Fig. S2). Since the ROCK-inhibitor did not increase the sensitivity of the assay we did not include it in the following experiments. A Standardized Teratoma Assay for Pluripotency Using the Assay to Detect Teratoma Forming Cells within hESC-derived Differentiated Cell Populations We next analyzed the capability of the assay to evaluate the tumorigenic potential of hESC-derived differentiated populations. We evaluated the assay with two types of differentiated cell populations that differed with regard to their contamination by undifferentiated hESCs. A population of differentiated cells enriched for neural progenitors (NPs) was derived from hESCs (HES-1) as previously described [17]. Routine FACS analysis performed after 6 weeks of differentiation showed that the majority of cells (94.6 6 1.5%) expressed the early neural marker PSA-NCAM, while a minority of the cells (3.2 6 0.8%) expressed the pluripotent stem-cell marker Tra-1-81. Following transplantation of 56105 cells from 7 W NPs enriched population, mixed with 56105 feeders, 1 out of 4 mice (25%) developed a teratoma. These results confirmed the ability of the assay to detect residual pluripotent stem cells within differentiated progeny of hESCs. We next transplanted RPE cells that were derived from HES-1 cells according to our previously published protocol [18]. FACS analyses, performed on the day of transplantation, showed that the majority of transplanted cells (99%) expressed high levels of CD81, a cell surface marker associated with RPE cells [19], while cells expressing Tra-1-60 and Tra-1-81 were not detected. 15 animals were transplanted s.c., in two separate experiments, with 56105 hESC-derived RPE cells, mixed with 56105 MMC-treated foreskin fibroblasts, and Matrigel. After an observation period of 30 weeks, we did not detect teratomas in any of the transplanted animals. The transplanted RPE cells remained at the site of transplantation as small grafts that could be visualized due to their pigmentation (Fig. 3A). Histological analysis of the RPE-grafts revealed that they contained pigmented cells (Fig. 3B). Imuno- fluorescence staining of a representative RPE-graft showed that the majority of the cells within the graft expressed high levels of Bestrophin (Fig. 3C) and RPE65 (Fig. 3D), markers specific for RPE cells. Thus the transplanted RPE cells survived at the site of transplantation, and in line with the FACS analysis that did not show residual pluripotent stem cells, teratoma tumors were not observed. We further performed a spiking study to analyze the sensitivity of the assay to detect pluripotent stem cells within the RPE population. We spiked decreasing numbers of undifferentiated HES-1 cells into 56105 HES1-derived RPE cells mixed with 56105 MMC-treated foreskin fibroblasts. Spiking of 16104 undifferentiated HES-1 cells into 56105 RPE cells promoted teratoma formation adjacent to a small RPE graft (Fig. 4A–B) in 2 out of 5 transplanted animals. Spiking of lower numbers of hESCs (56103, 16103, and 16102) did not generate teratomas (Fig. 4C), and the transplanted RPE cells generated only the small pigmented grafts. Thus, the sensitivity of the assay was lower when pluripotent hESCs were transplanted in the presence of RPE cells. Discussion The field of pluripotent stem cells is rapidly evolving concom- itant with the establishment of multiple new hES and iPS cell lines worldwide. Defining uniform standards for the assessment of the pluripotency of new putative pluripotent stem cell lines is of major importance. The teratoma assay is considered by many as one of the minimal criteria that should be fulfilled in order to determine pluripotency [7,8,20]. However, currently there is lack of consistency in the performance and analysis of the assay in different laboratories. PLOS ONE | www.plosone.org 4 September 2012 | Volume 7 | Issue 9 | e45532