tumor forming cells within a therapeutic cell preparation would be highly valuable. Previous studies reported inconsistent detection sensitivities of various teratoma assays. The sensitivities ranged from 16104 hESCs after intra-testicular [10], or intra-muscular [11] trans- plantation, 16103 after injection into human fetal tissue grafts in SCID mice [12], and 245 hESCs after intra-muscular co-injection of hESCs with their feeder fibroblasts [13]. In view of the importance of standardizing the teratoma assay, we present here a detailed characterization of an efficient, quantitative, sensitive and easy-to-perform teratoma assay. Our data lay the foundation, for the standardized use of this assay for the analysis of pluripotency of hESC and iPS cell lines. We further demonstrate the use of the assay for safety analysis of the tumorigenic potential of hESC-derived differentiated populations intended for the clinic. Results Key Features of the Teratoma Assay To establish a teratoma assay that is sensitive, quantitative, easy to perform and to monitor, we included the following components in our protocol: (1) Quantification of the number of transplanted cells. Prior to inoculation, hESC colonies were dissociated into single cell suspensions, to enable transplantation of defined numbers of cells. (2) Characterizing the phenotype and genotype of transplanted cells. Prior to transplantation, we quantified by FACS the percentage of cells expressing pluripotency-associated cell surface markers (Tra-1-60, Tra-1-81, and SSEA-4). We also confirmed that the karyotype of the transplanted cells was normal. (3) The mode and site of transplantation. To increase the sensitivity of the assay, hESCs were co-transplanted with mitotically inactivated human foreskin- fibroblast-feeders. The beneficial effect of co-transplantation of hESCs with their own feeders was observed by Hentze et al [13]. To further increase the sensitivity, the transplanted cells were also mixed with Matrigel. The cells were injected subcutaneously (s.c.) since transplantation to this site is easy to perform, does not involve an invasive surgical procedure, and allows simple monitoring of teratoma develop- ment. Subcutaneous transplantation of hESCs with Matrigel was reported to induce teratomas with high efficiency [14]. (4) Recipient animals. To minimize the risk of immune rejection, the cells were transplanted into NOD/SCID mice. These mice were previously shown to tolerate hESC grafts well in comparison to immune-competent mice [15]. Since NOD/SCID mice have a tendency to develop spontaneous tumors, and to rule out the unlikely formation of tumors by the co-transplanted feeders, in each experiment we included a control group of mice transplanted with mitotically-inactivated feeders. (5) Duration of monitoring of tumor formation. We monitored the development of tumors in the transplanted animals for a period of 30 weeks. The prolonged follow-up increased the sensitivity of the assay by allowing the detection of late appearing tumors that developed from a small number of cells. (6) Defined histological criteria for teratoma. We defined a tumor as a teratoma only if it contained tissues representing all three germ layers. The histological analysis was performed by a pathologist. We adhered to these guidelines throughout the study. We characterized the sensitivity of detection and the time course of tumor formation with this teratoma assay protocol by transplant- ing various numbers of hESCs, ranging from 56105 to 16101. We A Standardized Teratoma Assay for Pluripotency also studied whether the efficiency and sensitivity of the assay could be improved by supplementing the transplanted cell preparation with the p-160-Rho-associated coiled-coil kinase (ROCK) inhibitor, Y-27632. This inhibitor was shown to enhance hESC survival under unfavorable conditions [16]. We thus hypothesized that its addition to the dissociated hESCs prior to transplantation might have a beneficial effect on their survival and improve the efficiency of teratoma formation from a small number of transplanted hESCs. Evaluation of the Assay with a Relatively Large Number of Transplanted Cells In initial experiments we evaluated the assay by transplantation of relatively large numbers of undifferentiated hESCs (56105 and 16105). Prior to transplantation we confirmed that the trans- planted hESCs (HES-1 cell line; [2]) carried a normal karyotype (46, XX). FACS analyses, performed on the day of trans- plantation, showed that the majority of transplanted cells expressed high levels of cell surface markers associated with undifferentiated hESCs (Tra-1-60, Tra-1-81, and SSEA-4: Fig. S1A). Five week old NOD/SCID mice (n = 10) were transplanted subcutaneously with 56105 hESCs combined with 56105 mitomycin (MMC)-treated foreskin cells, and Matrigel, in the presence or absence of the ROCK inhibitor Y-27632 (5 animals in each group). In all transplanted animals, tumors were observed 3– 4 weeks after transplantation. By 8–10 weeks the tumors were larger than 1 cm3 (average tumor volume of 2.260.4 cm3) and the animals were sacrificed (Fig. 1). Macroscopic analysis of the tumors revealed that they were encapsulated, and sometimes cystic. Assessment of the peritoneal cavity, liver, spleen and lungs tissues of the sacrificed animals did not show additional sites of tumor formation. Histological analysis of each of the tumors revealed the presence of cells and structures representing the three germ layers. The three germ layer structures were randomly arranged within the teratomas (Fig. 2 A–C). In contrast, none of the control animals (n = 10) transplanted with 16106 MMC- treated foreskin fibroblasts, either in the presence or absence of ROCK inhibitor, developed tumors. Notably, the addition of ROCK inhibitor had no effect on the length of time from injection to tumor detection, nor did it promote the development of larger teratomas (Fig. S2). Transplantation of 16105 hESCs gave similar results. All animals (n = 15) transplanted with hESCs developed teratomas while none of the control animals (n = 6) developed tumors. The tumors were first observed 3–5 weeks after transplantation and reached volumes larger than 1 cm3 by 8–9 weeks (average tumor volume of 1.360.1 cm3) (Fig. 1). Addition of ROCK inhibitor had no effect on the timing of tumor detection or on its size (Fig. S2). Transplantation of 16105 hESCs from a different line (HES-2, [2]) also gave rise to teratomas in all animals (n = 3) (Table S1). These results demonstrated that subcutaneous transplantation of 56105 or 16105 undifferentiated hESCs, combined with their feeders and Matrigel, into NOD/SCID mice was highly efficient, leading to teratoma formation in 100% of the transplanted mice. Moreover, tumor progression was rapid, as shown by the detection of tumors in all animals within 3.760.3 weeks, and the development of teratomas to a size larger than 1 cm3 within 9.060.2 weeks after transplantation. Determination of Assay Sensitivity After demonstrating that our assay could efficiently promote teratoma formation from relatively large numbers of hESCs, we sought to evaluate the sensitivity of the assay. For this purpose we transplanted decreasing numbers of undifferentiated HES-1 PLOS ONE | www.plosone.org 2 September 2012 | Volume 7 | Issue 9 | e45532