A Standardized Teratoma Assay for Pluripotency Figure 4. Histological Analysis of Teratomas formed after Transplantation of RPE Cells Spiked with Undifferentiated HES-1 Cells. (A–B): H&E staining of a section of a teratoma and an RPE graft generated after transplantation of 56105 HES-1-derived RPE cells spiked with 16104 undifferentiated HES-1 cells. (C): Efficiency of teratoma formation after transplantation of 56105 HES-1-derived RPE cells spiked with decreasing numbers of undifferentiated HES-1 cells. doi:10.1371/journal.pone.0045532.g004 vast majority of transplanted cells in the various experimental groups expressed pluripotency-associated cell-surface markers. Quantitative analysis by FACS of the percentage of undifferen- tiated cells within transplanted cell preparations should be routinely included as part of the teratoma assay. Prior to transplantation we also confirmed the normal karyptype of the transplanted cells. During prolonged propagation in culture hESC and iPS cell lines were shown to acquire chromosomal abnormal- ities of which some are similar to those observed in their malignant counterparts, embryonal carcinoma (EC) cells [21–24]. Similar to EC cells, karyotypically abnormal hESCs and iPS cells may generate teratocarcinomas that contain undifferentiated cells [24,25] and show more limited differentiation capacity. Hence, it is essential to ascertain that stem cells that are evaluated by the teratoma assay carry a normal diploid karyotype. Immune deficient NOD/SCID mice were used in our assay since they tolerate hESC grafts and support the development of teratoma tumors [2,15]. A potential disadvantage of NOD/SCID mice is their tendency to develop thymus tumors. However, within ten weeks of follow up, none of the transplanted mice developed these tumors. Moreover, none of the control mice that were grafted only with fibroblasts developed tumors. These data support the suitability of this animal model for the standardized teratoma assay of pluripotency. A qualitative histological analysis of the tumors was performed by a certified pathologist. This ensured accurate identification of progeny representing the three germ layers. As part of assay development, we determined the sensitivity of the assay that included all the above parameters. We carried out quantitative analysis of the ability to detect teratomas after transplantation of decreasing hESC numbers. We show that transplantation of 1610525 hESCs reproducibly gave rise to teratoma tumors that developed into a size of 1 cm3 within 10 weeks in all transplanted animals. Given the 100% reproducibility of the assay with this cell number, if the number of cells to be transplanted is within or above this range, a limited number of animals may be used for the analysis of pluripotency. Based on the data presented, Figure 5 shows our recommenda- tions for a standard teratoma assay for pluripotency. The standardized assay presented here is based on s.c. injection and is easy to perform and monitor. It is highly sensitive, allowing the detection of as few as 16102 undifferentiated hESCs. A quantitative limiting dilution analysis of the data in Fig. 1A suggested that the detection limit of the assay is 28 hESCs. This sensitivity is higher than the reported sensitivity in previous publications [11–13] and may be attributed to the site of transplantation as well as the inclusion of both feeders [13] and Matrigel [14] in the transplanted cell-mixture. In light of the low number of hESCs that generated teratomas in our xenograft transplantation assay, the possibility that even a lower number of residual pluripotent cells may give rise to teratomas in an autologous iPS transplantation should be considered. In the mouse ES cell system, the minimal number of ES cells that could generate teratomas was reported to be 2 cells [26]. The higher sensitivity of the mESC assay could be due to a higher tumorigenic potential of mESCs [24], or due to a higher survival rate of mESCs as single cells. The fact that the inclusion of ROCK inhibitor in the transplants did not increase the appearance of tumors, suggests that the former may be the more likely reason. Assessment of the bio-safety of stem cell-based products for clinical studies and future therapy is of major importance, and several publications have proposed guidelines for the design of PLOS ONE | www.plosone.org 7 September 2012 | Volume 7 | Issue 9 | e45532