Figure 5. Proposed protocol for a standardized teratoma assay of pluripotency. The scheme describes our recommendations for the performance and analysis of a standardized teratoma assay of pluripotency. doi:10.1371/journal.pone.0045532.g005 safety assays in animal models [27,28]. It is largely accepted that safety analysis should include evaluation of the tumorigenic potential of the cells after transplantation into the intended site of clinical transplantation, using methods of delivery that mimic, as best as possible, the planned clinical study [27]. Nonetheless, a universal, sensitive, quantitative and easy to perform teratoma assay such as described here may be highly valuable for on-line assessment of the tumorigenic potential of hESC-derived differ- entiated cell preparations during the course of product de- velopment. Indeed, we found a good correlation between the ability or inability of differentiated populations of NP or RPE, A Standardized Teratoma Assay for Pluripotency respectively, to bring about teratoma formation under these assay conditions and other characteristics of each population pointing to the presence, or absence, of pluripotent cells. Our data show that tumors arising from the transplantation of low numbers of undifferentiated hESC may be detected after a prolonged monitoring period and at relatively low frequencies. Therefore, long-term follow-up, and expanded animal experimen- tal groups are needed to detect low numbers of undifferentiated tumor forming cells within differentiated cell preparations. Several lines of evidence indicate that the potential of undifferentiated hESCs to give rise to teratoma tumors was reduced when they were transplanted in the presence of a mixture of differentiated cells and feeders compared to feeders only. This was observed for the residual, about 3%, undifferentiated hESCs present within a cell population enriched for NPs. In this experiment the efficiency of tumor formation by about 15,000 hESCs among the 56105 NP cells (25%) was lower than anticipated (about 70%). It was further observed in the RPE spiking experiments in which the presence of 16104 undifferentiated hESCs added to 56105 RPE cells gave rise to teratoma formation in 40% of the cases but the presence of a smaller number of hESCs, such as 56103, did not yield any teratomas. The reduced potential of the hESC present in largely differentiated populations to form teratomas could be due to signals arising from the differentiated cells leading to hESC death or differentiation and may be an additional important consideration in the assessment of the safety of these populations. In the two cases above the decrease in sensitivity appears to be of a similar magnitude However, it is possible that the sensitivity of the assay may vary depending on the specific differentiated cell population that is being analyzed and hence should be specifically determined for various differentiated cell populations. In conclusion, we have presented detailed characterization of a quantitative, sensitive, easy-to-perform and monitor teratoma assay. Our results may serve as a basis for a standardized and uniform teratoma assay for the analysis of pluripotency. The assay may be also used for preclinical bio-safety analysis of pluripotent stem cell-derived differentiated therapeutic populations during the course of their development. Materials and Methods Cell Culture hESC lines HES-1 and HES-2 [2] were cultured on human foreskin feeders in serum free medium as described [29]. The cells were passaged routinely as small clusters by treatment with 1 mg/ ml collagenase type IV (Invitrogen, Carlsbad, CA). Foreskin fibroblasts [29] were cultured in 90% DMEM (Invitrogen) supplemented with 10% Fetal Calf Serum (FCS, Biological industries), 0.2 mM L-glutamine, 50 U/ml penicillin, and 50 mg/ml streptomycin. The cells were inactivated by treatment with 10 microgram/ml mitomycin C (MMC), dissociated into a single cell suspension with 0.05% trypsin-EDTA (Invitrogen), and frozen. HES-1 derived retinal pigment epithelium (RPE) cells were cultured as previously described [18]. Characterization of Transplanted hESCs Prior to transplantation a cell-sample was collected for karyotype analysis. In addition, FACS analyses were performed for detection of cell surface markers that are expressed in undifferentiated hESCs, using the following antibodies: Tra-1-60 (mouse monoclonal, clone MAB4360, Chemicon, Temecula, CA, 1:100), Tra-1-81 (mouse monoclonal, clone MAB4381, Chemicon, 1:100), and SSEA-4 (mouse monoclonal, clone MAB4304, Chemicon, 1:100). PLOS ONE | www.plosone.org 8 September 2012 | Volume 7 | Issue 9 | e45532