A Standardized Teratoma Assay for Pluripotency Figure 2. Histological Analysis of Teratomas Formed after Transplantation of undifferentiated HES-1 Cells. Images of sections of teratomas, formed after s.c. transplantation of undifferentiated hESCs with their feeders and Matrigel into NOD/SCID mice. (A–C): A teratoma formed from transplantation of 56105 hESCs. (A) A lower magnification (6 4) of a section of a teratoma showing derivatives of all three germ layers. (B–C) A higher magnification (6 20) of regions of (A) showing ectodermal, and endodermal structures (B) and mesodermal and endodermal structures (C). (D–G): A teratoma formed from transplantation of 100 hESCs. (D) A lower magnification (61.25) of a section of a teratoma showing derivatives of all three germ layers (E–G) a higher magnification (620) of regions of (D) showing ectodermal structures (E), mesodermal structures (F) and endodermal structures (G). doi:10.1371/journal.pone.0045532.g002 Here we present a quantitative analysis of a teratoma assay in which defined numbers of characterized hESCs were co- transplanted subcutaneously with feeders and Matrigel into NOD/SCID mice. We show that the assay was highly re- producible and 100% efficient when 16105256105 hESCs were transplanted, with decreasing efficiency when smaller numbers of hESCs were transplanted. We further show that the assay is sensitive down to 16102 hESCs. Importantly, we demonstrate the potential usefulness of this assay for bio-safety detection of residual teratoma forming cells within differentiated cell populations, albeit with a lower sensitivity than when pure hESCs are transplanted. Our data pave the way towards standardization of the teratoma assay for analysis of pluripotency and for analysis of residual teratoma forming ability in therapeutic cell populations during product development. In the design of the assay we specifically addressed parameters which we considered important for standardization of the teratoma assay. These included characterization of the trans- planted stem cells, quantification of the number of engrafted cells, the mode and site of transplantation, monitoring of tumor formation and growth, and its pathological analysis. The assay was designed on the basis of pooled published data with emphasis on the approach that it should be easy to perform and monitor and consistently promote efficient formation of teratomas. The phenotype and genotype of the transplanted cells may affect the results of the teratoma assay. hESC cultures are most commonly heterogeneous with variable levels of background differentiation. Transplantation of relatively homogenous plurip- otent stem cell populations is expected to promote higher efficiencies of teratoma formation. Therefore, since the goal of this study was standardization of the assay, we confirmed that the Figure 3. Analysis of HES-1-derived RPE Cell Grafts. Following transplantation of 56105 HES-derived differentiated RPE cells, the transplanted cells remained as small RPE-grafts at the site of injection. (A): The grafts could be visualized because RPE cells are pigmented. The arrow indicates the pigmented graft. (B): H&E staining of a section of a RPE-graft revealed pigmented cells. (C–D): Immunofluorescence staining of a section of the graft for markers specific for RPE cells, Bestrophin (C, red), and RPE65 (D, red), together with DAPI (blue) showed that the majority of cells within the graft expressed these markers. doi:10.1371/journal.pone.0045532.g003 PLOS ONE | www.plosone.org 6 September 2012 | Volume 7 | Issue 9 | e45532