Preparation of hESCs for Transplantation On the day of transplantation hESCs were harvested and dissociated into a single cell suspension using 0.05% EDTA (Biological Industries, Beit-Haemek, Israel) .To ensure a single cell suspension the cells were filtered through a sterile filter. The filtered cells were centrifuged and the pellet was re-suspended in PBS (Invitrogen). To determine the number of viable cells, an aliquot of the hESCs was counted. Frozen MMC-treated foreskin fibroblasts were thawed and centrifuged. The pellet was re- suspended in PBS (Invitrogen), and viable cells were counted. For transplantation, various numbers of hESCs were combined with MMC-treated foreskin cells to a total of 16106. The cell-mixture was centrifuged and the pellet was re-suspended in PBS to a total volume of 50 ml. For negative controls, 16106 MMC-treated foreskin cells were transplanted. When the ROCK inhibitor Y- 27632 (Calbiochem, Darmstadt, Germany) was included in the experiment, it was added to the cell mixture to a final concentration of 10 mM. The cells were kept for a minimal period at room temperature and transferred to the animal facility. The cell-mixture was combined with 50 ml undiluted cold Matrigel Basement membrane matrix (BD #354234, NJ, USA) immedi- ately before transplantation. Pre-cooled pipettes, tips and syringes were used. Transplantation and Animal Observation Five week old immune-deficient NOD/SCID mice were used for transplantation. They were maintained under non-specific pathogen-free (SPF) conditions, at the Hadassah Animal Facility. The cell-Matrigel mixture (100 ml) was injected into the subcutis of the right hind leg of the mice. The transplanted animals were observed routinely once a week, and tumor growth was measured with a caliper. They were sacrificed after development of tumors larger than 1 cm3 or following an observation period of 30 weeks. The sacrificed animals were subjected to general inspection. When tumors were observed at the injection site, they were collected. In addition, the liver, lungs and spleen tissues of the all animals were inspected for tumor invasion into other organs. Histological Analysis The tumor containing tissues were fixed in 4% PFA, embedded in paraffin and serially sectioned into 6 micron sections. Every other section was mounted on slides (2–3 sections per slide, and 20 slides for each tumor). 4 slides, containing 8–12 sections from various parts of the tumor (slides #3, 8, 13 and 18) were stained with hematoxylin and eosin, and subjected to histological analysis by a certified pathologist. Large tumors were cut into halves before fixation, and the two halves were placed in the paraffin block so that the central parts of tumor (with the larger surface area) were faced up. To enable analysis of the different parts of large tumors, the sectioning was performed from the upper part of the paraffin block, containing the central parts of both halves of the tumor, towards the tumor’s edges. References 1. Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, et al. (1998) Embryonic stem cell lines derived from human blastocysts. Science 282: 1145–1147. 2. Reubinoff BE, Pera MF, Fong CY, Trounson A, Bongso A (2000) Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro. Nat Biotechnol 18: 399–404. 3. Lensch MW, Ince TA (2007) The terminology of teratocarcinomas and teratomas. Nat Biotechnol 25: 1211. 4. Blum B, Benvenisty N (2008) The tumorigenicity of human embryonic stem cells. Adv Cancer Res 100: 133–158. A Standardized Teratoma Assay for Pluripotency Statistical Analysis Data is presented as mean 6 SEM (standard error of mean). Kaplan-Meier analysis was performed using the GraphPad PRISM version 4.0 software. Ethics Statement This study was performed in accordance with the recommenda- tions of the Authority for Biological and Biomedical Models of the Hebrew University, Jerusalem. The protocol was approved by the Committee on the Ethics of Animal Experiments (Permit Number: MD-09-11663-4). Supporting Information Figure S1 Expression of pluripotency-associated cell surface markers in transplanted HES-1 cells. (A): A representative FACS analysis of the expression levels of pluripo- tency-associated cell-surface markers, Tra-1-60, Tra-1-81, and SSEA-4 in transplanted HES-1 cells. (B): Average expression levels of Tra-1-60, Tra-1-81, and SSEA-4 in transplanted HES-1 cells from twelve transplantation experiments. Data presented as mean 6 SEM. (TIFF) Figure S2 Effect of the ROCK-inhibitor Y-27632 on Teratoma Formation. Kinetics of teratoma formation after transplantation of decreasing numbers of undifferentiated HES-1 cells in the presence (dark gray) or absence (light gray) of the ROCK-inhibitor Y-27632. (A): Efficiency of teratoma formation (B): The average volume of the teratomas at the time of animals sacrifice. (C): The average time interval between transplantation and the detection of tumors. (D): The average time interval between transplantation and the experiment endpoint. Data presented as mean 6 SEM. (TIFF) Table S1 Teratoma Formation Kinetics after Trans- plantation of Various Defined Numbers of Undifferen- tiated HES-2 Cells. Various numbers of undifferentiated HES- 2 cells were mixed with MMC-treated foreskin fibroblasts (to a total of 16106 cells) and Matrigel, and transplanted s.c. into NOD/ SCID mice. The transplanted animals were weekly monitored for the appearance of tumors, and for the progression of tumor size. The endpoint of the experiments was when the tumors reached a size of $ 1 cm3 or 30 weeks after transplantation. Data presented as mean 6 SEM. (DOC) Author Contributions Conceived and designed the experiments: Michal Gropp BER NBZ. Performed the experiments: Michal Gropp VS Miri Gov YG. Analyzed the data: Michal Gropp VS GV JK NBZ BER. Contributed reagents/ materials/analysis tools: HK LM MI. Wrote the manuscript: Michal Gropp BER. 5. Brivanlou AH, Gage FH, Jaenisch R, Jessell T, Melton D, et al. (2003) Stem cells. Setting standards for human embryonic stem cells. Science 300: 913–916. 6. (2009) Consensus guidance for banking and supply of human embryonic stem cell lines for research purposes. Stem Cell Rev 5: 301–314. 7. Maherali N, Hochedlinger K (2008) Guidelines and techniques for the generation of induced pluripotent stem cells. Cell Stem Cell 3: 595–605. 8. Muller FJ, Goldmann J, Loser P, Loring JF (2010) A call to standardize teratoma assays used to define human pluripotent cell lines. Cell Stem Cell 6: 412–414. 9. Mayor S (2010) First patient enters trial to test safety of stem cells in spinal injury. Bmj 341: c5724. PLOS ONE | www.plosone.org 9 September 2012 | Volume 7 | Issue 9 | e45532