d iPS Cells primed with oligo1218. PCR was performed using Taq DNA Polymerase. ” Primer sequences were the same as previously described. TRAP Assay TRAP Assay was performed by using TRAPEZEH RT Telomerase Detection Kit with Taq polymerase, according to the manufacturer’s instructions. 500 cells were extracted by CHAPS lysis buffer, extracts were analyzed by PCR with Taq DNA Polymerase and separated by 10% polyacrylamide TBE Precast Gel. C, hES H9 cells treated with Dispase followed by the ROCK inhibitor Y-27632. Panels B and D, hES H9 cells treated with Accutase treatment followed by the ROCK inhibitor Y-27632. Panels A and B show the flow cytometry of GFP+ 
cells. Panels C and D show fluorescence microscopy of individual colonies, 406 magnification. EB formation Human iPS cells were harvested by cell scraper and plated on Ultra low adhesion plate in DMEM/ F12 consisting of 15% fetal bovine serum, 15% knockout serum replacement, 0.1 mM nonessential amino acids and 0.5% penicillin and streptomycin. Media was changed every two day. Ten days postdifferentiation, EBs in the supernatant were harvested by centrifugation and RNA was isolated using the RNeasy Micro Kit. Total RNA was reverse-transcribed using Superscript III Reverse Transcriptase primed with oligo1218 and used as template in subsequent PCR with Taq DNA Polymerase. List of primers for amplification of endoderm, ectoderm, and mesoderm markers are included in Text S1 Optimization of gene transduction and expression using VSV-G pseudotyped lentiviral vectors on the H9 human ES cell line. JW 55 Author Contributions Conceived and designed the experiments: DTW CWL XZ MJR. Performed the experiments: DTW YS. Analyzed the data: DTW XZ YS CWL MJR. Contributed reagents/materials/analysis tools: YS. Wrote the paper: DTW MJR. Supporting Information References 1. Niakan KK, Ji H, Maehr R, Vokes SA, Rodolfa KT, et al. Sox17 promotes differentiation in mouse embryonic stem cells by directly regulating extraembryonic gene expression and indirectly antagonizing self-renewal. Genes & Dev 24: 312326. ” Seguin CA, Draper JS, Nagy A, Rossant J Establishment of endoderm progenitors by SOX transcription factor expression in human embryonic stem cells. Cell Stem Cell 3: 182195. Morizono K, Xie Y, Ringpis G-E, Johnson M, Nassanian H, et al. Lentiviral vector retargeting to P-glycoprotein on metastatic melanoma through intravenous injection. Nature Medicine 11: 346352. Yang L, Bailey L, Baltimore D, Wang P Targeting lentiviral vectors to specific cell types in vivo. Proc Natl Acad Sci U S A 103: 1147911484. Ohno K, Sawai K, lijima Y, Levin B, Meruelo D Cell-specific targeting of Sindbis virus vectors displaying IgG-binding domains of protein A. Nat Biotech 15: 763767. Pariente N, Morizono K, Virk MS, Petrigliano FA, Reiter RE, et al. A novel dual-targeted lentiviral vector leads to specific transduction of prostate cancer bone metastases in vivo after systemic administration. Mol Ther 15: 19731981. Pariente N, Mao SH, Morizono K, Chen ISY Efficient targeted transduction of primary human endothelial cells with dual-targeted lentiviral vectors. J Gene Medicine 10: 242248. Liang M, Pariente N, Morizono K, Chen ISY Targeted transduction of CD34+ hematopoietic progenitor cells in nonpurified human mobilized peripheral blood mononuclear cells. J Gene Med 11: 185196. Zhang X, Roth MJ Antibody-directed lentiviral gene transduction in early immature hematopoietic progenitor cells. J Gene Med 12: 945955. Yu J, Vodyanik MA, Sm