Retronectin

Cryopreserved BM or peripheral blood mononuclear cells were thawed and cultured in X-VIVO 15 media with 1% nonessential amino acids (NEAA), 1 mM L-glutamine and 0.1 mM β-mercaptoethanol (2ME) and supplemented with 100 ng/ml stem cell factor (SCF), 100 ng/ml Flt3 ligand (Flt3L), 100 ng/ml thrombopoietin (TPO) and 20 ng/ml IL-3 for 1–4 days. The excisable lentiviral vector CMV-fSV2A expressing OCT4, KLF4, c-MYC and SOX2 (derived from pLM-fSV2A^{12 (link)} after replacing CMV for hPGK) was used and produced as described^{13 (link)}. For induction of reprogramming, 10,000–250,000 cells were plated on retronectin-coated 24-well dishes and transduced at an MOI of 30 or higher for 16h. Two days later, the cells were harvested and plated on mitotically inactivated MEFs (GlobalStem) in 6-well plates and the plates were centrifuged at 500g for 30 min at RT. The next day and every day thereafter, half of the medium was changed to hESC medium with 0.5 mM valproic acid (VPA). Colonies with hPSC morphology were manually picked and expanded, as described^{13 (link)}.
Culture of hPSCs on MEFs or in feeder-free conditions was performed as previously described^{13 (link)}. Characterization of pluripotency (flow cytometry, OCT4 promoter methylation analysis, teratoma formation assays) and Cre-mediated vector excision were done as previously described^{12 (link), 13 (link), 47 (link)}. Patient samples were obtained with informed consent under protocols approved by an Institutional Review Board at Memorial Sloan-Kettering Cancer Center and the Fred Hutchinson Cancer Research Center.

Nine shRNA pools (~5 shRNAs per gene) were created and subcloned into the pLKO-Thy1.1 lentiviral vector. Each pool also included 85 negative-control shRNAs. OT-I T cells were cultured with IL-7 (5ng/mL) and IL-15 (100ng/mL); on day 2 cells were spin-infected with lentiviral pools supplemented with protamine sulfate (5µg/mL) in retronectin-coated 24-well plates (5µg/mL) at a multiplicity of infection (MOI) of 15. Following infection, OT-1 cells were cultured with IL-7 (2.5ng/mL), IL-15 (50ng/mL) and IL-2 (2ng/mL). On day 5, shRNA-transduced T cells were enriched by positive selection using the Thy1.1 surface reporter (Stemcell Technologies). T cells (5×10⁶) were injected i.v. into C57BL/6 mice bearing day 14 B16-Ova tumors (15 mice per shRNA pool). Seven days later, shRNA-expressing T cells (CD8⁺Vα2⁺Vβ5⁺Thy1.1⁺) were isolated by flow cytometry from tumors, spleens, tumor-draining lymph nodes and irrelevant lymph nodes. Genomic DNA was purified (Qiagen) and deep-sequencing templates were generated by PCR amplification of the shRNA cassette. Representation of shRNAs in each pool was analyzed by deep sequencing using an Illumina Genome Analyzer^{47 (link)}.
Secondary screens were performed using focused pools containing ~15 shRNAs/gene as well as 85 negative controls. Cut-off in the secondary screen was defined as >3 shRNAs with >4 fold enrichment in tumor relative to spleen. Screening results were validated at a cellular level by introducing individual shRNAs into T cells, along with a reporter protein (GFP, TFP, RFP or Ametrine fluorescent proteins, Thy1.1). This approach enabled simultaneous testing of five shRNAs in an animal (three mice per group). Proliferation of shRNA-transduced T cells was visualized based on CFSE dilution after 24 hours as well as 3, 5 and 7 days.