Lakhasly

iPS cell generation with lentiviral transduction from PBMCs and BM stromal cells in SCD patients

We generated iPS cell lines using PBMCs and BM stromal cells in SCD patients, as previously described.25,26 All human subject materials were collected under protocols approved by the Institutional Review Board of NHLBI (07-H-0113, 08-H-0156, and 03-H-0015).The iPS cells were evaluated by immunostaining (Nanog, Oct4, SSEA4, Tra1-60, and Tra1-81), alkaline phosphatase stain, karyotyping, and teratoma assay.

iPS cell generation with lentiviral transduction from PBMCs and BM stromal cells in SCD patients

We generated iPS cell lines using PBMCs and BM stromal cells in SCD patients, as previously described.25,26 All human subject materials were collected under protocols approved by the Institutional Review Board of NHLBI (07-H-0113, 08-H-0156, and 03-H-0015). The PBMCs and BM stromal cells were transduced with an Oct4, Klf4, Sox2, and c-Myc encoding lentiviral vector (hSTEMCCA-loxP) and the transduced cells were cultured on irradiated mouse embryonic fibroblast feeder cells (CF1-MEF; GlobalStem, Gaithersburg, MD) in Dulbecco's modified Eagle medium/Nutrient Mixture F-12 (Life Technologies) containing 20% KnockOut Serum Replacement (Life Technologies), 10 ng/mL basic fibroblast growth factor (PeproTech, Rocky Hill, NJ), 0.1 mM nonessential amino acids (Life Technologies), 1mM l-glutamine (Life Technologies), and 0.1 mM 2-mercaptoethanol (Life Technologies). At 2–5 weeks later, we picked iPS cell–like colonies, and the reprogramming cassette was later excised by Cre recombinase. The iPS cells were evaluated by immunostaining (Nanog, Oct4, SSEA4, Tra1-60, and Tra1-81), alkaline phosphatase stain, karyotyping, and teratoma assay.