Human stemcca cre excisable constitutive polycistronic oksm lentivirus reprogramming kit

All patient samples were collected from patients enrolled in a study that had been approved by the respective institutional review board. Control and ACDC-derived human dermal fibroblasts (HDFs) were generated from explants of 4-mm punch biopsy skin specimen and grown in Dulbecco’s modified Eagle’s medium containing 10% fetal calf serum (FCS) and 1% penicillin-streptomycin as previously described (46 (link)). After 1 to 2 weeks, fibroblast outgrowths from the explants were passaged. HDFs were transduced with Human STEMCCA Cre-Excisable Constitutive Polycistronic (OKSM) Lentivirus Reprogramming Kit [Millipore; (47 (link))]. Cells were harvested 3 to 4 days after transduction and replated on plates coated with Matrigel (BD Biosciences). On the following day, E8 medium without transforming growth factor–β (TGF-β) supplemented with 1 μM hydrocortisone and 100 μM butyrate was added to cells and replaced every other day. After 2 weeks of transduction, those cells were fed in full E8 medium. The Tra-1-60–positive iPSC colonies were collected 18 days after transduction and maintained in full E8 medium (STEMCELL Technologies) and passaged with 0.5 mM EDTA (K&D Medical). Twenty-five iPSC lines from five individuals (five lines for each patient) in two families and seven nonrelated control subjects were generated. Two ACDC lines were used in the TALEN rescue experiments.

Fibroblasts from individuals with the homozygous CCR5Δ32 variant were reprogrammed to generate iPSC lines by transduction with the Human STEMCCA Cre-Excisable Constitutive Polycistronic (OKSM) Lentivirus Reprogramming Kit (Millipore). iPSC colonies were collected at 21 days post-transduction, and expanded in a typical hESC/iPSC culture condition (Jin et al., 2016 (link)).