Epi5 episomal ipsc reprogramming kit

LCLs (n = 13) were reprogrammed into hiPSCs using the Epi5™ Episomal iPSC reprogramming Kit (Thermo Fisher Scientific, Waltham, MA), containing Oct4, Sox2, Klf4, L-Myc, and Lin28. Nucleofection was conducted using the Cell Line Optimization Nucleofector™ X Kit for the 4D-Nucleofector™ System (program EW113, Lonza, Basal, Switzerland). Briefly, 2 × 10⁶ cells from each subject were nucleofected with Epi5™, stabilized in RPMI1640 medium with 10% FBS (Gibco) and 1x Penicillin Streptomycin (PenStrep, Gibco-Thermo Fisher Scientific, Waltham, MA) for 3 days, and then transferred to 60 mm dishes coated with Membrane Matrix (Corning Matrigel hESC-Qualified Matrix (Corning) or Geltrex™ LDEV-Free Reduced Growth Factor Basement Membrane Matrix (Gibco-Thermo Fisher Scientific, Waltham, MA)). RPMI medium was then replaced by TeSR™-E8™ (StemCell Technologies, Vancouver, Canada) and 1x PenStrep (Gibco) every day until the appearance of colonies (15–30 days), at which time clones were manually chosen and expanded. iPSC clones from each of the cell lines were stained for the pluripotency markers SSEA4 and Oct4, and karyotyping analysis by standard G-banding technique was carried out by KaryoLogic, Inc. (Research Triangle Park, NC, USA). Details regarding clinical information for each donor and cell lines used for experiments described below are found in Supplementary Table S1.

Expanded EP cells were collected and nucleofected using an Epi5 Episomal iPSC Reprogramming kit (Thermo Fisher) containing an optimized mixture of five reprogramming factors (Oct-4, Sox2, Lin28, L-Myc and Klf4). Transfection (electroporation) was performed with episomal vectors (virus free, nonintegrating) from a Lonza Nucleofactor kit. After transfection, cells were plated in ReproTeSR-specific culture medium for reprogramming (Stem Cell Technologies) under standard cell culture conditions (37°C, 95% CO₂, and 5% O₂).

Human PBMC's were isolated following the ‘STEMCELL Integrated Workflow for the Isolation, Expansion, and Reprogramming of CD34+ Progenitor Cells’. Briefly, blood samples were collected in Heparin-vacutainer tubes from donors ranging from 8–20 mL samples. CD34+ hematopoietic stem and progenitor cells were isolated from peripheral blood using the EasySep RosetteSep kit (STEMCELL) and expanded in vitro in CD34+ expansion media made of StemSpan SFEM II and CD34+ expansion supplements (STEMCELL) (Fig. S1A). After 7–10 days of culture, 1×10⁶ cells were collected for reprogramming by electroporation using the Epi5 Episomal iPSC Reprogramming Kit (ThermoFisher Scientific) and Human CD34+ Cell Nucleofector Kit (Lonza) using a Nucleofector 2b Device (Lonza). After electroporation cells were cultured on Matrigel coated six-well plates (100 µg/ml, Corning) in CD34+ expansion media. After 3 days, ReproTeSR (STEMCELL) was added to culture media for 2 more days. At day 7, cell were cultured in ReproTeSR media only. Media was changed on a daily basis. After 2–3 weeks, IPS cell colonies were isolated manually (Fig. S1B) and transferred to Matrigel coated six-well plates containing mTeSR plus media (STEMCELL). Three subclones were created for each IPS cell line.