Fibroblast Reprogramming to iPSCs

Human fibroblasts from three apparently healthy individuals (GM00038, GM08680, GM05756) were purchased from Coriell. Reprogramming was performed using the CytoTune-iPS 2.0 Sendai reprogramming kit according to the manufacturer’s instructions (Thermo Fisher Scientific). Briefly, fibroblasts were plated at 20,000 cells/cm^{2 (link)} and cultured until they were 30–60% confluent. Cells were transduced with the Sendai reprogramming vectors at an MOI of 5:5:3 (KOS:hc-Myc:hKlf4). Cells were maintained in fibroblast media until passaged onto VN-coated plates on day 7. On day 8, medium was changed to E8 Medium and cells were cultured for additional 20 days. On day 28, colonies were picked using a 25-gauge needle to cut single colonies into 6–8 pieces and transferred onto VN-coated plates containing E8 Medium only or supplemented with Y-27632 or CEPT for 48 h. After 48 h, cells were kept in E8 Medium only. Confluency was assessed 8 days after colony picking using the Celigo Imaging Cytometer (Nexcelom Biosciences).