Reprogramming PBMCs to iPSCs

Peripheral blood mononuclear cells (PBMCs) were isolated from patients’ blood by Percoll^R gradient separation. PBMCs were purified and replated as previously described (Liu et al., 2021 (link)). Briefly, PBMCs were cultured in 1 ml of Stem-Pro™-34 medium (100 ng/ml FLT3, 20 ng/ml IL-6, 20 ng/ml EPO, 20 ng/ml IL-3, and 100 ng/ml SCF). PBMCs were resuspended in 300ul of Stem-Pro™-34 medium and transduced with Sendai virus reprogramming cocktail (CytoTune®-iPSC Sendai Reprogramming Kit). After 24 h, cells were replated, and the medium was replaced every two days. On Day 7, 1 ml of supplemented StemMACS™ iPSC-Brew XF medium (Miltenyi Biotec) was added on top of Stem-Pro™-34 medium. On Day 8, the medium was replaced completely with StemMACS™ iPSC-Brew XF medium. Fresh StemMACS™ iPSC-Brew XF medium was replaced on Days 10–15 when colonies appeared.