Efficient Generation of CAR-NK Cells from iPSCs

The human UKKi011-A iPS cell line (66540010, Sigma) was maintained according to the instructions. To produce mature and functional natural killer (NK) cells from iPSC lines, we used a previously published protocol with minor changes [11 (link), 12 (link)]. In brief, 6000 TrypLE-adapted iPSCs were seeded in 96-well low attachment plates in BPEL (bovine serum albumin, polyvinyl alcohol, essential lipids) containing 40 ng/mL stem cell factor (SCF) (R&D Systems), 20 ng/mL vascular endothelial growth factor, and 20 ng/mL bone morphogenic protein 4 (R&D Systems). On Day 11 of hematopoietic differentiation, embryoid bodies (EBs) were directly transferred into each well of uncoated 24-well plates. Cells were then further differentiated into NK cells using 5 ng/mL IL-3, 10 ng/mL IL-15, 20 ng/mL IL-7, 20 ng/mL SCF), and 10 ng/mL Flt3 ligand (R&D Systems) for 30 days. As described above, our major improvement was transducing the CD22-CAR into the iPS cells first, and then inducing the CD22-CAR-iPS cells to differentiate into CD22-CAR-NK cells. This strategy efficiently raised the living ratio of the CAR-NK cells and reduced the suffering damages or toxicities from transduction regents for CAR-NK cells.