Af 300 19

The iNK cells were cultured in GMP Serum-free Stem Cell Growth Medium (20802, CellGenix) supplemented with 10% fetal bovine serum (FBS, 10091, Gibco). The cells were cultured at an initial density of 1×10⁶ cells/mL. Cord blood CD34⁺ HSCs were usually cultured in 24-well cell culture plates at the beginning, while bone marrow CD34⁺ HSCs were cultured in 48- or 96-well cell culture plates and pNK cells were in 25T flasks. For CB-iNK cells and BM-iNK cells, SCF (20 ng/mL, AF−300−07, PeproTech), Flt3L (30 ng/mL, AF−300−19, PeproTech) were added to the medium during the first two weeks, and IL-15 (20 ng/mL, AF−200−15, PeproTech) was added on day 4 of culturing. At the end of day 14, the dosage of Flt3L and SCF gradually decreased until it was no longer added, while afterward only IL-15(30 ng/mL) was added from day 21. Cells were induced and cultured for approximately five weeks at 37°C in a humid atmosphere with 5% CO₂. Two times a week, half of the medium was removed and replaced with fresh medium and cytokines. For the pNK-iNK group, TGF-β1 (5 ng/mL, AF−100−21C, PeproTech), hCG (10 IU/mL, LIVZON Group, China), and IL-15 (10 ng/mL) were added to the medium. Equivalent fresh medium was added after three or four days of culture, after which the plates were incubated under 1-2% O₂ for the next 4~6 days.