2.3 Generation of mature ILC3 from stem cell precursors

UCB-derived CD34⁺ HPCs were cultured for 48 h in complete Icove’s Modified Dulbecco’s Medium (Thermo Fisher Scientific, Waltham, MA) containing 10% Fetal Calf Serum (FCS; Biowest, Nuaillé, France), 100 U/ml penicillin, 100 µg/ml streptomycin and 2mM glutamine (all from Thermo Fisher Scientific) and supplemented with 20 ng/ml thrombopoietin (TPO), 100 ng/ml stem cell factor (SCF) (Peprotech, London, UK) and 100 ng/ml FMS-like tyrosine kinase 3 ligand (FLT3-L) (R&D Systems, Minneapolis, MN). Thereafter, these cells were transferred to RetroNectin (12 µg/ml)-coated plates and transduced with retroviral supernatant by spinning at 950 g for 90 minutes at 32°C. After 48 h of transduction, CD34⁺Lin(CD3, CD14, CD19, CD56)^-eGFP⁺ HPCs were sorted using a BD FACSAria™ Fusion cell sorter (BD Biosciences, San Jose, CA) and were cultured on inactivated EL08.1D2 feeder cells (23, 24) in differentiation medium consisting of Dulbecco’s modified Eagle medium and Ham’s F-12 medium (2:1 ratio) (all from Thermo Fisher Scientific), supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin, 2 mM glutamine, 10 mM sodium pyruvate (Thermo Fisher Scientific), 20% heat-inactivated human AB serum (Biowest), 24 μM β-mercaptoethanol, 20 μg/mL ascorbic acid and 50 ng/mL sodium selenite (all from Sigma-Aldrich, Saint-Louis, MO). To promote ILC3 differentiation, IL-3 (5 ng/mL, R&D systems), IL-7 (20 ng/mL), IL-15 (10 ng/mL) (Miltenyi Biotec), SCF (20 ng/mL), and Flt3-L (10 ng/mL) were added to the culture medium. On day 7 the culture medium was refreshed by doubling the medium supplemented with the above mentioned cytokines, excluding IL-3. Between day 12 and 14, depending on cell density, cells were harvested and transferred to new inactivated feeder cells in fresh medium containing cytokines.