Fms related tyrosine kinase 3 ligand flt3

Mobilized human CD34⁺ HSPCs (AllCells, Alameda, CA, USA) were thawed and cultured for 48 h at a density of 2.5 × 10⁵ cells/mL in StemSpan SFEM II (StemCell Technologies, Vancouver, Canada), supplemented with 100 U/mL penicillin and 0.1 mg/mL streptomycin (Biological Industries, Beit Haemek, Israel), stem cell factor (SCF), thrombopoietin (TPO), Fms-related tyrosine kinase 3 ligand (Flt3), and interleukin (IL)-6 (100 ng/mL each; PeproTech, Rocky Hill, NJ, USA). Cells were cultured at 37°C, 5% CO₂, and 5% O₂. 0.5 × 10⁵ CD34⁺ HSPCs were reconstituted in P3 Primary Cell electroporation solution, according to the manufacturer’s instructions (Lonza, Basel, Switzerland) and mixed with RNP complexes at a final concentration of 1 or 4 μM. Cells were then supplemented with either 3.85 μM Alt-R EE (IDT, Coralville, IA, USA)¹⁴ (link) or an equivalent volume of PBS. The supplemented cell solution (final cell concentration of 2 × 10⁶ /mL) was transferred into the Lonza 4D-Nucleofector and electroporated using the DZ-100 program. Recovered cells were cultured for 48–72 h prior to gDNA extraction with QuickExtract (Lucigen, Middleton, WI, USA). RNP complexes were formed and delivered in the same manner with the Alt-R HiFi Cas9 variant (IDT, Coralville, IA, USA).