Isolation and Transduction of Murine Sca-1+ HSPCs

Sca-1⁺ isolation from murine bone marrow was performed by immunomagnetic bead selection as previously described.⁸⁰ (link) Briefly, bone marrow from CD45.1⁺ donor mice was harvested and incubated with a biotinylated anti-Sca-1 antibody (Biolegend, San Diego, CA, USA) followed by incubation with anti-biotin microbeads (Miltenyi, Gaithersburg, MD, USA). Labeled cells were then passed through an LS column loaded on a MidiMACS Separator (Miltenyi). Sca-1⁺ cells were harvested and cultured in serum-free StemSpan medium (Stem Cell Technologies, Vancouver, BC, Canada) for 3 days in the presence of mouse SCF (100 ng/mL), mouse IL-3 (20 ng/mL), human IL-11 (100 ng/mL), and human Flt-3 ligand (100 ng/mL). All cytokines were purchased from R&D Systems (Minneapolis, MN, USA). Sca-1⁺ cells were transduced on day −1 and day 0 with half volume CD68-ECO-ET3-LV (LV production described in detail by Lytle et al.⁸ (link)) at a density of 2.0 × 10⁶ Sca-1⁺ cells/mL and multiplicity of infection (MOI) of 13–63. Transduced CD45.1⁺ Sca-1⁺ HSPCs were harvested, washed, and resuspended in PBS and then transplanted by retro-orbital injection into preconditioned HA mice.