Differentiation Assay for Hematopoietic Progenitors

DC differentiation assay was performed as described (23 (link)–25 (link)) with minor adaptation. Briefly, 1×10⁴ purified progenitors, DC, and monocyte subsets were cultured in 96-well flat bottom plates layered with 4×10⁴ murine MS5 stromal cells (DSMZ, Germany) in the presence of human FLT3 ligand (FL; 100 ng/ml; Miltenyi Biotec), recombinant human SCF (20 ng/ml; R&D Systems), and recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) (10 ng/ml; Peprotech). MS5 stromal cells were seeded 24 hours prior to co-culture. Growth factors were replenished on day 3 of culture. Cells were in culture for up to 7 days prior to harvesting by physical dissociation on ice. Cells were then stained on ice either for flow cytometry analysis (see output panel in table S12) or single-cell index sorting of CD45⁺ cells for scRNA-seq of culture output analysis.