Collagen solution

The cells were cultured in Dulbecco’s modified Eagle’s medium (Life Technologies, Carlsbad, CA) containing 10% fetal bovine serum, 100 U/mL penicillin, and 100 mg/mL streptomycin and maintained at 37 °C with 5% CO₂. Culture plates were coated with Collagen Solution (STEMCELL Technologies, Inc., Vancouver, Canada).

Primary human CD34⁺ cells, obtained from the Yale Cooperative Center of Excellence in Hematology, were stained with antibodies according to Supplementary Table 15. Human MEP (DAPI^–Lin^–CD34⁺CD41a^–CD45RA⁻CD135^–CD110^–CD38^mid)^{40 (link)}, MEP CD133^low, MEP CD133^mid and MEP CD133^hi cells were sorted on a BD FACSAria. The c.f.u. analysis was performed as previously described^{41 (link)}. Briefly, MEPs, MEP CD133^low, MEP CD133^mid and MEP CD133^hi cells were cultured in MegaCult-C Medium Plus Lipids (04850, STEMCELL Technologies) mixed with collagen solution (04902, STEMCELL Technologies) to a final concentration of 1.2 mg ml^–1 with 3.0 U ml^–1 rhEPO, 10 ng ml^–1 rhIL-3, 10 ng ml^–1 rhIL-6, 25 ng ml^–1 rhSCF, 50 ng ml^–1 rhTPO, 20 ng ml^–1 rhG-CSF, 20 ng ml^–1 rhM-CSF and 20 ng ml^–1 rhGM-CSF at 37 °C with 5% CO₂. At day 6 after plating, colonies were stained in situ with c.f.u. staining panel antibodies in Supplementary Table 15 diluted in 300 μl of PBS (1:100 dilution) per well of a six-well plate. At day 7, six-well plates were imaged using a Molecular Devices ImageXpress Micro 4 microscope to produce high-resolution whole-well scans at ×40 magnification. All images were then processed using ImageJ, and all colonies were counted manually from processed images.

Total nucleated cells were isolated from unprocessed bone marrow product by layering 1:1 PBS:cell suspension over 15-ml Ficoll Paque Plus in SepMate-50 tubes, according to the manufacturer’s instructions. RBCs were lysed with PharmLyse (555899, BD). Cells were then labeled according to Supplementary Table 15 and sorted by a BD FACSAria II directly into culture medium consisting of MegaCult-C Medium Plus Lipids (04850, STEMCELL Technologies) supplemented with 3.0 U ml^–1 recombinant human (rh) erythropoietin (rhEPO), 10 ng ml^–1 rh interleukin-3 (rhIL-3), 10 ng ml^–1 rhIL-6, 25 ng ml^–1 rh stem cell factor (rhSCF), 50 ng ml^–1 rh thrombopoietin (rhTPO), 20 ng ml^–1 rh granulocyte colony-stimulating factor (rhG-CSF), 20 ng ml^–1 rh macrophage colony-stimulating factor (M-CSF) and 20 ng ml^–1 rhGM-CSF. The sorted cells were then mixed with collagen solution (04902, STEMCELL Technologies) to a final concentration of 1.2 mg ml^–1, plated in six-well plates and incubated at 37 °C with 5% CO₂ for 1 week. Colonies were stained in situ 6 days after plating using antibodies according to Supplementary Table 15. Colony assays were imaged the next day using an ImageExpress-4 (Molecular Devices) to produce high-resolution scans of each well, which were then processed in ImageJ for subsequent colony scoring.