Human Hematopoietic Stem Cell Isolation From Cord Blood

Human cord blood was collected at the Mary Greeley Medical Center in Ames, Iowa, into 50 mL conical tubes containing 8 mL of anticoagulant citrate dextrose solution (38 mM citric acid, 85.25 mM sodium citrate, 136 mM dextrose). Mononuclear cells (MNCs) were isolated from cord blood by diluting blood 1:2 in HBSS and then layering over Ficoll-Paque (GE Healthcare). Buffy coats were collected and washed in HBSS. Prior to stem cell isolation, MNCs were resuspended in an isolation HBSS (iHBSS) consisting of 0.5% FBS and 2 mM EDTA in HBSS.

To isolate human CD34⁺ cells, we used a CD34 MicroBead kit from Miltenyi Biotech. Briefly, MNCs were incubated in iHBSS with CD34 microbeads and FcR blocking reagent for 30 min at 4°C on a rocker. After the incubation period, cells were washed in iHBSS and then passed through a LS column in a Miltenyi magnet (Miltenyi Biotech) to capture human CD34⁺ cells. The column was then removed from the magnet and cells were flushed, washed once more in HBSS, and then frozen in 10% DMSO and 90% FBS at −80°C until use.