Adult bone marrow CD34+ cell isolation and culture

Bone marrow aspirates were collected under FHCRC IRB protocol 0999.209, with consent obtained for use of left-over specimens for research purposes. The material was deidentified prior to being provided for research use. The collected aspirates were washed twice in 1× PBS, Ficoll fractionated and the mononuclear cell fraction collected. Enrichment for CD34+ cells was done by magnetic bead isolation using the CD34 MicroBead Kit (Miltenyi Biotec). CD34+ HSPCs were grown in StemSpan SFEM II (Stemcell Technologies) supplemented with the following growth factor cocktails: expansion (SCF 100 ng/mL, FLT-3 ligand 10 ng/mL, TPO 100 ng/mL, IL-6 20 ng/mL), erythroid (SCF 20 ng/mL, IL-3 10 ng/mL, and EPO 4 U/mL), myeloid (SCF 100 ng/mL, FLT-3 ligand 10 ng/mL, IL-3 20 ng/mL, IL-6 20 ng/mL, GM-CSF 20 ng/mL, M-CSF 20 ng/mL, and G-CSF 20 ng/mL), megakaryocyte (SCF 10 ng/mL, IL-6 20 ng/mL, IL-9 12.5 ng/mL, and TPO 100 ng/mL). EPO was purchased from PeproTech and all other cytokines were purchased from Shenandoah Biotechnology.