Phenotypic analysis of bone marrow CD34⁺cells following ex vivo culture.

Purified human CD34⁺ cells were plated on confluent HUBEC monolayers, stroma-free liquid suspension cultures, nonbrain endothelial cell monolayers, and noncontact HUBEC cultures in the presence of optimal concentrations of GM-CSF plus IL-3 plus IL-6 plus SCF plus flt-3 ligand for 7 days. (A) The phenotype of untreated purified bone marrow CD34⁺ cells at day 0. (B) HUBEC-cultured bone marrow cells at day 7, demonstrating a high percentage of CD34⁺ and CD34⁺CD38⁻cells. (C-D) Stroma-free liquid suspension cultures (C) and nonbrain endothelial cell cocultures, demonstrating loss of CD34⁺CD38⁻ phenotype cells (D). (E) HUBEC noncontact cultures, showing preservation of cells with CD34⁺CD38⁻ phenotype. Harvested nonadherent cells were stained with FITC-conjugated CD34 mAb and PE-conjugated CD38 mAb and analyzed by FACS. Log fluorescence distribution of CD34 expression is shown along the x-axis, and CD38 expression along the y-axis. Cursor lines indicate the nonspecific staining levels of isotype-matched control mAbs.