Isolation of hemopoietic progenitor cells from human umbilical cord blood

A two-stage procedure to enrich hemopoietic progenitor cells from human umbilical cord blood is described. Mononuclear cells isolated from 60% Percoll gradients were first treated with a panel of monoclonal antibodies and complement to remove lymphoid and myeloid cells and granulocyte-macrophage colony-forming cells (CFU-GM) and their immature progeny. Cells stained with monoclonal antibody RFB-1 were separated on the fluorescence-activated cell sorter and cultured in the mixed-colony assay. Progenitor cells were isolated in a high RFB-1 antigen density cell fraction containing greater than 95% undifferentiated blast cells and 32% +/- 12% colony-forming cells. Pluripotential progenitor cells (CFU mix) were enriched 229-fold and accounted for 3.2% +/- 1.2% of the isolated cells. Erythroid progenitor cells (BFU-E) were enriched 204-fold and accounted for 20.8% +/- 8.4% of the isolated cells. The procedure recovered 114% +/- 32% of CFU mix but the number of CFU mix in unseparated cord blood appears to be underestimated due to the presence of granulocytic cells identified by monoclonal antibody CMRF-7 (CD15) that decrease the cloning efficiency of CFU mix. Removal of CD15+ cells yielded a four- to fivefold improvement in the plating efficiency of CFU mix. Mixed-colony formation was completed in 9-11 days in cultures inoculated with enriched progenitor cell fractions, compared with 14-16 days in cultures of unseparated cord blood. The enriched progenitor cells could be useful for studying the regulation of hemopoiesis by recombinant growth factors.