Immunologic selection of hemopoietic precursor cells utilizing antibody-mediated plate binding ("panning").
We utilized the property of antibody adherence to plastic to separate and obtain enriched fractions of human myeloid (CFU-GM), erythroid (BFU-E) and pluripotent (CFU-GEMM) hemopoietic precursor cells. Nonadherent buoyant human marrow cells coated with mouse anti-human HLA-DR monoclonal antibody (Mc ab), an anti-pan T lymphocyte Mc ab (Leu 1/17F12) or a granulocyte--monocyte-specific Mc ab (MCS2) were incubated on polystyrene Petri plates coated with affinity purified goat anti-mouse immunoglobulin G (IgG). Cells bound to the coated plates and nonbound cells were separately recovered ("panned") by differential elution. Analysis of the nonadherent buoyant marrow cells demonstrated 12% to possess HLA-DR, 6% T, 40% MCS2 antigens on their surface by indirect immunofluorescence (IMF). After panning, 15% +/- 8%, 14% +/- 4% and 8% +/- 6% cells were plate-bound by their respective antibodies, demonstrating differing binding efficiencies. A substantial degree of purity of the recovered cell fractions was shown for bound 74% +/- 6% and 75% +/- 5% IMF positive cells) and nonbound cells (3% +/- 1% and 0.1% +/- 0.8% positive cells) coated with anti-HLA-DR or anti-T Mc ab respectively, with lesser purity for MCS2 panned cells. Seventy-three percent to 126% CFU recovery was noted, with a sevenfold enrichment of the HLA-DR bound cells for CFU-GM and CFU-GEMM, and 3.5-fold enrichment for BFU-E. Sequential panning, obtaining T nonbound-DR bound-surface immunoglobulin nonbound fractions, resulted in tenfold CFU-GM enrichment (107/10(4) cells, approximately equal to 1/100). Anti-MCS2 antibody was ineffective for panning, but use of this antibody in fluorescence-activated cell sorting (FACS) indicated the absence of the MCS2 antigen on the vast majority of CFU-GM. This study describes a relatively rapid and inexpensive method for obtaining enriched antigenically defined hemopoietic precursors in high yield. These techniques should prove useful for more clearly evaluating cellular and humoral interactions with hemopoietic precursor cells.
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D000954:Antigens, Surface / Q000032:analysis; D001666:Binding Sites, Antibody; D001854:Bone Marrow Cells; D002469:Cell Separation / Q000379:methods*; D003114:Colony-Forming Units Assay; D006098:Granulocytes / Q000276:immunology; D006684:HLA-DR Antigens; D006412:Hematopoietic Stem Cells / Q000276:immunology* / Q000378:metabolism / Q000502:physiology; D000949:Histocompatibility Antigens Class II / Q000032:analysis; D006801:Humans; D008214:Lymphocytes / Q000276:immunology; D051379:Mice
D000911:Antibodies, Monoclonal; D000954:Antigens, Surface; D006684:HLA-DR Antigens; D000949:Histocompatibility Antigens Class II