title: Blood-derived macrophage layers in the presence of hydrocortisone support myeloid progenitors in long-term cultures of CD34+ cord blood and bone marrow cells.
abstract: Monocytes/macrophages secrete various cytokines that induce proliferation of colony-forming unit granulocyte-macrophage (CFU-GM) in short-term assays. To determine whether macrophages also support proliferation of more primitive progenitors, i.e., cells that give rise to colony forming cells in a 5-week long-term culture (LTC), we established plastic-adherent macrophage layers from human peripheral blood (PB) and filgrastim (G-CSF)-mobilized progenitor cell collections in the presence of hydrocortisone, and compared these layers with bone marrow (BM) stroma regarding their suitability to support proliferation and differentiation of CD34+ BM and cord blood (CB) cells in 5-week LTCs. CD34+ cells were seeded onto irradiated macrophage and BM stromal layers, as well as without any preformed layer. After 5 weeks, colony formation (CFU-GM, BFU-E/CFU-E) and cell expansion were determined. CD34+ cells from BM and CB yielded more CFU-GM and total nucleated cells at 5 weeks in the presence of both types of adherent layer compared with cultures without a layer (p<0.05). For CD34+ BM cells, macrophage layers were superior to BM stroma in enhancing CFU-GM and CFU-E/BFU-E output (p < 0.05). In contrast, BM stroma was favorable compared with macrophages concerning nucleated cell expansion from CD34+ CB cells (p = 0.027). The macrophage nature of PB-derived adherent cells was confirmed immunocytochemically by positive staining for CD68, Ki-Mlp, CD31, CD54, inconstant staining for CD14, and negative staining for CD1a, CD3, CD15, CD34, and CD62E. Cytochemical reactions were positive for alpha-naphthyl acetate esterase and negative for peroxidase and periodic acid-Schiff, consistent with the immunophenotype. In conclusion, the results show that blood-derived macrophages support CFU-GM generation from CD34+ CB and BM progenitors for 5 weeks in vitro. Differential effects on proliferation and maturation of BM versus CB progenitors are discussed.
authors.affiliation: Bone Marrow Transplantation Center, University Hospital Eppendorf, Hamburg, Germany.
authors.forename: J
authors.initials: J
authors.lastname: Clausen
authors.forename: M
authors.initials: M
authors.lastname: Stockschläder
authors.forename: N
authors.initials: N
authors.lastname: Fehse
authors.forename: H T
authors.initials: HT
authors.lastname: Hassan
authors.forename: C
authors.initials: C
authors.lastname: Gabl
authors.forename: A R
authors.initials: AR
authors.lastname: Zander
mesh_terms: D018952:Antigens, CD34 / Q000032:analysis*; D001854:Bone Marrow Cells / Q000166:cytology / Q000276:immunology*; D002478:Cells, Cultured; D005312:Fetal Blood / Q000166:cytology / Q000276:immunology*; D006412:Hematopoietic Stem Cells / Q000166:cytology*; D006801:Humans; D006854:Hydrocortisone; D007150:Immunohistochemistry; D008264:Macrophages / Q000166:cytology*
chemical_list: D018952:Antigens, CD34; D006854:Hydrocortisone