Phenotypic analysis and characterization of CD34+ cells from normal human bone marrow, cord blood, peripheral blood, and mobilized peripheral blood from patients undergoing autologous stem cell transplantation.
Single- and multicolor flow cytometry were used to define progenitor subsets in normal human bone marrow and peripheral blood, cord blood, and blood following mobilization of CD34+ progenitor cells by cyclophosphamide or cyclophosphamide/etoposide/G-CSF treatment. CD34 cells were quantitated and subsets of CD34+ cells were defined by coexpression of CD33, CD13, CD10, CD19, CD45RA, and CD71. Myeloid and erythroid progenitors were quantitated by sorting single CD34+ cells into individual wells of 96-well plates containing methylcellulose, IL-3, GM-CSF, G-CSF, IL-6, and erythropoietin. Comparative studies of CD34 cells showed that the percentage of CD34+ mononuclear cells was greatest in blood samples from patients following mobilization treatment with cyclophosphamide/etoposide/G-CSF averaging 2%. By comparison, the remaining sample groups ranged from 1.68 to 0.15% CD34 cells in this order, bone marrow > cord blood > cyclophosphamide mobilized blood > peripheral blood. Comparison of CD34 cells per milliliter of bone marrow or blood showed a range of 22.4 x 10(4) to 0.65 x 10(4)/ml in the following order, bone marrow > chemotherapy/etoposide/G-CSF > cord blood > cyclophosphamide-mobilized blood. Comparative analysis of CD34 subsets from different sources showed significant differences, particularly bone marrow and blood samples. A distinct population of CD34+ CD19+ (Leu 12) CD10+ (CALLA) pre-B lymphocyte cells was defined in bone marrow with lower side and forward light scatter characteristics and was variable between donors (29.8 +/- 16.9%, mean +/- 1 SD; range, 3-54%; n = 8). This population was not found to a significant degree in blood and also expressed CD45RA (Leu 18). Coexpression studies of CD45RA and CD71 (transferrin receptor) expression on CD34+ cells defined a CD45RA- CD71+ population containing 89 +/- 6.3% (n = 4) BFU-E and a CD45RA+ CD71+ population that contained all CFU-GM (n = 4). LeuM7 (CD13) stained a larger percentage to a greater intensity than MY7 (CD13). Coexpression of CD45RA (Leu 18) and CD13 (LeuM7) defined a subset of CD13+ CD45RA+ cells enriched for CFU-GM and CFU-M with a cloning efficiency of 31%. Coexpression of CD33 (MY9) and CD13 (MY7) defined a population that was predominantly CFU-GM with a cloning efficiency of 38%. These studies define CD34+ phenotypes containing pure populations of B lymphocyte, granulocyte-macrophage, or erythroid progenitors and demonstrate the utility of multiparameter flow cytometry to define lineage-committed CD34+ cells.
Bender
J G
JG
Immunotherapy Division, Baxter Healthcare Corporation, Round Lake, Illinois 60073.
Unverzagt
K
K
Walker
D E
DE
Lee
W
W
Smith
S
S
Williams
S
S
Van Epps
D E
DE
D000328:Adult; D015703:Antigens, CD / Q000032:analysis*; D018941:Antigens, CD19; D018952:Antigens, CD34; D000944:Antigens, Differentiation, B-Lymphocyte / Q000032:analysis; D015214:Antigens, Differentiation, Myelomonocytic / Q000032:analysis; D001853:Bone Marrow / Q000276:immunology; D001854:Bone Marrow Cells; D016026:Bone Marrow Transplantation; D018826:CD13 Antigens; D005312:Fetal Blood / Q000276:immunology; D018380:Hematopoietic Stem Cell Transplantation; D006801:Humans; D017493:Leukocyte Common Antigens / Q000032:analysis; D007963:Leukocytes, Mononuclear / Q000276:immunology*; D015260:Neprilysin / Q000032:analysis; D010641:Phenotype; D011990:Receptors, Transferrin; D063268:Sialic Acid Binding Ig-like Lectin 3; D014182:Transplantation, Autologous
D015703:Antigens, CD; D018941:Antigens, CD19; D018952:Antigens, CD34; D000944:Antigens, Differentiation, B-Lymphocyte; D015214:Antigens, Differentiation, Myelomonocytic; C571163:CD33 protein, human; C068126:CD71 antigen; D011990:Receptors, Transferrin; D063268:Sialic Acid Binding Ig-like Lectin 3; D017493:Leukocyte Common Antigens; D018826:CD13 Antigens; D015260:Neprilysin