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

Abstract

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 × 10 4 to 0.65 × 10 4/ml in the following order, bone marrow > chemotherapy/etoposide/G-CSF > cord blood > cyclophosphamidemobilized 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.