Jeffrey Martinson

Immunocytochemistry and flow cytometry evaluation of human megakaryocytes in fresh samples and cultures of CD34+ cells.

Adhering platelets on the cell surface can give misleading results when doing flow cytometry analysis of platelet/megakaryocyte-specific glycoprotein (GP) antigens to enumerate megakaryocytes (MK) in mobilized peripheral blood (PB), apheresis products, or normal bone marrow (BM). For adequate quantification and characterization of human MK, we examined samples with parallel flow cytometry and immunocytochemistry. MK expression of GP IIb/IIIa (CD41a), GP Ib (CD42b), GP IIIa (CD61), CD45, CD33, and CD11b, and their light scatter properties were evaluated. Fresh samples of low density mononuclear cells (MNC) or purified CD34+ cells contained 10-45% of platelet-coated cells. Platelet-coated cells decreased dramatically after several days of incubation in a serum-free medium supplemented with stem cell factor, IL-3, IL-6, and/or GM-CSF. Between d 9-12, flow cytometry detected a distinct CD41a+ MK population, 8.3 +/- 1.3% in BM CD34 cell cultures (n = 7) and 13.1 +/- 2.1% in PB CD34 cell cultures (n = 14), comparable to immunocytochemistry data (7.8 +/- 1.9% and 16.4 +/- 2.6%, respectively). CD41a stained a higher proportion of MK than CD42b or CD61, while CD42b+ or CD61+ cells contained more morphologically mature MK than CD41a+ cells in cultures containing aplastic serum. When fluorescence emission of CD41a was plotted against forward-light scatter (FSC), subpopulations of small and large MK were observed. Such subpopulations overlapped in CD41a intensity and side-light scatter (SSC) property. Most MK co-expressed CD45 (98.8% positive) but not CD33 (80.7% negative) or CD11b (88.9% negative). Our data indicate that flow cytometry can be used effectively to identify MK. However, caution should be taken with samples containing adherent platelets.

Characterization of a culture-derived CD15+CD11b- promyelocytic population from CD34+ peripheral blood cells.

Selected CD34+ cells from mobilized apheresis products were cultured in serum‐free or serum‐containing media supplemented with granulocyte colony‐stimulating factor (G‐CSF), granulocyte‐macrophage colony‐stimulating factor (GM‐CSF), interleukin‐3 (IL‐3), and stem cell factor (SCF; c‐kit ligand). We examined the emergence of a CD15+ CD11b‐ population, which appeared morphologically to be promyelocytes. This CD15+CD11b‐ population can be further expanded in culture into morphologically mature granulocytes. In an attempt to characterize this culture‐derived CD15+CD11b‐ promyelocytic population, single cells were clone sorted into wells of a Terasaki plate containing various growth factors. We compared the growth factor requirements and kinetics of this apheresis culture‐derived CD15+ CD11b‐ population to the CD15+CD11b‐ population from fresh bone marrow samples. Our studies indicate that the CD15+CD11b‐ promyelocytic population from bone marrow and blood are equivalent in their ability to proliferate and in their requirements for growth factors. The CD15+CD11b‐ population in vitro shows a high proliferative capacity when compared with the other CD15/CD11b populations (CD15‐CD11b‐, CD15+CD11b+, CD15‐CD11b+). Thus, we can manipulate CD34+ cells in vitro to proliferate and differentiate toward a mature neutrophil lineage. The CD15+ CDllb‐ promyelocytic population derived from this culture may represent the most effective cultured cell population for therapeutic reduction of neutropenia in vivo based on both its stage of differentiation and its proliferative potential. J. Leukoc. Biol. 62: 480–484; 1997.

Identification of a human erythroid progenitor cell population which expresses the CD34 antigen and binds the plant lectin Ulex europaeus I.

Two and three color flow cytometry of normal human bone marrow was used to identify CD34+ progenitor cells and examine their binding to the plant lectin Ulex europaeus I (Ulex). In normal bone marrow, 48.48 +/- 17.4% of the CD34+ cells bind to Ulex. Two color flow cytometry was used to sort CD34 + cells, and subsets of CD34+ cells, CD34+ Ulex+ and CD34+ Ulex-. These populations were sorted into colony assays to assess myeloid (CFU-GM) and erythroid (BFU-E) progenitors. The CD34+ Ulex+ subset was 84 +/- 14% BFU-E colonies (mean +/- S.D.) and had the highest cloning efficiency of 28 +/- 13%. Three color analysis of CD34+ Ulex+ cells showed staining with other erythroid (CD71, GlyA) antibodies and lack of stain. ing with myeloid (CD13, CD45RA) antibodies. These studies confirmed the erythroid characteristics of this subpopulation.