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Four-color flow cytometry was used with a cocktail of antibodies to identify and isolate CD34+ hematopoietic progenitors from normal human peripheral blood (PB) and bone marrow (BM). Mature cells that did not contain colony forming cells were resolved from immature cells using antibodies for T lymphocytes (CD3), B lymphocytes (CD20), monocytes (CD14), and granulocytes (CD11b). Immature cells were subdivided based on the expression of antigens found on hematopoietic progenitors (CD34, HLA-DR, CD33, CD19, CD45, CD71, CD10, and CD7). CD34+ cells were present in the circulation in about one-tenth the concentration of BM (0.2% v 1.8%) and had a different spectrum of antigen expression. A higher proportion of PB-CD34+ cells expressed the CD33 myeloid antigen (84% v 43%) and expressed higher levels of the pan leukocyte antigen CD45 than BM-CD34+ cells. Only a small fraction of PB-CD34+ cells expressed CD71 (transferrin receptors) (17%) while 94% of BM-CD34+ expressed CD71+. The proportion of PB-CD34+ cells expressing the B-cell antigens CD19 (10%) and CD10 (3%) was not significantly different from BM-CD34+ cells (14% and 17%, respectively). Few CD34+ cells in BM (2.7%) or PB (7%) expressed the T-cell antigen CD7. CD34+ cells were found to be predominantly HLA-DR+, with a wide range of intensity. These studies show that CD34+ cells and their subsets can be identified in normal PB and that the relative frequency of these cells and their subpopulations differs in PB versus BM.

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. © 1996 Wiley‐Liss, Inc.

Twenty two patients were treated with cyclophosphamide therapy to mobilize progenitors into the blood. Progenitor cells were quantitated in peripheral blood or leukapheresis products using colony assays and flow cytometric measurement of CD34 + cells. Prompt engraftment of > 500 granulocytes/ul at a median of 13 days was observed in all patients reconstituted with mobilized cells. In four patients where complete sets of serial samples were obtained, the appearance of CD34+ cells preceded the increase in CFU‐GM by 24 to 48 hours. Peak levels of CD34+ cells ranged from 0.6–5% and coincided with the peak increase in CFU‐GM. Mobilized CD34+ cells were predominantly CD33+, CD13+, CD45R+, CD38+, HLA‐DR+ and CD41+. In contrast to bone marrow CD34+ cells, few mobilized CD34 + cells expressed CD71, CD19 or CD10. Long term culture initiating cells (LTC‐IC), capable of reconstituting hematopoiesis in vitro on irradiated stromal layers, were also measured in serial samples from a single patient with high peak levels of CD34 (∼︁5%). LTC‐IC were present in all samples and the ratio of LTC‐IC to CD34 + cells remained similar throughout the post cyclophosphamide recovery phase. In this patient, preferential mobilization of LTC‐IC earlier in the recovery phase was not observed suggesting that during recovery both primitive and committed progenitors are mobilized together. These data indicate that mobilized CD34+ cells represent subpopulations of CD34+ cells with a myeloid phenotype and that the presence of primitive progenitors among these cells confirms clinical reports that they are capable of long term reconstitution.

Hematopoietic recovery after high-dose chemotherapy is characterized by an obligate period of neutropenia of approximately 8-10 days. It is postulated that if a pool of neutrophil precursors and progenitors were expanded in vitro and reinfused, the duration of neutropenia may be substantially shortened by these cells capable of providing mature neutrophils within days of reinfusion. In this study, peripheral blood progenitor cell products were obtained from six normal donors mobilized with rhG-CSF and two patients mobilized with cyclophosphamide and rhG-CSF. CD34+ cells were isolated using the Isolex immunomagnetic bead method. A mean of 8.26 x 10(7) CD34+ cells with a mean purity of 74.5% were seeded at a concentration of 1 x 10(5)/ml into a 12 day stroma-free liquid culture using gas-permeable bags. A serum-free growth medium supplemented with PIXY321 was used. On day 7, there was a mean cellular expansion of fourfold, at which time the cells were resuspended at the initial concentration, yielding a mean culture volume of 3L (1-6 L). On day 12, there was an additional mean fold cellular expansion of 10 x, achieving an overall mean fold expansion of 41 +/- 16. Cellular characterization of the expanded cells revealed predominantly neutrophil precursors by morphology (mean 70.1%) and flow cytometric analysis. A mean of 52.3% of the expanded cells expressed CD15. Immunohistochemical staining revealed a mean of 7.1% CD41a+ megakaryocytic progenitors in the final cultured cell product. Detectable CD34+ cells were maintained only in those cultures initiated with greater than 90% CD34+ cells. Colony-forming units-granulocyte-macrophage (CFU-GM) were maintained in the 12 day culture at a level similar to the preculture number, whereas CFU mixed were depleted in all samples. On day 0, there were few CFU clusters (colonies containing fewer than 50 cells) identified, but by day 12, a mean total of 8.3 x 10(6) CFU clusters were identified. On day 12, the expanded cells were harvested and pooled using the Fenwal CS3000 Plus blood cell separator and resuspended in Plasma-Lyte-A with 1% human serum albumin. The mean harvest recovery of expanded progenitors was 91%, with a mean viability of 86%.

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.

Bone marrow (BM) or peripheral blood (PB) CD34+ cells were cultured for 12 days in serum-free culture medium containing PIXY321 (IL-3/ GM-CSF fusion protein) with or without periodic supplements of granulocyte-colony stimulating factor (G-CSF). The cultures were evaluated at day 12 for total cell proliferation (fold increase from day 0), neutrophil differentiation by flow cytometry, using dual staining with CD15-FITC and CD11b-PE, and morphology using Wright-Giemsa and granule staining. In cultures containing PIXY321 where 6000 U/ml of G-CSF was added days 0 and 6, there was no significant difference (p > or = 0.05) in cell proliferation or the percent of CD15+/CD11b+ cells when compared with cultures with PIXY321 alone. ELISA analysis showed G-CSF levels had declined by 90% after 3 days of culture. Further studies were performed to assess the benefit of supplementing lower concentrations of G-CSF (600 U/ml) at more frequent intervals. A significant increase (p < or = 0.05) in cell proliferation and percent CD15+/CD11b+ was observed when G-CSF was added on days 0, 3, 6, and 9 (every 3 days) as compared with those cultures with PIXY321 alone. CD34+ cell proliferation without G-CSF was 19.6 +/- 4.8-fold, with G-CSF added on days 0 and 6 was 28.7 +/- 6.4-fold, and with G-CSF added on days 0, 3, 6, and 9 was 45.9 +/- 10.6-fold. Percent of CD15+/CD11b+ cells was 19.0 +/- 4.6%, 38.2 +/- 7.2%, and 58.5 +/- 6.5%, respectively, in these cultures. We observed more CD15+/CD11b+ cells, myelocytes/metamyelocytes, and secondary granule staining in cultures with G-CSF added on day, 0, 3, 6, and 9 as compared with cultures with G-CSF added on days 0 and 6 or no G-CSF added. We conclude that PIXY321 and G-CSF act synergistically on the in vitro proliferation and neutrophil differentiation of BM and PB CD34+ cells and that frequent supplements of G-CSF facilitate neutrophil differentiation.

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