Neutrophil Maturation of CD34+ Cells from Peripheral Blood and Bone Marrow in Serum-Free Culture Medium with PIXY321 and Granulocyte-Colony Stimulating Factor (G-CSF)

Abstract

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.

The Lactate Issue Revisited: Novel Feeding Protocols To Examine Inhibition of Cell Proliferation and Glucose Metabolism in Hematopoietic Cell Cultures

“…However, 1 M NaOH was added daily to adjust the pH to match that of the Fed cultures after feeding. In {Not Fed} and {Not Fed + NaOH} cultures, 10 ng/mL G-CSF was added every other day to prevent depletion of G-CSF due to instability (25).…”

Heteroclitic CD33 Peptide With Enhanced Anti-Acute Myeloid Leukemic Immunogenicity

“…Jilani et al (42) reported that the level of CD33 expression on CD34 ϩ cells from AML patients was similar to levels observed on CD34 ϩ cells from normal donors. The CD34 ϩ cells used as target cells in our cytotoxicity and proliferation assays were isolated from G-CSF-mobilized normal donors and were therefore primed for differentiation into the myeloid lineage (43,44). We did not observe a substantial lysis of the CD34 ϩ cells by CD33 1Y2L-CTL clone (Fig.…”

Cytokine-Dependent Proliferation of Human CD34+ Progenitor Cells in the Absence of Serum Is Suppressed by Their Progeny's Production of Serine Proteinases

“…Use of defined media and supplements is critical for interpreting the results of experiments, improving reproducibility of studies, and allowing production of cells under good manufacturing practice (GMP) conditions for clinical applications. In many cases, however, there is a discrepancy in the outcome of hematopoietic cell cultures between cultures in serum‐free media and cultures in serum‐supplemented media regarding the concentration of supplements needed for a cellular response [5–8]. Previously, we found that hematopoietic growth factor (HGF)–dependent proliferation of hematopoietic progenitor cells (HPCs) in the absence of serum was suppressed by the presence of accessory bone marrow cells [9].…”