Gene Expression Profiles for Normal Human Bone Marrow-Derived CD34+ Stem Cells and CD34−/CD33+ Myeloid-Committed Progenitor Cells in Response to Daily Granulocyte-Colony-Stimulating Factor (G-CSF) Treatment.

Abstract

G-CSF is widely used to accelerate marrow recovery after cancer chemotherapy, to facilitate collection of hematopoietic progenitor cells, and to treat severe chronic neutropenia. Although G-CSF was originally defined as a stimulus for myeloid cell proliferation, it has potent anti-apoptotic properties, affects synthesis of proteins stored in neutrophil granules, and has many other effects on cells of the myeloid lineage. To improve understanding of the molecular and cellular effects of G-CSF, particularly related to its use for the treatment of severe chronic neutropenia, we performed gene expression profile studies using Affymetrix oligonucleotide arrays and purified bone marrow cell sub-populations from normal volunteers treated with daily subcutaneous G-CSF (300 mcg/sc/qd) for five days. Under local anaesthesia, paired marrow aspirates were obtained from the posterior iliac crest before and after 5 daily doses of G-CSF. CD34+ and CD34−/CD33+ cells were purified using Miltenyi immunomagnetic beads. Two rounds of amplification of total RNA isolated from purified CD34+ or CD33+cells was used to obtain sufficient cRNA for hybridization. Expression data from scanned chips were first analyzed using the RMA algorithm. The limma package of the Bioconductor project was used to identify differentially expressed genes. Limma uses an empirical Bayes method to moderate the standard errors of the estimated log-fold changes. The statistical analysis of CD33+ cells revealed that 150 of more than 12,000 genes examined were up- or down-regulated >2-fold in response to G-CSF treatment. The top 10 genes with up- or down-regulated level of expression include clusterin, neutrophil elastase, two transcription factors, gelsolin, Grb2, phospholipase D3, protein kinase C, the major vault protein, and serine-threonine kinase. In the myeloid-committed CD34-/CD33+ progenitor cells, genes with altered expression level represent those with gene products involved in the cell cycle, regulation of apoptosis, the cytoskeleton, the inflammatory response, or serine proteases and transcription factors. Most of the genes up-regulated in CD33+ cells (e.g. neutrophil elastase, phospholipase D, protein kinase C) were down-regulated in CD34-positive cells in response to G-CSF. The results of the comparative analyses revealed the normal signature gene expression profiles for CD34+ and CD34−/CD33+ cells and identified genes that may mediate specific G-CSF effects.