Mobilisation with G-CSF in Helathy Donors Promotes a High but Temporal Deregulation of Genes.
Authors: Castilla C. , Gutierrez N. C. , Isidro I. M. , Delgado M. , Rivas J. , Ferminan E. , Canizo M. C. , Hernandez J. M. , Miguel J.F. San 
Published: April 2020

Abstract
G-CSF is commonly used for stem cell mobilisation in healthy donors. G-CSF stimulates proliferation and differentiation of myeloid lineage. However, other effects and their possible repercussions in healthy donors are less widely analyzed. In order to evaluate G-CSF effects in transcriptome of peripheral blood cells (PB) nine healthy donors mobilized with G-CSF (5 microg/kg/q12h for 4 days) were studied. PB samples were collected before starting G-CSF (day 0), the day of stem cell collection (day +5), 2 months and 6 months after mobilisation. Total RNA (5–10 microg) was labelled and hybridized to a high-density oligonucleotide microarray (Human Genome U133A microarray Affymetrix). Cluster analysis showed a dendrogram with 2 major branches: one included the samples before G-CSF as well as 2 and 6 months after the mobilisation while the second branch only included the samples on day +5 after G-CSF. Supervised analysis identified 761 significant genes that distinguished the two groups: 374 increased their expression level while 387 decreased their expression level after G-CSF. The most altered gene categories involved protein byosinthesis, signal transduction, transcription, immune response, cell adhesion, cellular cycle and apoptosis. A large number of genes involved in protein biosynthesis and protein folding were down-regulated. Most of them encode for ribosomal proteins while others, such as initiation factor IF2, are essential for the initiation of protein synthesis. Regarding cell adhesion ICAM2 was downregulated following G-SCF and some small GTPases and Cdc42 were overexpressed, all these changes would favour cell migration of stem cells. Most of the genes involved in the immune response were down-regulated, including T-cell related genes and HLA complex as well as GATA3, a master key in T-cell development. One of the side-effects in patients receiving G-CSF is a mild thrombocytopenia, and this could be explained by the observation of underexpression of profile of genes related to megakaryotcyitc proliferation (PF4 and PTPN4). In contrast, genes involved in the erythropoiesis, such as TIMP1 and ETS-2 were up-regulated. These results could help to understand the synergistic effect on red cell counts in patients treated with G-CSF plus EPO. As far as genes involved in blood coagulation is concerned, only two genes (FV and Annexin A5) were found to be overexpressed. The latter protein shows an anticoagulant effect. According to these results, the prothrombotic status suggested after G-CSF administration would not be supported by our gene analysis. Nevertheless, we have found an important overexpression of genes associated with neutrophil activation (elastase, CD11b) which may favour a transient mild hipercoagulable status after G-CSF. Regarding proliferation and cell cycle, several genes were deregulated. Interestingly BTG1, ATM and MYC were down-regulated. These inhibitions could induce cell cycle arrest, promote maturation block in addition to differentiation, as well as an increased sensitivity to apoptosis. The deregulation of these genes involved in cell cycle could explain the increased levels of mature leukocytes showed after G-CSF. In summary, the use of G-CSF in healthy donors promotes changes in expression levels of multiple genes involved in important mononuclear blood cell functions. These gene expression changes are temporary, returned to normal profile after two months and remained normal by month +6.