Granulocyte colony-stimulating factor produces long-term changes in gene and microRNA expression profiles in CD34+ cells from healthy donors

Abstract

© F e r r a t a S t o r t i F o u n d a t i o ngene and miRNA expression profiles in HPC from healthy donors, and to determine whether any changes in expression signatures persist in the long-term or return to the original status. Methods SamplesCD34 + progenitor cells from peripheral blood of six healthy donors were collected before and at 5, 30 and 365 days after the mobilization with G-CSF (mobilization regimen: 10-15 mg/kg of G-CSF daily for 5 days). All donors were included in the transplant program of the Hematology Department of the University Hospital Virgen del Rocío (Seville, Spain). The local ethics committee of the same hospital provided institutional review boardapproval for this study, and informed consent was obtained from all donors in accordance with the Declaration of Helsinki. Isolation of hematopoietic progenitor cellsMononuclear cells were collected from all samples by density gradient centrifugation with Ficoll-Paque solution (Amersham Biosciences, Uppsala, Sweden). The CD34 + cells were isolated in an AutoMACS pro separator (Miltenyi Biotec, Bergisch Gladbach, Germany) by positive immunomagnetic selection using the CD34 MACS microbead Human Kit (Miltenyi Biotec, Bergisch Gladbach, Germany) and, after magnetic enrichment, CD34 + cells were sorted by flow cytometry. The purity of the isolated CD34 + cells was greater than 95% in all cases. RNA extractionTotal RNA was extracted by TRIsure (Bioline, Luckenwalde, Germany) in all samples. The quality and integrity of the RNA were verified by a Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA). MicroRNA and gene expressionThe expression profiles of 384 miRNA were analyzed in all samples using TaqMan Human MicroRNA v2.0 Arrays (Applied Biosystems, Foster City, CA. USA) which were analyzed on a 7900 HT Fast Real Time Polymerase Chain Reaction (PCR) System (Applied Biosystems, Foster City, CA, USA). The SDS 2.3 and RQ Manager 1.2 software (both Applied Biosystems, Foster City, CA, USA) were used for the analysis. Data were normalized using the average of the endogenous small-nucleolar RNU48 and the noncoding small nuclear U6, both included in the array.The expression profile of 45,000 genes was analyzed in the same samples using the Whole Human Genome Oligo microarray kit 4x44K (Agilent Technologies, Santa Clara, CA, USA). The microarrays were scanned in a GenePix reader (Molecular Devices, Sunnyvale, CA, USA). Samples from non-mobilized CD34 + cells were used as the reference group in both types of expression analysis.The expression of significant genes was validated by quantitative real-time PCR using Quantitec Primer Assays and the Quantitec SYBR Green Kit (both from Qiagen, Hilden, Germany) in a 7900 HT Fast Real Time PCR System (Applied Biosystems, Foster City, CA, USA). Data were normalized to the housekeeping gene ACTB and the group of samples from non-mobilized CD34 + cells was used as a control. The relative gene expression levels were calculated by the 2 -ΔΔCT method. Statistical analysisUnsupervised hierarchical clust...

Granulocyte colony-stimulating factor alters the systemic metabolomic profile in healthy donors

“…We have not performed any functional assays to evaluate the potential association between metabolites altered by G-CSF and immunomodulatory effects; however, G-CSF has been shown to induce several cellular and immunological changes in donor cells (Anderlini et al 1996; Shaw et al 2015), and several of the altered metabolites found in our study have known immunoregulatory and/or angioregulatory effects, or can be markers of altered regulation of epigenetic/gene expression that may contribute to the previously described long-lasting effects after G-CSF therapy (Baez et al 2014). Altered metabolite levels may reflect the direct effects of G-CSF on different immune cell types, affecting cell proliferation, differentiation and function, but also indirect effects through e.g.…”

“…Changes in global gene expression profiles were then described both at early time points after G-CSF administration (up to 5 days) (Hernandez et al 2005) and after 2–10 months (Amariglio et al 2007), and even a year after G-CSF administration in CD34 + progenitor cells (Baez et al 2014). These studies have revealed that there seems to be both early responses to G-CSF, transient changes that are normalized over time and more long-lasting changes.…”

“…These studies have revealed that there seems to be both early responses to G-CSF, transient changes that are normalized over time and more long-lasting changes. In the recent study by Bàez et al, G-CSF-mobilized hematopoietic progenitors had a difference in the expression of six microRNAs and even one year after G-CSF administration over 2424 genes maintained their altered expression (Baez et al 2014). Among the differentially expressed genes were genes involved in cellular growth, cell death and survival, protein synthesis, gene expression and nucleic acid metabolism.…”

A review of the genetic and long-term effects of G-CSF injections in healthy donors: a reassuring lack of evidence for the development of haematological malignancies

“…Although not all studies can be directly compared with each other due to differences in the cellular population, time points at testing, or testing methodology, the majority have shown only transient abnormalities in either chromosomal number (or other abnormalities) or gene expression profiles. The exception is a study by Baez et al, 31 which does show persistent changes at 1 year post exposure. Further analyses of this sort are warranted to investigate the significance of these findings and correlation with the clinical condition for these donors is necessary.…”

“…30 A few recent studies have addressed the issues of whether these changes in donor cells are persistent invivo. Baez et al 31 examined isolated CD34+ cells from six healthy donors before G-CSF exposure and on day 5, 30 and 365 after G-CSF mobilisation. They found that changes in both gene expression profiles and miRNAs were present and persisted until at least 1 year after the administration of G-CSF.…”