Pegylated G-CSF Mobilizes CD34+ Cells with Different Stem and Progenitor Cell Subsets and Distinct Functional Properties in Comparison with Unconjugated G-CSF.

Current regimens for peripheral blood stem cell (PBSC) mobilization in patients with multiple myeloma are based on daily subcutaneous injections of G-CSF starting shortly after cytotoxic therapy. Recently a polyethylenglycole (PEG)-conjugated G-CSF has been introduced which has a substantially longer half-life than the original formula and therefore provides the basis for long-lasting G-CSF serum-levels after a single injection. In this study, we compared gene expression patterns, subset composition and functional properties of CD34+ cells and highly purified HSC mobilized with cyclophosphamide and either Peg-G-CSF or G-CSF. Cells were derived from peripheral blood of patients with multiple myeloma. After the end of chemotherapy, 7 patients got a single injection of Peg-G-CSF whereas 9 patients received daily G-CSF resulting in an equal cumulative dose. Gene expression analysis was performed using Affymetrix HG Focus GeneChips. Key functional genes were verified by RT-PCR. Subset analysis and fluorescence based cell sorting has been conducted to assess the effects of stimulation with either pegylated or unconjugated G-CSF on CD34+ subset composition and to obtain HSCs. Cell cycle and apoptosis assays as well as clonogenic assays were for functional corroboration. The same patients with multiple myeloma who had donated CD34+ cells for the molecular and biological studies were transplantated with Peg-G-CSF- or G-CSF-mobilized PBSC.

Peg-G-CSF-mobilized cells showed lower expression of genes characteristic for erythroid and later stages of myeloid differentiation as well as a lower BFU-E/CFU-GM ratio compared to G-CSF-mobilized cells. In turn, we found higher expression levels of genes indicative of early hematopoiesis including HOXA9, MEIS1, MLL and GATA3. Subset analyses revealed a greater number of HSC and CMP (common myeloid progenitors) and a lower number of MEP (megakaryocyte-erthrocyte progenitors) in Peg-G-CSF-mobilized CD34+ cells.

Cell cycle-promoting genes including cyclins and kinases were higher expressed in Peg-G-CSF-mobilized cells. On the other hand human HTm4, which causes cell cycle arrest in hematopoietic cells, was lower expressed compared to G-CSF-mobilized cells. This is emphasized by a significant higher proportion of actively cycling CD34+ cells after pegfilgrastim-mobilization. Higher gene expression levels of HOXA9, MEIS1 and GATA3 were also found in sorted Peg-G-CSF-mobilized HSC in comparison to G-CSF-mobilized HSC. Moreover, Peg-G-CSF-mobilized HSC showed a lower apoptosis rate and a greater proportion of cells in S- and G2/M phase of cell cycle. After transplantation of Peg-G-CSF-mobilized stem- and progenitor cells we observed earlier leukocyte recovery compared to G-CSF-mobilized transplants.

Our data demonstrate that Peg-G-CSF and G-CSF stimulation differentially affects the expression of key regulatory genes and functional properties of mobilized HSC as well as their progeny, which might be important for their application in stem cell transplantation.