Characterization of Human CD34+ Hematopoietic Stem Cells Following Administration of G-CSF or Plerixafor

Background: Current clinical protocols use granulocyte colony-stimulating factor (G-CSF) to mobilize normal hematopoietic stem cells (HSCs) from the bone marrow (BM) to the peripheral blood. Unfortunately, this process requires from 4 to 6 days of G-CSF injection and is associated with significant morbidity, most notably bone pain. We are evaluating a novel method for the mobilization of HSCs using a direct antagonist of the CXCR4/SDF-1 interaction called Plerixafor (AMD3100).

Methods: Human CD34⁺ cells were collected from three different studies at our institution. In the first study, fifteen healthy allogeneic related donors were initially mobilized with increasing doses of intravenous (IV) AMD3100 (80, 160, 240, or 320 μg/kg). After 4 days of drug clearance, the same donors were then mobilized with a single subcutaneous (s.c.) dose of 240 μg/kg AMD3100 and collected cells were used as a source of HSCs for transplantation. In the second study, ten healthy donors were mobilized with 5 days of s.c. injection of G-CSF (10 μg/kg/day), and leukapheresed on day 5. In the final study, eight individual normal donors were mobilized sequentially with AMD3100 and G-CSF. Donors received 1 s.c. injection of 240 μg/kg AMD3100, followed by leukaphersis beginning 4 h after drug treatment. After 10 days of drug clearance, the same donors were mobilized with 5 days s.c. injection of 10 μg/kg/day G-CSF, and leukapheresed on day 5. Human CD34⁺ cells were purified by positive selection with a Magnetic Affinity Cell Selection (MACS) CD34 isolation kit and total RNA was isolated using RNeasy Mini Kit columns (Qiagen). The purity (>85% for all experiments) and phenotype of isolated CD34⁺ cells was quantified by flow cytometry. RNA profiling analyses were performed using Affymatrix U133+2 arrays.

Results: Peak mobilization of CD34⁺ cells occurred 4 to 6 hours after both IV and s.c. dosing, however, patients given IV doses had higher peak levels of CD34/μl at every time point. There was a clear doseresponse relationship of IV AMD3100 on mobilization of CD34⁺ HSCs in normal donors, with the 320 μg/kg dose yielding a maximum increase in circulating CD34⁺ cells from 3.3 ± 1.8 CD34⁺/μl at baseline to 28.8 ± 4.7 CD34⁺/μl at 6 h after injection. Although the magnitude of neutrophil, monocyte, and T lymphocyte mobilization by IV AMD3100 was less than that observed for CD34⁺ cells (2 to 3 fold increase over baseline), the kinetics of their mobilization were similar to the CD34⁺ HSCs (peak mobilization 4 to 6 h after AMD3100). In contrast, B-lymphocytes were mobilized more rapidly (4.5 ± 1.7-fold at 15 min post-AMD3100) and efficiently (6.6 ± 2.6-fold at 2 h post-AMD3100) by IV AMD3100. This rapid mobilization of B-lymphocytes correlates with our pharmacokinetic studies, which showed that peak levels of AMD3100 occur between 15 and 30 minutes after IV infusion. The gene signature of AMD3100-mobilized human CD34⁺ HSCs is distinct from that of G-CSF-mobilized CD34⁺ cells. Of note, EMR1, GIMAP8, PIM1, S100A8, SOCS3, and TMEM49 were expressed more abundantly in all GCSF-mobilized CD34⁺ cells while BCL-2, CLC, CXCR4, C200rf118, DNTT, IRF8, PRG2, RASD1, RNASE6, and UHRF1 were more abundantly expressed in all AMD3100-mobilized CD34⁺ cells. Interestingly, the RNA profile of CD34⁺ HSCs obtained from the BM of three healthy donors clustered with AMD3100-mobilized CD34⁺ HSCs rather than GCSF mobilized HSCs. Using flow cytometry, we identified a CD34^dimCD45RA⁺ hematopoietic precursor cell that is uniquely enriched in nearly 60% of the AMD3100 products evaluated to date (6/10 patients). In contrast to G-CSF mobilized products, where <2% of CD34⁺ cells are CD34^dimCD45RA⁺, up to 20% of CD34⁺ cells in AMD3100 treated donors are CD34^dimCD45RA⁺. Preliminary flow cytometry data suggest that this CD34^dimCD45RA⁺ population represents a pro-DC2 (for progenitor of pre-dendritic cell type 2) progenitor compartment as indicated by their IL-3Rα^brightCD62L^brightα4β7^dimCD4^dimCD25⁻c-kit⁻CD13⁻ phenotype.

Conclusions: These observations suggest that IV AMD3100 may be a more effective mobilization agent with a low side effect profile. The gene signature and phenotype of AMD3100-mobilized CD34⁺ HSCs is distinct from that of G-CSF-mobilized HSCs and resemble CD34⁺ HSCs present in an unmanipulated BM.