Functional characterization of CD34 subsets. (A-B) Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis of CXCR4 mRNA. (A) Expression of CXCR4 mRNA in CD34⁺ HSPCs. Total CD34⁺ HSPCs were purified by immunomagnetic selection from normal healthy bone marrow (n = 4), G-CSF (10 μg/kg per day × 5 days) mobilized leukapheresis products (n = 7), or plerixafor-mobilized leukapheresis products (n = 9). qRT-PCR of CXCR4 mRNA in the different cell subsets was normalized to GAPDH. (B) Expression of CXCR4 mRNA in CD34⁺ HSPC subsets. CD34⁺ HSPCs (n = 7) from plerixafor-mobilized leukapheresis products were purified by magnetic-activated cell sorting and CD34⁺CD45RA⁻CD123^+/− HSPCs (n = 9); CD34^dimCD45RA⁺CD123⁺⁺ pro-DC2 cells (n = 9) were isolated by flow cytometry. (C) CD34⁺ HSPCs from plerixafor (n = 4) or G-CSF (n = 3) mobilized leukapheresis products were purified by immunomagnetic selection and added into the upper chamber of a 5-μm pore size transwell. After 4 hours, migrated cells recovered from the lower chamber were counted and CD34⁺CD45RA⁻CD123^+/−, CD34⁺CD45RA⁺CD123^+/−, and CD34⁺CD45RA⁺CD123⁺ were enumerated by flow cytometry (D) after treatment with ODN2216. Flow cytometry–purified CD34⁺CD45RA⁺CD123⁺⁺CD303⁺ pro-DC2 cells were treated with vehicle alone or ODN2216 (a CpG TLR9 agonist) in the presence of IL-3 and CD40 ligand-transfected L cells. After 48 hours, the amount of IFN-α in the supernatant was determined using an enzyme-linked immunosorbent assay. Purified CD34⁺CD45RA⁻ HSPCs and CD34⁻CD45RA⁺CD123⁺⁺CD303⁺ pDCs were included in these studies as negative and positive controls, respectively. (E) CD34⁺ HSPCs from plerixafor-mobilized (n = 4) or G-CSF–mobilized (n = 3) leukapheresis products were purified by immunomagnetic selection; KI67 expression was evaluated by flow cytometry. The percentage of KI67 positive cells (E) and KI67 MFIR (F) is shown. (G) MethoCult colony-forming unit assay of CD34 subsets. Statistical comparisons were performed using an unpaired 2-tailed Student t test. *P < .05, **P < .01, ***P < .001.