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. 2017 Mar 14;129(19):2680–2692. doi: 10.1182/blood-2016-09-739722

Figure 5.

Figure 5.

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); CD34dimCD45RA+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.