Properties of expanded HSPCs. (A) Schematic presentation of competitive transplantation analysis of HSPCs from POL5551 in comparison with G-CSF treated or sham-operated (sham-op) mice. (B-C) Competitive repopulation with differentially treated BM cells. (B) Test BM (n = 3 donor mice; 5 × 10⁵ cells per recipient; CD45.1⁺) from control mice (sham-op) or mice treated with G-CSF (100 μg/kg per dose, 9 doses every 12 hours) or POL5551 (100 mg/kg per day for 2 weeks via pump) was mixed with competitor BM (n = 4 donor mice; 5 × 10⁵ cells per recipient; CD45.1/2⁺) and transplanted into lethally irradiated recipients (CD45.2⁺). PB chimerism analysis of the primary recipients (left) was performed at the indicated time points after transplantation. The percentage of CD45.1⁺ cells within the graft-derived (ie, CD45.1⁺ or CD45.1/2⁺) CD45⁺CD3^– population is shown (data are from 1 of 7 independently performed experiments and represent the mean ± SEM of 5 mice per cohort). At 20 weeks after primary transplantation, BM of the recipients was harvested, pooled, and injected into lethally irradiated secondary (CD45.2⁺) recipients (2.5 × 10⁶ cells per recipient). Right panel shows PB chimerism measurement in secondary recipients (data represent the mean ± SEM of 5 mice per cohort). (C) The frequency of repopulating units in different test BM suspensions was calculated on the basis of the contribution within the B-cell lymphoid and myeloid (non–T-cell) fraction 16-20 weeks after primary transplant. Data from 7 independently performed transplant experiments (including those presented in Figure 5H) are shown for the control and POL5551-treated BM recipients (mean ± SEM of 34-41 mice per cohort for control and POL treated BM recipients; mean ± SEM of 5 mice in G-CSF BM recipient group). (D-E) Kinetics of noncompetitive engraftment. BM from POL5551 (100 mg/kg per day for 2 weeks of continuous infusion) or sham-op–treated C57BL/6 mice (CD45.2⁺; 4 donor mice per group) was transplanted noncompetitively into lethally irradiated recipients (CD45.1⁺; 1 × 10⁶ cells per recipient). Hematopoietic reconstitution was assessed by serial blood count analysis. (D) Neutrophil (NE) and (E) platelet (Plt) counts are shown (data represent the mean ± SEM of 5 recipients per group). (F-G) Competitive repopulating unit (CRU) assay: Small volumes (2, 4, or 8 μL) of POL5551 (100 mg/kg per day for 2 weeks of continuous infusion; pooled from 4 donor mice) or G-CSF (100 μg/kg per dose, 9 doses every 12 hours; pooled from 4 donor mice) mobilized blood (CD45.1⁺) were cotransplanted with 2.5 × 10⁵ competitor BM cells (CD45.1/2⁺) into lethally irradiated recipients (CD45.2⁺; 8-9 mice per dose per mobilizing agent). At 12 weeks after transplantation, CRU engraftment (≥0.5% engraftment in all lineages) was quantified. (F) Percentages of nonengrafted mice were plotted against blood graft volume. Data are represented by f(x) = –5.13+ 103.06 (R² = 0.91) for G-CSF and f(x) = –7.97x+100.83 for POL5551 (R² = 0.99). (G) Competitive repopulating unit frequency was determined by using Poisson’s statistic (LCALC software, Stem Cell Technologies) (mean ± upper or lower frequency). (H-I) Genetic blockade of CXCR4 signaling. CXCR4^del/del and wild-type (wt) control mice (test BM donors) were generated by injecting tamoxifen (2 sets of 3 consecutive doses) into CXCR4^flox/floxERT2Cre⁺ and CXCR4^wt/wtERT2Cre⁺ mice (both CD45.2⁺), respectively. (H) CXCR4 expression was quantified via real-time polymerase chain reaction in unfractionated BM (wBM) and sorted HSCs (LSKCD135^–CD34^–) from test as well as CD45.1/2⁺ competitor (comp.) BM donors. (I) Two groups of recipients (CD45.1⁺) received a graft consisting of 5 × 10⁵ test (control or CXCR4^del/del; pooled from 3 donor mice each) and competitor BM cells. The 3:1 group (CD45.1⁺) received a graft composed of 7.5 × 10⁵ CXCR4^del/del and 2.5 × 10⁵ competitor cells. Shown is the CD45.2⁺ percentage within the graft-derived (ie, CD45.2⁺ or CD45.1/2⁺) CD45⁺CD3^– population (data represent the mean ± SEM of 3-4 mice per cohort). ***P < .001; **P < .01; *P < .05. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; RU, relative unit; Tx, treatment.