Anti-human CD117 mAb SR-1 blocks SCF binding to CD117 on human HSCs and inhibits human HSC proliferation in vitro. (A) Relative CD117 expression of HSCs (Lin⁻CD34⁺CD38⁻CD90⁺CD45RA⁻) and progenitors (Lin⁻CD34⁺) from normal BM samples and MDS BM samples from all IPSS-R risk groups. CD117 expression was assessed by mean fluorescence intensity (MFI) by flow cytometry using a commercially available anti-human CD117 mAb, clone 104D2. MFI was normalized to BM CD3⁺ T cells from healthy subjects, which served as staining control. *P < .001 compared with control CD3⁺ T cells (Student t test). (B) Pretreatment with SR-1 mAb inhibits binding of fluorescently labeled SCF (SCF-A488) to human CD117. A mixture of CD117-expressing and untransfected fibroblasts were stained with SR-1 mAb (left), SCF-A488 alone (center), or first stained with SR-1 and then subsequently stained with SCF-A488 (right). SR-1 mAb was detected using a fluorescently labeled goat-anti-mouse secondary mAb. (C) SR-1 mAb inhibits proliferation of FACS-purified human UCB-derived HSCs (Lin⁻CD34⁺CD38⁻CD90⁺CD45RA⁻) in liquid culture. UCB HSCs were plated via plate-sorting at a density of 25 cells per well in StemSpan media supplemented with SCF, TPO, FLT3L, IL-3, and IL-6, and treated with varying concentrations of SR-1. Cell counts were performed on days 2, 4, and 5 postplating. *P < .001 comparing 100 μg/mL, 10 μg/mL, and 1 μg/mL conditions compared with untreated on day 7 (Student t test). Error bars indicate 1 standard error of the mean. (D) SR-1 mAb inhibits proliferation of FACS-purified human BM-derived HSCs (Lin⁻CD34⁺CD38⁻CD90⁺CD45RA⁻) in liquid culture. BM HSCs were plated via plate sorting at a density of 25 cells per well in StemSpan media supplemented with SCF, TPO, FLT3L, IL-3, and IL-6, and treated with varying concentrations of SR-1. Cell counts were performed on days 2, 4, 5, and 7 postplating. *P < .001 comparing 100 μg/mL, 10 μg/mL, 1 μg mL, and 0.1 μg/mL conditions compared with untreated on day 7 (Student t test). Error bars indicate 1 standard error of the mean. (E) SR-1 mAb inhibits colony formation of FACS-purified human UCB-derived HSCs (Lin⁻CD34⁺CD38⁻CD90⁺CD45RA⁻) in methylcellulose supplemented with G-CSF and treated with indicated concentrations of SR-1. CFU-GM colony counts were performed 2 weeks after culture initiation. Representative images of the colonies present at 2 weeks are shown (for untreated and 1 μg/mL SR-1). *P < .001 compared with day 0 (Student t test). Error bars indicate 1 standard error of the mean.