Loss of HLF reduces the CD34⁺ GPR56⁺ LSC compartment in vivo. (A) Knockdown level of HLF mRNA in CD34⁺ cord blood cells with 2 different shRNAs compared with shRNA against luciferase (shLuc) determined by quantitative PCR. HLF expression as percentage of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression was normalized to shLuc controls. Values indicate means (shHLF.3441, mean fraction [range] of shLuc expression 0.4 [0.2-0.67]; shHLF.630 mean [range] fraction of shLuc expression 0.06 [0.03-0.12]). Bars and error bars represent means and standard deviation of 3 individual CD34⁺ cord blood infections. (B, left) FACS plots showing differentiation of cord blood CD34⁺ cells 5 and 27 days after infection with shRNA.3441 against HLF or luciferase. Shown is 1 of 4 replicates derived from 2 independent experiments. Protein expression of CD34 and CD45RA were tracked during a period of 27 days. Values indicate percentages. (Right) Box plots showing fractions of CD34⁺CD45RA⁻ cells on day 5 (upper, median percentage 30.85% vs 25.65%; P = .02) and CD34⁺CD45RA⁺ cells on day 27 (lower, 20.80% vs 7.2%; P = .02) after infection with shHLF.3441 or shLuc (4 replicates of 2 independent infections, Mann-Whitney U test, *P < .05). (C) Engraftment of cord blood CD34⁺ cells in NRGS mice after infection with shRNAs against HLF or luciferase, using tagRFP as fluorescent marker. Horizontal lines indicate means; symbols represent individual mice. Shown are the percentages of human CD45⁺tagRFP⁺ cells in mouse bone marrow. At weeks 4 and 12, bone marrow was collected by aspiration from 1 femur, whereas bones from tibia, femur, pelvis, and spine were analyzed after sacrificing the mice in week 17. Mean engraftment levels at week 4: 24.8% vs 59.5% (n = 6 per group; P = .01); week 12: 0.6% vs 10.1% (n = 4, shHLF group, no aspiration material for 2 mice; n = 5, shLuc group, no aspiration material for 1 mouse; P = .046); week 17: 2.9% vs 11% (n = 5, shHLF, 1 mouse died before week 17; n = 6, shLuc; P = .029). (D) Cartoon illustrating experimental setup of in vitro and in vivo experiments. See Materials and methods for details. (E) Western blot showing human HLF protein expression in triple-mutated primary human AML cells (AML#04H112) harvested from mice 14 weeks after injection of cells electroporated with either sgRNA against HLF (sgHLF) or GFP (sgGFP, negative control) and Cas9 recombinant protein. GAPDH was used as loading control. Numbers below western blot indicate the knockout efficiency (%) for HLF, determined by Sanger sequencing on the genomic DNA from the same cells (see supplemental Figure 6B for gDNA results). Mouse IDs in bold indicate mice used for secondary transplantations. (F) Engraftment levels of total human CD45⁺ cells (left; mean percentages, from left to right, 99%, 98%, 99%), and fractions of CD34⁺GPR56⁺ (middle; mean percentages, from left to right, 15.80%, 38.5%, 34.9%), and CD34⁻GPR56⁻ (right; mean percentages, from left to right, 36%, 14%, 12%) cells of human CD45⁺ cells in primary recipient mice. Shown are individual mice and means. *P < .05; **P < .05, unpaired Student t test. (G) Representative FACS plots showing CD34 and GPR56 expression in sgGFP cells (left; HLF protein not lost), sgHLF cells with confirmed loss of HLF protein (middle), and those with sgHLF but no loss of HLF protein (see supplemental Figure 6 for complete data). Mice #247 and #198 were used for secondary transplantations. Values indicate percentages; #s indicate mouse IDs. (H) Proliferation curves for 5 sgGFP samples (blue) and 3 sgHLF samples with confirmed loss of HLF protein (red). Shown is fold-increase in absolute cell counts per well normalized to the start date of the culture. From each bone marrow sample, 6 replicate cultures were started. The average cell counts of the 6 cultures of each sample were used to compare the 5 sgGFP vs the 3 sgHLF samples (mean fold-change on day 3 was 2.3 vs 1 and on day 5 was 3.2 vs 0.8). Cells were counted by HTS-FACS. *P < .05; **P < .005, unpaired Student t test. (I) Overall human engraftment levels and CD34 and GPR56 surface expression in secondary recipients injected with 2 × 10⁶ sgGFP (#246) or sgHLF (#198) cells. Shown are means and individual values for week 4 and 7 bone marrow aspirates and final bone marrow analysis post mortem in week 8 for total human CD45⁺ levels (left; mean percentages, week 4: 4.8% vs 29%; week 7: 87% vs 98%; final: 96% vs 99%), CD34⁺GPR56⁺ fractions of human CD45⁺ cells (middle; mean percentages, week 4: 13% vs 8%; week 7: 26% vs 0.7%; final: 37% vs 1.6%), and CD34⁻GPR56⁻ fractions of human CD45⁺ cells (right; mean percentages, week 4: 9% vs 18%; week 7: 6% vs 45%; final: 5.5% vs 34%). *P < .05; **P < .005; ***P < .0005, unpaired Student t test. The third sgGFP mouse died before final bone marrow analysis, so data for this mouse are only available from week 4 and 7. (J) Representative FACS profiles showing CD34 and GPR56 expression of human sgGFP and sgHLF cells engrafted in secondary recipients 8 weeks after injection of 2 × 10⁶ cells. Numbers in quadrants indicate percentages. (K) Proliferation curves of sgHLF and sgGFP (control) cells in vitro after harvest from secondary recipients 8 weeks after injection of 2 × 10⁶ (2M) cells. Six cultures were started in 96-well plate formats from each mouse. Average cell counts of the 6 cultures per sample were used to compare the groups and were normalized to the start date of the culture. Cells were counted by HTS-FACS. *P < .05; **P < .005, unpaired Student t test. +, positive; AM, ametrine; BM, bone marrow; i.v., intravenous; K, thousand; M, million; n.s., not significant.