LSK cell levels in the BM of FVIIIKO vs wild-type (WT) mice. (A) Percentage of LSK cells in FVIIIKO (white) compared with WT (C57BL/6) mice (black) (0.976% ± 0.007% vs 0.946% ± 0.019% (n = 10, P = .671). (B) Representative FACS histograms comparing isotype control staining to CD34 staining on C57BL and FVIIIKO LSK are shown. (C) In 4 independent experiments, a total of 8 C57BL/6 and 7 FVIIIKO samples were stained for CD34 and the data analyzed as follows: first, the average CD34 median of the C57BL samples in each experiment was calculated. This value was then used as a reference for comparison against individual median CD34 values in the same experiment (ie, individual median of each mouse was divided by the average median staining of the C57BL group in the same experiment). FVIIIKO mice (gray) exhibited a significantly higher value (1.43 ± 0.226, N = 7) compared with the median value of C57BL (black) (1.0 ± 0.067, N = 8), P < .001. Error bars are means ± SD. (D-G) FVIII KO HSCs exhibit similar short-term but reduced LTR capacity. (d) Schematic representation of the competitive repopulation assay. Donor-type chimerism was defined by FACS in each recipient, and the average level was calculated based on the values found in the 6 recipients of every individual donor. In control experiments, the BM mixture was composed of similar numbers of B6SJL (CD45.1) and C57BL (CD45.2) WT BM cells. (E) Chimerism levels at 6 and 22 weeks after transplantation of C57BL/6 BM (black) and FVIIIKO BM (white). (F) Chimerism levels at different time points after transplantation of FVIIIKO BM (dashed line) and C57BL BM (black). Each line is based on the average values found in 6 recipients receiving BM from the same donor. Total number of mice was 30. (G) Average linear trend line slopes of donor-type chimerism over time in mice receiving FVIIIKO BM (square) vs mice receiving C57BL/6 BM (diamond). Error bars are means ± SD (n = 30 mice).