Cord blood hematopoietic stem/progenitor cell expansion ex vivo by the BET inhibitor CPI203. (A) Flow plots of 5-day expanded UCB CD133⁺ cells in vehicle/cytokines (top) and CPI203/cytokines (bottom), showing the expression of Lineage (Lin), CD34, CD38, CD45RA, CD90, and CD49f markers. Sequential gating strategy from left to right. (B) Absolute numbers of phenotypically defined (p) Lin⁻CD34⁺CD38⁻CD45RA⁻CD90⁺CD49f⁺ HSCs increased significantly in CPI203/cytokine condition compared with the vehicle/cytokine control (**P ≤ .01; ***P ≤ .005; each well seeded with 5000 cells, n = 4). (C) Absolute number of LTC-IC per well in 5-day CPI203/cytokine vs vehicle/cytokine expansion cultures based on limiting dilution analysis estimation. (n = 4, **P ≤ .01; ***P ≤ .005). (D) Percentage of Annexin V⁺PI⁻ (apoptotic) and Annexin V⁺PI⁺ (dead) cells in cytokine containing medium supplemented with CPI203 or the vehicle control did not differ on day 5 of expansion (n = 3, NS P = .82. (E) Bone marrow cells harvested from mice injected with 500 unexpanded UCB CD133⁺ cells or progeny of 500 culture initiating UCB CD133⁺ cells show significantly more human cells in CPI203/cytokine-expanded conditions than unexpanded or vehicle/cytokines conditions (left, primary transplantation; right, secondary transplantation, all replicates shown in supplemental Figures 3-6 and supplemental Tables 2-5). (F) Dot plots show the percentage of hCD45⁺ human leukocytes in all bone marrow cells from recipient mice in different conditions for (top) primary transplants and (bottom) secondary transplants (n = 3-4; *P ≤ .05; **P ≤ .01, 1-way analysis of variance with multiple comparison [Fisher’s least significant difference]).