CPI203 promotes megakaryocyte-accompanied ex vivo expansion of CB HSCs. (A) RNA sequencing was performed on UCB CD133⁺ cells that had been expanded for 5 days in culture with CPI203/cytokines (B) or vehicle/cytokines (V). Top differentially expressed genes associated with HSCs, megakaryocytes, myeloid cells, erythroid cells, and lymphoid cells are shown in a heatmap (false discovery rate < 0.01, gene expression level from low to high shown as blue to orange). (B) Flow plots showing the total CD41⁺ or CD41⁺CD42⁺ cells detected in all cells expanded in CPI203/cytokines condition compared with vehicle/cytokines on day 5. (C) Increased total CD41⁺ (top) and CD41⁺CD42⁺ (bottom) megakaryocytic cells expanded in CPI203/cytokine conditions compared with vehicle/cytokine for 5 days, followed by a further 5 days’ expansion in MK expansion medium (TPO, SCF) without CPI203 or vehicle (n = 3; *P ≤ .05). (D-E) Increased ploidy of megakaryocytes generated in MK expansion medium from d5-CPI203/cytokine-expanded cultures compared with d5-vehicle/cytokine-expanded cultures (n = 3; *P ≤ .05; ***P ≤ .005). Data shown are from day 10 of extended cultures: 5 days in CPI203/cytokines or vehicle/cytokines followed by 5 days in MK expansion medium (SCF, TPO) without CPI203 or vehicle.