Inhibition of nSMase-2 improves long-term transplantation outcomes after ex vivo expansion. (A) Schematic of primary competitive transplantation experiment into lethally irradiated CD45.1 recipient mice (n = 8) using ex vivo cultured GW4869-treated cells (HSPC^GW) vs dimethyl sulfoxide (DMSO)–treated cells (HSPC^Con). (B) Relative in vivo chimerism over time after competitive transplantation of ex vivo cultured HSPC^GW vs HSPC^Con. (C) BM and spleen homing analysis of lethally irradiated recipients at 20 hours after HSPC injection. Flow cytometric–based Ki-67/PI cell-cycle analysis of murine HSPCs at (D) 24 hours or (E) 6 days after initiation of 24-hour GW4869 treatment. (F) Schematic of serial (secondary) competitive transplantation experiment into lethally irradiated recipient mice (n = 12). (G) Relative chimerism after serial whole–BM transplantation of primary recipients. (H) Relative proportions of mature lineage cells within each donor compartment in peripheral blood at 28 weeks after secondary transplantation. (I) Relative gene expression of Smpd3 in Cas9/RNP-mediated KO compared with control (Rosa26-KO) cells. (J) Representative methylcellulose colony–forming assays performed after Smpd3-KO in murine HSPCs and total colony quantification. (K) Relative chimerism measured over weeks after competitive transplantation of Smpd3-KO vs Rosa26-KO murine HSPCs (n = 6). Schematic of xenografted NBSGW mice with HLA-mismatched human HSPCs (L), and BM chimerism measured at 6 weeks after transplantation (n = 10) (M). ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; NS, nonsignificant. Refer to supplemental Figure 1.