American Society of Hematology

Figure 1.

$ASXL1-MT and HHEX enhanced proliferation and impaired differentiation of myeloid progenitor cells in vitro. (A-C) c-Kit+ BM cells derived from control or ASXL1-MT-KI mice were transduced with control/HHEX (coexpressing NGFR). NGFR-positive cells were sorted 48 hr after transduction. (A) A total of 104 cells were cultured in M3234 Methocult containing myeloid-enriched cytokines (n = 4). Shown are weekly colony counts per 104 replated cells (left), and the number of cells per well (right). (B, C) Cells were cultured in media containing myeloid-enriched cytokines. (B) Shown are proliferation of the cells from 2 independent experiments (left), and the relative proliferation rate at the end of culture (right; n = 4). (C) Mast cell maturation was assessed by the ratio of FceIRa and CD117 double-positive cells (enriched with mature mast cells) at the end of culture (n = 4). (D-J) Human CD34+ CB cells were transduced with vector/ASXL1-MT (coexpressing GFP) and vector/HHEX (coexpressing NGFR). (D, E) GFP/NGFR double-positive (DP) cells were sorted 48 hr after transduction. A total of 5000 GFP/NGFR DP cells were cultured in H4434 Methocult classic. Shown are the number of BFU-E, CFU-E, and CFU-GEMM at day 10 (D), and the colony-replating ability of CFU-GM per 5000 cells (E). (F-J) After retroviral transduction to CD34+ CB cells, cells were cultured in media containing myeloid-enriched cytokines. (F) Shown are the changes of GFP/NGFR DP cell frequency from duplicated wells. Two independent representative experiments (left) and the statistical analysis (right) are shown. (G, H) Apoptosis (G) and cell cycle (H) status in each GFP/NGFR double-positive fraction of the cells described in panel F at day 7 (n = 4 each). (I, J) Expression of the indicated surface markers (I; n = 4) and cytospin preparation (J; scale bars, 20 μm) at day 20. Statistical analyses were performed by 1-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test. Data are shown as mean ± standard error of the mean (SEM). *P < .05; **P < .01; ***P < .001; ****P < .0001.$

ASXL1-MT and HHEX enhanced proliferation and impaired differentiation of myeloid progenitor cells in vitro. (A-C) c-Kit⁺ BM cells derived from control or ASXL1-MT-KI mice were transduced with control/HHEX (coexpressing NGFR). NGFR-positive cells were sorted 48 hr after transduction. (A) A total of 10⁴ cells were cultured in M3234 Methocult containing myeloid-enriched cytokines (n = 4). Shown are weekly colony counts per 10⁴ replated cells (left), and the number of cells per well (right). (B, C) Cells were cultured in media containing myeloid-enriched cytokines. (B) Shown are proliferation of the cells from 2 independent experiments (left), and the relative proliferation rate at the end of culture (right; n = 4). (C) Mast cell maturation was assessed by the ratio of FceIRa and CD117 double-positive cells (enriched with mature mast cells) at the end of culture (n = 4). (D-J) Human CD34⁺ CB cells were transduced with vector/ASXL1-MT (coexpressing GFP) and vector/HHEX (coexpressing NGFR). (D, E) GFP/NGFR double-positive (DP) cells were sorted 48 hr after transduction. A total of 5000 GFP/NGFR DP cells were cultured in H4434 Methocult classic. Shown are the number of BFU-E, CFU-E, and CFU-GEMM at day 10 (D), and the colony-replating ability of CFU-GM per 5000 cells (E). (F-J) After retroviral transduction to CD34⁺ CB cells, cells were cultured in media containing myeloid-enriched cytokines. (F) Shown are the changes of GFP/NGFR DP cell frequency from duplicated wells. Two independent representative experiments (left) and the statistical analysis (right) are shown. (G, H) Apoptosis (G) and cell cycle (H) status in each GFP/NGFR double-positive fraction of the cells described in panel F at day 7 (n = 4 each). (I, J) Expression of the indicated surface markers (I; n = 4) and cytospin preparation (J; scale bars, 20 μm) at day 20. Statistical analyses were performed by 1-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test. Data are shown as mean ± standard error of the mean (SEM). *P < .05; **P < .01; ***P < .001; ****P < .0001.

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