Fig. 4
ASPP 049-treated cells promote self-renewal and multi-lineage differentiation potential with unbiased lineage distribution. A The blood cell colony-forming potentials of nonadherent (5 × 104) cells from day 12 differentiation were harvested into single-cell suspension and cultured in Methocult™ medium for 2 weeks, evaluating the multi-lineage differentiation potentials. Representative morphologies of blood cell colony: CFU-GM (left), BFU-E (middle), CFU-GEMM (right). Scale bars, 50 μm. B May Grunwald-Giemsa staining of CFU-GM, BFU-E, and CFU-GEMM from harvested colonies. Green arrowhead: granulocytic neutrophils, black arrowhead: macrophages, red arrowhead: orthochromatic erythrocytes. Scale bars, 100 μm. C Statistical analysis of colony formation was classified and scored under a bright-field microscope according to color, colony size, and cellular compositions: CFU-GM (grey), BFU-E (red), CFU-GEMM (dark red), and total CFUs. D Blood lineage distribution represents the ratio of all CFUs between nonadherent cells harvested from ASPP 049 (1 μM)- and DMSO-supplemented conditions. Data are represented as mean ± SEM from three biological replicates. Remarks: * represents statistics of total CFUs, # represents statistics of CFU-GM, $ represents statistics of BFU-E, and % represents statistics of CFU-GEMM. **p < 0.01, ##p < 0.01, $p < 0.05, %p < 0.05 compared with DMSO vehicle control (paired student's t-test). E Limiting dilution analysis of long-term culture-initiating cells using different cell doses indicates the effect of ASPP 049 on maintaining a frequency of HSC-like cells after 5 weeks of LTC on AFT024 plus 2 weeks for MethoCult™ clonogenic ability, compared with positive control UCB-derived CD34+ cells. F At the highest doses of tested cells (hiPSC-derived CD34+ (1 × 104) cells and UCB-derived CD34+ (5 × 103) cells), frequency of blood colony formation was scored from positively responded LTC among hiPSC-derived HSPCs, hiPSC-derived HSPCs + ASPP 049, and UCB-derived HSPC. Each dot represents the number of clonogenic cells in a single LTC plus MethoCult™ wells with individual data from technical replicates: n = 9, n = 11, and n = 11, respectively. Remarks: * represents statistics compared with hiPSC-derived HSPCs, and # represents statistics compared with hiPSC-derived HSPCs + ASPP 049. *p < 0.01, ***p < 0.01, #p < 0.05 (one-way ANOVA with Dunnett’s multiple comparison)