Figure 3

Characteristics of colony formation and genetic analysis of CMML iPSC-derived HPCs. (a) CMML iPSC-derived HPCs retained their serial re-plating capacity and generated colonies even after the fourth plating, whereas Normal iPSC-derived HPCs lost the ability to generate colonies after the second plating. CMML iPSC-derived HPCs showed an enhanced self-renewal capacity (n = 3, independent experiments, ^*p < 0.05, Normal-iPSCs1 and 2 from a healthy donor, and CMML-iPSCs1 and 2 from a CMML patient, Statistical analyses were performed with ANOVA and the Dunnett post-test for multiple comparisons. ^*p < 0.05). (b) The CMML iPSC-derived HPCs generated hematopoietic colonies under cytokine-deprived conditions. The CMML iPSC-derived HPCs could form hematopoietic colonies, while Normal iPSC-derived cells gave rise to few detectable colonies (n = 3, independent experiments, ^***p < 0.001, Normal-iPSCs1 and 2 from a healthy donor, and CMML-iPSCs1 and 2 from a CMML patient, paired two-sided t-test). (c) Using pStat5 intracellular flow cytometric analysis, we observed increased phosphorylation of STAT5 in CMML iPSC-derived CD34⁺ CD43⁺ HPCs. (g–i) The profiles of the surface antigens in the colonies from CMML iPSC-derived HPCs and controls were analyzed using FACS. (d) The percentage of pSTAT5 on CMML iPSC-derived CD34⁺ CD43⁺ HPCs was significantly higher than Normal- iPSC-derived HPCs (n = 3, independent experiments, ^***p < 0.001, Normal-iPSCs1 from a healthy donor, and CMML-iPSCs1 from a CMML patient, paired two-sided t-test). (e) Hierarchical clustering analysis of gene expression patterns. Using hierarchical clustering, these cells were distinguished into two groups: the Normal/CMML iPSC group and Normal/CMML iPSC-derived HPC group. (f-1 and f-2) Expression data were analyzed to identify genes in CMML-iPSC-derived HPCs and CMML-iPSCs that positively affected NF1 using GSEA.(p < 0.001). (g-1 and g-2) Expression data were analyzed to identify genes in CMML-iPSC-derived HPCs and CMML-iPSCs that positively affect EZH2 using GSEA. (p < 0.001). Horizontal lines represent the means ± s.d.