Figure 1

Generation of CMML patient-derived iPSCs. (a) Protocol for the generation of CMML patient-derived iPSCs. CD34⁺ cells from patient samples were isolated from BM mononuclear cells. OCT3/4, SOX2, KLF4, L-MYC, LIN 28, and shP53 were transduced using episomal vectors under hypoxic conditions in the presence of a Rho kinase (ROCK) inhibitor and butyrate acid. Three clones of CMML iPSCs from one patient with CMML-1 were established. (b) Immunofluorescence staining of pluripotency marker antigens (SSEA-4 and Tra-1-60) in Normal and CMML iPSCs. (c) Semi-quantitative RT-PCR of pluripotency markers. The endogenous expression of pluripotent stem cell-specific genes (OCT3/4, SOX2, KLF4, C-MYC, NANOG, REX1, and TERT) was confirmed. Each of the images is cropped from different gels. (d) EZH2, RUNX1, and NRAS mutations were identified in CMML iPSCs. (e) Representative karyotypes of CMML iPSCs showing derivative chromosome (1;7)(q10;p10), an unbalanced translocation, and Normal-iPSCs. (f) Histological analyses of teratomas from CMML iPSCs. A teratoma with three germ layers, the ectoderm (neural tube), mesoderm (cartilage), and endoderm (intestinal tract), was observed following H&E staining. (g) Bisulfite sequence analysis of the NANOG gene promoter; the black circles represent methylated CpG, while the white circles represent unmethylated CpG. (h) CMML iPSCs grew rapidly and displayed a five-fold higher proliferation rate compared to control iPSCs (n = 3 independent experiments, ^***p < 0.001, CMML-iPSCs 2 clones and control iPSCs 2 clones derived from same donor samples, paired two-sided t-test,). (i–j) Cell cycle analysis showed a relative increase in CMML iPSCs in the G2/M phase. Statistical analysis was performed (n = 3 independent experiments, ^**p < 0.01, CMML-iPSCs 1 clones and control iPSCs 1 clones derived from same donor samples, Student’s t test). Horizontal lines represent the means ± s.d.