Page 125 - 2020_01-Haematologica-web

Intraclonal heterogeneity in CMML
ABC
D
GH
E
F
Figure 2. Hematopoietic cells derived from chronic myelomonocytic leukemia induced pluripotent stem cells are biased toward the monocytic lineage. (A) CD34+CD43+ hematopoietic stem cells derived from control and chronic myelomonocytic leukemia (CMML) induced pluripotent stem cells (iPSC) were sorted and plated for 14 days in methylcellulose in the presence of 50 ng/mL stem cell factor (SCF), 10 ng/mL interleukin-3 (IL-3), 1 U/mL erythropoietin (EPO), and 10 ng/mL granulocyte-macrophage colony-stimulating factor (GM-CSF) before analyzing the generated colonies (CFU). (B) Total number of colonies generated by plating 5,000 cells in methylcellulose for 14 days. Co and A represent the five control and five patient’s clones respectively. (C) Fraction of clusters, as defined by colonies <50 cells, among total colonies, separating results obtained with KRASv-wildtype (A1, A2, A4) and KRAS-mutated (A3, A5) iPSC; Kruskal-Wallis test. (D) Representative colony-forming unit – granulocyte-macrophage (CFU-GM) and colony-forming unit – macrophage (CFU-M) generated by the indicated clones and visualized by light microscopy. Scale bars indicate magnification. (E) Fractions of CFU-M, CFU-GM and colony-forming unit – granulocyte (CFU-G) among colonies; Kruskal-Wallis test. (F) Fractions of erythroid colonies (CFU-E: colony-forming unit - erythroid; BFU-E: burst-forming unit - erythroid) and CFU-GEMM (colony-forming unit - granulocyte-ery- throid-monocyte-megakaryocyte) among colonies; Kruskal-Wallis test. (G) Flow cytometry analysis of CD14+ cells in colonies generated by the indicated clones in methylcellulose; Kruskal-Wallis test. (H) Summary of the colonies generated by hematopoietic progenitors derived from five healthy donor and five CMML patient’s iPSC. (B, C, E-G) Green dots, control iPSC; blue squares, KRAS wildtype CMML-iPSC; red squares, KRAS(G12D) CMML iPSC; bars, mean ± standard deviation. *P<0.05; **P<0.01; ***P<0,001 ****P<0.0001.
haematologica | 2020; 105(1)
115