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A. Beke et al.
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immunodeficient mice (Online Supplementary Figure S1C), without reprogramming-induced cytogenetic abnormali- ties (Online Supplementary Figure S1D). We also used an additional, independently generated control clone (Figure 1A).16 Whole exome sequencing and Sanger sequencing of patient-derived iPSC indicated that we had captured a part of the genetic heterogeneity of her leukemic clone, i.e., three clones (A1, A2, A4) recapitulated the founding clone while the other two (A3, A5) were reprogrammed from a KRAS(G12D) subclone (Figure 1A, B and Online Supplementary Figure S1E). In contrast with other studies,18 we did not reprogram any wildtype CD34+ cells, probably due to the early clonal dominance that characterizes CMML clonal architecture, with very few residual wild- type cells in the stem cell compartment.7
Hematopoietic cells derived from chronic myelomonocytic leukemia induced pluripotent stem cells recapitulate the disease features
iPSC were induced to differentiate into CD34+CD43+ hematopoietic progenitors (Online Supplementary Figure S2A), which were plated for 10 days in methylcellulose in the presence of stem cell factor, interleukin-3, erythropoi- etin, and granulocyte-macrophage colony-stimulating fac- tor (Figure 2A). The total numbers of colonies generated
Figure 1. Generation and genetic characteriza- tion of chronic myelomonocytic leukemia- and control-induced pluripotent stem cells. (A) CD34+ cells from a patient with chronic myelomonocytic leukemia and an age-matched healthy donor were reprogrammed through infection with Sendai virus encoding the tran- scription factors Sox2, Klf4, Oct4, and c-Myc (SKOM) before characterization and selection of indicated induced-pluripotent stem clones (iPSC). An additional control iPSC (Co6) was kindly provided by Dr. Weiss. (B) Whole exome sequencing of DNA collected from sorted periph- eral blood monocytes (in black) and from five iPSC selected from patient A (each color indi- cates a specific clone), showing the detection of two genotypes with nine and 12 somatic vari- ants, respectively.
by healthy donor- and CMML iPSC-derived hematopoiet- ic progenitors were similar (Figure 2B and Online Supplementary Figure S3A). The fraction of clusters (colonies <50 cells) generated by KRAS wildtype CMML iPSC was significantly higher than that generated by KRAS(G12D)-mutated CMML iPSC and control clones (Figure 2C and Online Supplementary Figure S3B, C). KRAS(G12D) clones produced larger granulocyte- macrophage (CFU-GM) and macrophage (CFU-M) colonies (Figure 2D) as well as a higher proportion of CFU-M colonies (Figure 2E). Compared to control clones, CMML-derived clones generated fewer granulocytic and multipotent progenitor colonies (Figure 2E, F and Online Supplementary Figure S3D) and more granulocyte- macrophage colonies (Figure 2E) whereas the proportions of erythroid colonies were not significantly different (Figure 2F and Online Supplementary Figure S3D). Colonies derived from CMML iPSC also demonstrated an increased fraction of CD14+ cells (Figure 2G) at the expense of CD33+, CD123+, CD235a+ or CD41+ populations (Online Supplementary Figure S3E, summary in Figure 2H).
Cells that formed CFU-M generated by CMML iPSC did not show the typical, fibroblast-like shape of macrophages generated by healthy donor-derived iPSC (Figure 3A, B) and expressed less CD16 and CD163 than
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