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A. Beke et al.
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Figure 3. Defective maturation of chronic myelomonocytic leukemia induced pluripotent stem cell-derived hematopoietic cells into macrophages and platelet-forming megakaryocytes. (A) Representative morphology of colony-forming unit – macrophage (CFU-M) generated by hematopoietic cells derived from control (Co6) and chronic myelomonocytic leukemia (CMML) (clone A5) induced pluripotent stem cells (iPSC) cultured in methylcellulose as described in Figure 2A. Light microscopy visualization; scale bar indicates magnification. The rectangle is a zoom on the fibroblast-like shape of macrophages in the Co6 culture, which was not observed in A5 colonies. (B) Fraction of cells with a fibroblast-like shape in CFU-M generated from hematopoietic cells derived from four control and five CMML iPSC; unpaired t test. (C) Flow cytom- etry analysis of cells expressing both CD16 and CD163 in CD14+ cells collected from colonies generated by the five control iPSC- and the five CMML iPSC-derived hematopoietic cells plated in methylcellulose or in liquid medium, Kruskal-Wallis test. (D) Control iPSC and CMML iPSC-derived CD34+CD43+ hematopoietic cells were sorted and plated in coagulum for 10 days in the presence of 50 ng/mL stem cell factor (SCF) and 10 ng/mL thrombopoietin (TPO) to generate colony-forming unit - megakaryocyte (CFU-Mk). (E) Total number of colonies generated by plating 1,500 hematopoietic cells derived from the indicated iPSC. (F) Representative experiments showing the differential morphology of CFU-Mk generated by plating healthy donor (Co1 clone) or CMML (A2 clone) iPSC-derived hematopoietic cells (scale bar, 100 mm). (G) Fractions of large colonies, >50 cells, among the total number of colonies shown in panel E; Kruskal-Wallis test. (H) Fractions of intermediate colonies, 10-50 cells, among the total number of colonies shown in panel E; Kruskal-Wallis test. (I) Fractions of CD41+ megakaryocytes generated in liquid culture with all cytokines for 10 days and expressing the cell surface marker CD42, as detected by flow cytometry; Kruskal-Wallis test. (J) Upper panels show May-Grünwald-Giemsa-stained cytospins of CD41+ cells generated in liquid culture, 5 days after cell sorting, with a normal (Co3) or dysplastic (A3) morphology. Lower panels: representative images of platelet- producing megakaryocytes generated by hematopoietic cells derived from indicated clones; scale bars, 50 mm. (K) Fractions of platelet-producing megakaryocytes in CD41+ cells sorted from liquid culture of CD34+CD43+ cells with all cytokines for 10 days, then cultured for 6 days with SCF and TPO; Kruskal-Wallis test. Mk: megakary- ocytes. Colors as in Figure 1. Bars: mean ± standard deviation. *P<0.05; **P<0.01; ****P<0.0001.
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haematologica | 2020; 105(1)