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A. Beke et al.
those from control iPSC-derived CFU-M (Figure 3C). A similar defect in the generation of CD14+ cells expressing both CD16 and CD163 was detected when iPSC-derived hematopoietic cells were induced to differentiate in liquid culture (Figure 3C).
Since the patient demonstrated megakaryocytic hyper- plasia and dysplasia, we performed coagulum assays in the presence of stem cell factor and thrombopoietin to analyze the generation of megakaryocytes and platelets (Figure 3D). All iPSC generated a similar number of colonies (Figure 3E) but those generated by CMML iPSC were much larger (Figure 3F-H). We observed a decrease in the fraction of CD42+ cells among CD41+ cells (Figure 3I) and the fraction of CMML iPSC-derived megakaryocytes that produced platelets was decreased (Figure 3J, K).
CD34+CD43+ cells generated from iPSC were also cul- tured in liquid medium in the presence of stem cell factor, interleukin-3, interleukin-6, erythropoietin, granulocyte- macrophage colony-stimulating factor, thrombopoietin, Fms-like tyrosine kinase 3 ligand, granulocyte colony- stimulating factor and monocyte colony-stimulating factor for 10 days (Figure 4A). The quantity of cells generated by each clone and the number of viable cells after 10 days were not significantly different, except for clone A1 that demonstrated more dead cells (Figure 4B, C). Multiparameter flow cytometry analysis was used to measure each cell population obtained in culture (Online Supplementary Figure S2B). CMML iPSC generated a major- ity (~40%) of CD14+ cells (Figure 4D, E). Although they both had a KRAS(G12D) mutation, the A5 clone generated more CD14+ cells (~80%) than the A3 clone. In fact, monocyte production by the A3 clone was not significant- ly different from that of KRAS wildtype clones (Figure 4E, insert). Compared to control clones, KRAS(G12D) CMML iPSC generated fewer CD33+, CD14-, CD41-, CD235a- cells (Figure 4F) and KRAS wildtype CMML clones gener- ated fewer CD123+, CD14-, CD41-, CD235a- cells (Figure 4G). The generation of CD235a+ erythroid cells was more heterogeneous and higher in KRAS wildtype compared to KRAS(G12D) CMML clones (Figure 4H). The generation of CD41+ cells was not significantly different between control and patient-derived iPSC (Figure 4I). These liquid cultures also revealed the defective differentiation of monocytes into macrophages (Figure 3C) and the defec- tive generation of platelets (Figure 3D).
As expected, patient-derived clones showed a bias in their differentiation towards monocyte production. However, hematopoietic differentiation of CMML iPSC also demonstrated significant intraclonal heterogeneity that could not be explained by the sole genetic alterations detected in coding regions. The A1, A2 and A4 clones, which have the same mutations in coding regions, showed heterogeneous differentiation into CD235a+ and CD14+ cells whereas A3 and A5, which are KRAS-mutated clones, showed a marked difference in their monocytic differentiation in liquid culture. A summary of this clonal heterogeneity is shown in Figure 4J.
While the viability of cells generated by control iPSC was high in all but one experiment, the viability of CMML iPSC, especially KRAS wildtype CMML iPSC, was much more heterogeneous than that of control clones, suggesting a higher sensitivity to small variations in culture conditions (Figure 5A). Of note, the number of generated cells (indi- cated by the diameter of the circles in Figure 5A) could remain high in cultures in which the cell death rate was
elevated. In contrast, a decrease in cell viability was associ- ated with an increase in the fraction of CD235a+ cells and a decrease in the fraction of CD14+ cells generated by KRAS wildtype CMML iPSC (Figure 5A). By eliminating this culture condition-related variability in cell production, using a cut-off of 90% viability, we observed a much more robust trend in the differentiation of A1, A2 and A4 clones (Online Supplementary Figure S4). In contrast, even with this cut-off on viability, the A5 clone consistently produced more CD14+ cells than the A3 clone (Figure 5B).
A
B
Figure 5. Impact of viability on differentiation pattern analysis. (A) Percentages of DAPI-negative viable cells (x axis) and numbers of hematopoietic cells gener- ated (dots size) are plotted against the fraction of CD14+ and CD235a+ cells gen- erated in liquid culture. The vertical hatched line is an arbitrary cut-off value established at 90% viable cells; the horizontal hatched line emphasizes the dis- crepancies between samples with <90% viable cells and the others. (B) Fractions of CD33+CD14+ cells for the indicated clones after removing experi- ments in which cell viability was below 90%; Kruskal-Wallis test. *P<0.05.
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haematologica | 2020; 105(1)