Figure 3.

Differentiation of iPSCs toward the Hematopoietic Lineage

(A) Methylcellulose colony assays were performed at the end of the hematopoietic differentiation procedure with cKit⁺ cells purified from cultures initiated by WT, oc/oc, or oc/oc BAC-corrected iPSCs. Pictures on the left represent erythroid (BFU-E), myeloid (CFU-GM and CFU-M), and mixed (CFU-GEMM) colonies obtained from differentiated WT iPSCs; pictures on the right show CFU-GM colonies from one oc/oc and one corrected iPSC clone (scale bars, 400 μm). Colony assays were performed with all nine clones, for a total of 16–30 independent replicates/group (WT, oc/oc, and BAC-corrected). Once scored, some individual colonies were cytospun and stained with May-Grunwald and Giemsa. The insert shows a representative example of a cytospin preparation of a CFU-GEMM colony showing macrophages (M), monocytes (Mo), granulocytes (Gr), and erythrocytes (Er) (scale bar, 50 μm). In other experiments, FACS analysis was performed on pooled colonies to confirm the presence of TER119⁺ erythroid cells, CD11b⁺F4/80⁺ macrophages, TER119^–CD11b⁺Ly6G⁺ polymorphonucleated (PMN) granulocytes. On the PMN^– gate monoblasts, Pro-monocytes and monocytes were defined as CD31⁺Ly6C^–, CD31⁺Ly6C⁺, and CD31^–Ly6C⁺, respectively. Three clones for each group (WT, oc/oc and BAC-corrected) were used, and results are pooled (five to 15 independent replicates/group) and shown in the bar graph.

(B) Representative FACS analyses showing the expression of the CD45 pan-hematopoietic and the CD11b myeloid cell marker at day 12 of culture of WT, oc/oc, and corrected iPSC-derived EBs induced to differentiate toward the hematopoietic lineage. Numbers in the FACS plots indicate percentages among alive cells. Negative control is represented in the left contour plot.

(C) Upper panels: representative time course FACS analysis showing the expression of CD41 and CD45 hematopoietic markers at different time points of the differentiation protocol of one BAC-corrected iPSC clone. Upper contour plots show the control. Results from all cell lines are summarized and quantified in the bar graphs, using all nine clones. For each time point from three to 14 independent experiments were performed.

(D) The graph shows the percentage of CD11b⁺ cells within CD45⁺-gated cells in all iPSC clones. For each time point from three to 14 independent experiments were performed.

Error bars in all bar graphs indicate SEM. Significance of differences was determined by Mann-Whitney test. ^∗p < 0.03; ^∗∗p < 0.005.