Hematopoietic differentiation and differential gene expression analysis of FPD and mutation-corrected iPSC lines. (A) Schematic of an improved hematopoietic differentiation method starting with embryoid body (EB) formation and ending with megakaryocyte (MK) formation. HPCs, hematopoietic progenitor cells. (B) Day 14 fluorescence-activated cell sorter analysis of differentiated cells demonstrating rescue of megakaryopoietic defects in the mutation-corrected iPSC lines (clones E and F) compared with the FPD patient iPSC line (III-5). The iPSC clone D was targeted in the wild-type RUNX1 allele, therefore the Y260X mutation was still present. (C-D) (Bar graphs) fold changes of the percentages of CD41⁺CD42⁺ MK cells in culture on day 14 for each targeted clone as compared with the patient iPSC line, III-5, in 2 independent experiments (panels C and D). (E) Gene set enrichment analysis plot distribution of the Reactome Platelet Activation Signaling and Aggregation gene set across the ranked microarray data for day 14 mutation-corrected clones E and F vs patient clone III-5. The normalized enrichment score = 1.75, the nominal P < .01, and the FDR q = .0115. A full list of genes within this gene set is presented in supplemental Table 2. (F) Selected physiological system development and functions that are significantly enriched in differentially expressed genes (DEGs) from mutation-corrected iPSC clones E and F on day 14, as identified by ingenuity pathway analysis (IPA). (Orange vertical line) cutoff threshold of the Benjamini-Hochberg multiple testing correction (B-H p-value) = .05. “Hematological System Development and Function” is the most significantly enriched pathway, “Hematological Disease” ranks sixth, and “Hematopoiesis” ranks 47th among 55 enriched pathways/categories. A full list of significantly enriched physiological system developments and functions can be found in supplemental Table 3. (G) Network representation of processes that are related to disease (green), or predicted to be either activated (orange), or inhibited (blue), because of differential expression of genes between mutation-corrected iPSC lines (clones E and F) and the FPD iPSC line III-5 from day 14. The network was constructed using IPA. A selected list of FPD-related categories and diseases are listed in supplemental Table 4. RUNX1 (highlighted in yellow), upregulated genes (shades of red), and most upregulated gene (dark red). (Arrows) indicate predicted activation (orange), inhibition (blue), or no prediction (gray).