Figure 2.

Restoration of MPO activity in MPN-specific iPSCs

(A) Schematic representation of an “EB-based” differentiation protocol of iPSCs toward hematopoietic progenitors. Representative images of the differentiation procedure from EB formation to HSPC production are shown. Typical morphology of iPSC colonies on day 0, embryoid bodies on day 5, mesoderm commitment and differentiated hemogenic endothelial layers on day 8, and HSPC production on day 25. Scale bars, 1,000 μm (white) and 400 μm (black).

(B) Flow cytometry gating strategy to identify cyMPO⁺ neutrophils on day 15 of “EB-based” differentiation. Exemplarily shown for iPSC-derived hematopoietic cells carrying het (orange) or hom (red) CALR mutation or unmutated CALR (green). Numbers represent frequencies of CD15⁺cyMPO⁺ populations in percentage of living single cells.

(C) Flow cytometry data for cell surface CD15 and intracellular MPO expression of iPSC-derived HSPCs on day 15 of “EB-based” differentiation. Data are shown as mean values ± SD; ^∗p < 0.05, ^∗∗∗p < 0.001, n = 3 independent experiments.

(D) Relative MPO mRNA expression was confirmed by qRT-PCR. Gene expression is depicted as percentage of MT-ATP6. Mean value ± SD of representative clones carrying indicated CALR genotypes are shown; ^∗∗p < 0.01, n = 2 independent experiments.

(E) MPO functional activity was assessed by cytochemical staining. Representative images of hematopoietic cells harboring indicated CALR genotypes are shown. The intensity of the black-brown dye indicates the peroxidase activity. Scale bars, 50 μm.