Overexpression of full-length FVIII in HA-iECs by piggyBac vectors. (A) Genetic map of (1) piggyBac transposon vector with full-length F8 (FL-F8) transgene with intact B domain (marked yellow) and (2) super piggyBac transposase expression vector. Underneath, a diagram overview of the transfection strategy of HA-iPSCs followed by differentiation into HA-FLF8-iEC. (B) Confirmation of full-length transgene insertion into HA-FLF8-iECs (right) through PCR of cDNA showing a presence of 3 fragments (∼245-bp DNA fragment for P1-P2 primers, ∼290 bp for P3-P4, and ∼3 kbp for P1-P4) compared with no endogenous bands in unedited HA-iECs (left) and a singular short fragment (∼401-bp DNA fragment for P1-P4) in control ECs that were piggyBac transfected to overexpress B-domain–deleted F8 (Control-BDD-F8-ECs; middle). (C) Quantitative reverse transcription PCR analysis confirming F8 mRNA overexpression in HA-FLF8-iPSCs and HA-FLF8-iECs in 5 independent clones (F8-C1-5). F8 expression was normalized to 10³ glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Unedited HA-iPSCs and HA-iECs served as controls. Bars represent mean ± SD; ^##P < .01, ^###P < .001 between unedited HA-iPSCs and edited HA-FLF8-iPSC clones; **P < .01, ***P < .001 between unedited HA-iECs and edited HA-FLF8-iEC clones; n = 3. (D) Linear relationship (R² = .79) between piggyBac (PB) insertion number (x-axis) and expression of F8 transgene (y-axis) in the 5 HA-FLF8-iEC clones and an unedited HA-iECs control. (E) Immunofluorescent costaining of FVIII (green) and VWF (red) protein in both HA-iECs and edited HA-FLF8-iECs showing overexpression of FVIII protein. Cell nuclei were stained with DAPI. Scale bar, 100 μm.