Fig. 1

Functional restoration of FVIII in human ECs and mice via targeted entire or partial B-domain deletion of the endogenous F8 gene. a Schematic representation of generation of B-domain targeted deletion and 8 N-linked glycosylation sites retained in B domain of the endogenous F8 gene. b PCR screening of BD-iPSCs using the primers BUF/BDR. Sizes of the PCR products: B-domain deletion, 341 bp; N-iPSCs, 3023 bp; HA-iPSCs, 3019 bp. c EPCs differentiation efficiency was determined by FACS analysis of CD31 and CD34 expression. d ELISA analysis of the FVIII antigen in iPSC-derived iECs. Data represent the mean ± SEM (n = 3 independent cultures). n.s., not significant compared with BD25-iECs. *p < 0.05, ***p < 0.001, vs. the BD25-iECs group. e Immunofluorescence staining of FVIII (red) and vWF (green) in iECs, DAPI was used for nuclear staining. FVIII-N, F8 protein N-terminus. FVIII-C, F8 protein C-terminus. f PCR screening of N8-iPSCs. The sizes of the PCR products using primers BUF/BDR for the N8-iPSCs was 1160 bp, N-iPSCs was 3023 bp, and HA-iPSCs was 3019 bp. g qRT-PCR analysis of F8 expression in iECs using primers targeting exons 23 and 26 (E23-26), with GAPDH used as a loading control. h Proportions of surviving mice after tail-clip challenge. HA mice, hemophilia A mice (n = 9); HA mice transplanted with HA-iEPCs (n = 9), BD21-iEPCs (n = 9), BD25-iEPCs (n = 9), N8-9-iEPCs (n = 9), N8-46-iEPCs (n = 9), N-iEPCs (n = 9), N-BD-iEPCs (n = 9), and N-N8-iEPCs (n = 9), respectively. n.s., not significant compared with HA mice. ***p < 0.001, vs. the HA-iEPCs group (log-rank test). i Average survival time of mice. Note that the mice that survived the challenge were excluded in this analysis. Data represent the mean ± SEM. n.s., not significant compared with HA mice. ***p < 0.001, **p < 0.01, vs. the HA-iEPC group. j Relative FVIII activity detected at 2-week post-transplantation in HA mice, HA mice without transplantation (n = 6); HA mice transplanted with DPBS (n = 6), HA-iEPCs (n = 6), BD21-iEPCs (n = 6), BD25-iEPCs (n = 6), N8-9-iEPCs (n = 6), N8-46-iEPCs (n = 6), N-iEPCs (n = 6), N-BD-iEPCs (n = 6), and N-N8-iEPCs (n = 6), respectively. Data represent the mean ± SEM. ***p < 0.001, vs. the HA-iEPC group. k Liver tissue sections of HA mice transplanted with BD-iEPCs were analyzed using immunofluorescence with anti-human vWF (green) and CD31 (red) antibodies. No signal was found in the DPBS group. DAPI was used for nuclear staining. l Tissue sections of other organs from mice transplanted with BD-iEPCs were analyzed by immunofluorescence. Cells positive for anti-human vWF staining were observed in the spleen and lung. CD31 (red), vWF (green), with DAPI used for nuclear staining. m No signal was found in the heart or kidney. CD31 (red), vWF (green), with DAPI used for nuclear staining