Fig. 4: Functional recovery from FVIII deficiency in ECs differentiated from knock-in iPSC lines. | Experimental & Molecular Medicine

Fig. 4: Functional recovery from FVIII deficiency in ECs differentiated from knock-in iPSC lines.

From: Long-term correction of hemophilia A via integration of a functionally enhanced FVIII gene into the AAVS1 locus by nickase in patient-derived iPSCs

Fig. 4

a Results of qPCR analysis showing the expression levels of EC markers (CD31, VWF, and VE-cadherin) and FVIII in ECs differentiated from parental and genetically modified iPSC lines. GAPDH was used to normalize gene expression. The data are presented as the means ± SEMs of three independent experiments. ND indicates “not detected”. b Immunofluorescence staining revealed the expression of endothelial marker proteins (CD31 and VWF) in ECs differentiated from genetically modified iPSC lines. Nuclei were labeled with DAPI (scale bars, 100 μm). c FVIII activity was measured after a 30-fold concentration of supernatants harvested from ECs differentiated from parental or genetically modified iPSC lines. The data represent the activity detected per 1 × 105 ECs. The data are presented as the means ± SEMs of three independent experiments. * p < 0.05, ** p < 0.01 and *** p < 0.001 compared with ECs differentiated from parental iPSCs (one-way ANOVA with post hoc Dunnett’s test) and #p < 0.05 compared with WT-KI ECs (one-way ANOVA with post hoc Tukey’s test). d Activity in the decay assay was determined after incubating EC-derived FVIII for 0 h, 8 h, 16 h, or 24 h at 37 °C. The data are presented as the means ± SEMs of three independent experiments. *p < 0.05 compared with WT-KI ECs (two-way ANOVA with post hoc Tukey’s test). e FVIII decay rate and relative half-life of FVIII harvested from ECs. The data are the means ± SEMs of three independent experiments. SDs for rate decay values are estimated via least squares curve fitting and are within approximately 10% of the mean values. *p < 0.01 compared with WT-KI ECs (one-way ANOVA with post hoc Bonferroni correction). f The total antigens of FVIII produced by the patient, WT-KI or FE-KI iPSC-derived ECs in conditioned media and cell lysates was quantified. The data indicate the total number of antigens of FVIII detected per 1 × 105 ECs. The data are presented as the means ± SEMs of three independent experiments. ns indicates nonsignificance (one-way ANOVA with post hoc Tukey’s test).

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