Fig. 4

Gene expression and FVIII activity in hESCs and transgenic clones. a F8 gene expression was determined by RT-qPCR analysis and performed at 24 h for F8 mRNA expression in parental hESCs cells, transgenic clones and transiently substrate vector-transfected hESCs. F8 mRNA expression was normalized to the level of invariant control human beta-actin and represented as fold change compared to parental hESCs. ES, cDNA from parental hESCs; F1, F9, B6, B8, cDNA from transgenic clones; + indicates transiently transfected hESCs with 1 μg of substrate vector. b FVIII activity in hESCs and transgenic clones. 48 h culture supernatants of parental hESCs cells, clones and transiently transfected hESCs were subjected to FVIII fluorometric activity assay to measure the secreted FVIII. The FVIII fold activity was normalized to cell viability and represented as fold change compared to values obtained with parental hESCs. Cell viability was measured using the MTT assay. ES, parental hESCs; F1, F9, B6, B8, clones; + indicates transiently transfected hESCs with 100 ng of substrate vector. c–f Gene expression in retinoic acid differentiated hESCs and clones. The RT-qPCR analysis was performed for F8 and pluripotency markers Oct4, Nanog, Sox2 mRNA expression in differentiated parental hESCs cells and transgenic clones on day 14 of differentiation. Corresponding gene expression in differentiated hESCs/clones was compared to that in undifferentiated hESCs/clones. mRNA expression was normalized to the level of invariant control human beta-actin and represented as fold change compared to respective parental/differentiated hESCs. g FVIII activity in differentiated hESCs and transgenic clone F1. Culture supernatants of differentiated hESCs and clone F1 were subjected to FVIII fluorometric activity assay to measure the secreted FVIII. The FVIII fold activity is represented as fold change compared to differentiated parental hESCs. ES, parental hESCs; F1, F9, B6, B8, transgenic clones; D denotes retinoic acid differentiated hESCs/clones