After fixation and staining of cell nuclei, automated image analysis for determination of cell numbers/well was applied to detect the potential toxicity of individual compounds (Figure?4F)
After fixation and staining of cell nuclei, automated image analysis for determination of cell numbers/well was applied to detect the potential toxicity of individual compounds (Figure?4F). VX-809, and seamless gene correction of the p.Phe508del mutation resulted in full restoration of function. The identification of a series of validated primary hits that improve the function of p.Phe508del from a library of 42,500 chemical compounds demonstrates that the advantages of complex iPSC-derived culture GSK256066 2,2,2-trifluoroacetic acid systems for disease modeling can also be utilized for drug screening in a true HT format. potentiators, which restore the channel activity by enhancing gating, and correctors, which are able to rescue trafficking of specific mutants to the cell surface mutant (p.Phe508del). By applying immortalized cell lines, the potentiator VX-770 and the correctors VX-661 and VX-809 were identified. VX-770 was reported to increase chloride secretion about 10-fold in primary human bronchial epithelial (HBE) cells heterozygous for the gating mutation p.Gly551Asp (Van Goor et?al., 2009), whereby VX-809 was able to enhance chloride secretion in HBE cells from homozygous p.Phe508del patients to 14% of wild-type activity (Van Goor et?al., 2011). Results from clinical trials of VX-809 on homozygous patients, however, were modest at best (Clancy et?al., 2012). Even with the combination of the potentiator VX-770 and the corrector VX-809 for homozygous p.Phe508del, CF patients showed an improvement in lung function to a relatively low extent GSK256066 2,2,2-trifluoroacetic acid (Graeber et?al., 2018, Wainwright et?al., 2015). The new triple combination of modulators (VX-661, VX-659, VX-770) so far promises considerably more effect (Davies et?al., 2018) but needs further evaluation. It is therefore clear that previous models for correctors are poor predictors of clinical efficacy, although the most promising compounds were even validated on primary human epithelial cells. This underlines the GSK256066 2,2,2-trifluoroacetic acid need for the identification of novel compounds with a screening system that closely recapitulates the situation and the complexity of CF disease more accurately and reliable. With patient-derived iPSCs, a suitable source of expandable CF patient-derived cells is now available that can be genetically engineered to establish appropriate reporter cell lines and can be differentiated toward different function to a different extent, underlining that even complex functional organotypic screens based on disease-specific iPSC derivatives can FLJ44612 be conducted in a true HT format. Further comprehensive analyses are now required to investigate the degree of rescue in primary airway cells to identify binding sites and to elucidate mechanisms of action of the individual compounds. Results Requirement for an Isogeneic Control Cell Line with Seamless Correction of the p.Phe508del Mutation Isogenic iPSC control lines with seamless correction of the respective disease-specific mutation are generally considered as ultimate control in iPSC-based disease modeling. Gene editing of iPSCs and the subsequent clonal selection procedure, however, may not only lead to introduction of new mutations but also to the selection of cell clones with (epi)genetic aberrations that show altered culture characteristics and differentiation behavior. In order to confirm the similarity of the p.Phe508del line MHHi002-A and its seamless corrected counterpart GSK256066 2,2,2-trifluoroacetic acid MHHi002-A-1 (Merkert et?al., 2017) to be used in our screen, we have compared the global gene expression of both cell lines before and after intestinal differentiation (Figure?1). Principal component analysis (PCA) revealed close clustering of the p.Phe508del line (donor 2 derived) with its isogenic gene-corrected counterpart, but more divergence from two unrelated human iPSC lines (donor 1 and donor 6 derived), either in the undifferentiated state (Figure?1A) or after intestinal differentiation (Figure?1B). This indicates, that despite gene editing and the single-cell cloning process, the seamless corrected subclone is still much more similar to the parental cell collection than additional unrelated iPSC lines, all generated in the same laboratory. This was confirmed by a more detailed assessment of differentially.