Figure 4
Functional correction of the Z-AAT phenotype in in vitro hepatic differentiated gene-corrected iPSC.
(A) Taqman-based qRT-PCR analysis for hepatic markers of day 17 hepatic differentiated DG#22-ex2, DG#22-ex4, DG#22-ex7, hPi and hPi-DG#5 cell lines, relative to GAPDH. (B) Co-staining of hepatic differentiated corrected (DG#22-ex2, DG#22-ex4, DG#22-ex7) and uncorrected parental hPi cell lines for polymeric AAT using a polymer-specific antibody (2C1) and for total AAT. (C) Neutrophil elastase inhibition assay of 24 h supernatants of day 17 hepatic differentiated gene corrected DG#22-ex2, parental hPi and non-excised bi-allelic targeted hPi-DG#5 cell lines. Hepatocyte culture medium (HCM) and HCM with 60 uM (final) SPCK inhibitor served as negative and positive controls, respectively. Progression of fluorescence was measured after 5, 15 and 30 min with n = 3. (D) Native Western blot for detection of high molecular AAT protein in hepatic differentiated corrected cell line DG22-ex2 compared to Z-AAT expressing hPi parental cell line. HepG2 hepatocarcinoma cells and hepatic differentiated AAT-knockout cell lines hPi-DG#5 and hPi-DG#22 served as positive and negative controls, respectively. Data are represented as mean ± SD with n = 3 biological replicates.
