Highly GFP-positive colonies were picked for generation of clonal cell lines. A1AT, which is known to cause chronic liver damage in affected patients. This knockdown cassette is traceable from clonal iPSC lines to differentiated hepatic progeny via an enhanced green fluorescence protein reporter expressed from the same RNA-polymerase II promoter. Importantly, the cytomegalovirus ML-324 i/e enhancer chicken actin (CAG) promoter-driven expression of this construct is sustained without transgene silencing during hepatic differentiation in vitro and in vivo. At low lentiviral copy numbers per genome we confirmed a functional relevant reduction (?66%) of intracellular PiZ protein in hepatic cells after differentiation of patient-specific iPSCs. In conclusion, we have demonstrated that lentiviral vector-mediated expression of shRNAs can be efficiently used to knock down and functionally evaluate disease-related genes in patient-specific iPSCs. for 10 seconds) to separate clumps from single cells, and the pellet was resuspended in trypsin/EDTA to obtain single cells. Trypsin was inactivated with fetal calf serum (FCS)-containing medium, and the cells were washed with PBS and counted. Ten thousand cells ML-324 were seeded on one well of a six-well plate prepared with CF1 feeder cells, in 1 ml of hES medium containing 10 M ROCK inhibitor and 4 g/ml protamine sulfate. Then, 1 106 active particles of concentrated lentiviral CG-P or CG-s were added to the cell suspension, and the medium was changed every day with hES medium with ROCK inhibitor for 7 days until compact colonies started to form. Highly GFP-positive colonies were picked for generation of clonal cell lines. For bulk populations, cells were prepared in the same way as described for transduction, and 50,000 cells of the GFP+ fraction were sorted using the MoFlo cytometer and expanded in one well of a six-well plate in hES medium with ROCK inhibitor for the first 7 days. Hepatic Differentiation of Murine and Human iPSCs Transduced and untransduced murine iPSCs were differentiated using a protocol based on the previously described hanging drop method . For fluorescence-activated cell sorting (FACS) analysis, 1 106 active particles of lentiviral vector AN-dTom were added per well of a six-well plate at day 5 + 9 + 3. Cells were analyzed at day 5 + 9 + 23. Hepatic differentiation of human iPSCs was performed based on Proc a recently published protocol . Briefly, iPSCs were passaged as large clumps for attachment on Matrigel (BD Biosciences) and cultured in two-thirds MEF-conditioned and one-third fresh hES medium. When colonies reached approximately 80% confluence (day 1), medium was changed to hepatic differentiation basal medium (HDBM) (RPMI 1640 [PAA Laboratories] with 5% KOSR, 1% l-glutamine with Pen/Strep, 1% nonessential amino acids, 0.5 mg/ml bovine serum albumin, 10 nM Ly294002 [Calbiochem, San Diego, CA, http://www.emdbiosciences.com; Merck Millipore, Billerica, MA, http://www.millipore.com]) with 100 ng/ml activin A. On day 2 medium was exchanged with HDBM with 0.1% insulin-transferrin-selenium (ITS) (PAA Laboratories) and on day 3 with HDBM with 1% ITS. On days 4C6, medium was changed daily with hepatic cultivation medium (HCM) (Lonza, Walkersville, MD, http://www.lonza.com) supplemented with 50% epidermal growth factor (EGF) ML-324 from the HCM Bullet Kit plus 30 ng/ml fibroblast growth factor 4, 20 ng/ml bone morphogenetic protein 2, and 10 nM SB431542 (Sigma-Aldrich). Day 5 cells were transduced with 3 106 active particles of lentiviral AN-dTom per well of a six-well plate. On days 7C10, medium was exchanged daily with HCM containing 50% EGF, 20 ng/ml hepatocyte growth factor (HGF), and 10 nM SB431542. For maturation, cells were kept on HCM without EGF but with 20 ng/ml HGF, 10 ng/ml oncostatin M (OSM), and 10?7 M dexamethasone for 4 days and expanded in HCM without EGF and with 20 ng/ml HGF for 3 more days. Albumin-positive cells were selected by addition of 1 1.5 mg/ml G418 (Invitrogen) on days 10C12 and 1 mg/ml G418 on days 13C18. Samples were analyzed on day 18. All cytokines with exception of bFGF and LIF (provided by the Leibniz University Hannover, Hannover, Germany) were purchased from Peprotech (Hamburg, Germany, http://www.peprotech.com). Western Blot Western blot for lysates from differentiated human PiZZ iPSCs was performed as described previously . Goat anti-human A1AT antibody (MP Biomedicals, Inc., Irvine, CA, http://www.mpbio.com) was used as primary antibody at a dilution of 1 1:500. For detection of human A1AT in E15.5 fetal mouse livers, goat anti-human A1AT (A80-122A; Bethyl) was used as primary antibody at a dilution of 1 1:500. Isolation of Fetal Liver GFP+ CD45? Cells from Chimeras E13.5 fetal livers were isolated manually from the rest of the embryo under a stereomicroscope, and at least six fetal livers per cell.