iPSC Generation, Culture, and Characterization

Primary skin fibroblasts from ASLD-affected subjects were provided by Baylor Genetics Laboratories. Fibroblasts were cultured in alpha MEM (Hyclone) with 10% FBS (Hyclone), 2 mMol/mL L-Glutamine (Hyclone), 50 U/mL penicillin, and 50 μg/mL streptomycin (Hyclone). The HSCC-003iPS (Ctrl 1) cell line was generated using dermal fibroblasts from a 22-year-old healthy male donor (ZenBio, lot # DFM062509),¹² Ctrl 2 cell line from a 61-year-old healthy male donor, and the HSCC-022iPS (Ctrl 4) cell line from a 16-year-old healthy male donor (CRL-2529, ATCC).

iPSC lines were generated from control and ASLD fibroblasts using two methods: by transduction with gamma-retroviruses encoding OCT4, SOX2, KLF4, and cMYC and by transduction with Sendai virus encoding OCT4, SOX2, KLF4, and cMYC (Table S2). Gamma-retrovirus reprogramming experiments were performed as previously described.¹³ Sendai virus reprogramming experiments were conducted using CytoTune-iPS 2.0 Sendai Reprogramming Kit (ThermoFisher Scientific) per manufacturer’s recommendations. Established iPSC lines were cultured and expanded in feeder-free TeSR-E8 medium (STEMCELL Technologies) on hESC-qualified Matrigel (Corning, 354277).

Chromosome G-banding analysis for karyotyping was performed by the T. C. Hsu Molecular Cytogenetics Facility, University of Texas MD Anderson (Houston, TX). STEMdiff Trilineage Differentiation Kit (STEMCELL Technologies) was used to differentiate established iPSC lines into three germ layers for assessment of pluripotency. Cells were harvested for gene expression analysis on day 5 for endoderm and mesoderm lineages and day 7 for the ectoderm lineage.