242 Chapter 9 for an additional 3 days. The cells were gradually transitioned into E8 stem cell medium in a step-wise manner over 5-6 days by replacing 20% of the media each day. Stem cell-like colonies were manually picked about two weeks post transduction and expanded in E8 stem cell media (Life Technologies) on plates coated with human ESC-qualified Matrigel (BD Biosciences) under hypoxic conditions (5% O2, 5% CO2) at 37ºC. Cells were dissociated with Gentle Cell Dissociation Reagent (StemCell Technologies) with E8 medium supplemented with 2.5 μM Y-27632 (SelleckChem). Immunofluorescence analysis of pluripotency markers. The hiPSCs were seeded in Matrigel-coated well in a 96-plate (Greiner Bio-One) and cultured for 4-5 days. The cells were washed with PBS, fixed with 4% paraformaldehyde. After permeabilizing with 0.3% Triton-X diluted in PBS supplemented with 2% BSA, 2% FBS, the cells were incubated with primary antibodies (Human Pluripotent Stem Cell Immunocytochemistry Kit, R&D System; Tra-1, Millipore) overnight at 4 ºC. After washing, cells were incubated with secondary antibodies Alex Fluor 488/555 / Hoechst 33342 (Invitrogen). Images were taken on an IC200 Kinetic Imaging Cytometer (Vala Sciences; 20x 0.75 N.A.) and further processed using ImageJ software (National Institutes of Health). Karyotyping. The hiPSCs were snap-frozen in liquid nitrogen and genomic DNA (gDNA) was extracted using the Blood and Tissue DNA extraction kit (Qiagen). SNP karyotyping analysis of >713,014 SNPs was performed using Genome-Wide HumanOmniExpress-24 BeadChips v1.1 on a HiScan sequencing platform per the manufacturer’s directions (Illumina) and analyzed using the KaryoStudio v1.4 software (Illumina). Cardiomyocyte differentiation. Differentiation towards cardiomyocytes was carried out following a small molecule Wnt-activation/inhibition protocol previously described37,38. Briefly, hiPSCs were first treated with CHIR99021 (4-6 μM; Tocris) in RPMI/B27 without insulin (Life Technologies) for 72 hours, and then with IWR (3 μM; Selleck Chemicals) for another 48 hours. The media was then replaced with RPMI/B27 with insulin (Life Technologies) and refreshed every 2 days. Spontaneously beating cells was typically observed 8-10 days postdifferentiation. On day 13 post-differentiation, hiPSC-CMs were metabolically selected in RPMI-B27 without D-glucose (Life Technologies) supplemented with 0.2% Sodium DL-lactate (Sigma) for 96 hours. Differentiated hiPSC-CMs were maintained with RPMI/B27 medium. All experiments were conducted by using hiPSC-CMs between 35 and 50 days after culture in maturation media39 (2D baseline contractility assessment, siRNA UPR experiment, XBP-1 splicing reporter) or RPMI/B27 (scRNA-seq, BiX experiments).
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