300 Chapter 12 METHODS hiPSC reprogramming and cell lines. Dermal fibroblasts were obtained from a skin biopsy of a 52-year-old PLN-R14del (D4iR14del) patient following informed consent (Stanford Institutional Review Board and Stem Cell Research Oversight Committee). Genome editing was performed in hiPSCs to correct the PLN-R14del mutation in hiPSCs by CRISPR-Cas9-mediated homology-directed repair (HDR), as described to generate the C31iCTR line.13 One previously established20 healthy individual hiPSC line was used for this study (SCVI-273 = 273iCTR, Sendai virus reprogrammed, PBMCs, 42-year-old female). Following informed consent (UNRAVEL, UMCU METC 12/387), peripheral blood mononuclear cells (PBMCs) were collected from one healthy proband relative of the D4iCTR (1CiCTR, 62-year old male), and 2 PLN-R14del patients (6BiR14del, 61-year old male, and 10BiR14del, 60-year old female). The blood was collected in heparin-coated tubes and diluted 1:1 with phosphate-buffered saline (PBS). Per 15 mL Ficoll (Thermo Scientific) 20 mL of blood-PBS was added and centrifuged for 30 minutes at 400×g. PBMCs were isolated by transferring the PBMC layer to a new tube, washed, and resuspended in StemPro-34 SFM media (Life Technologies) supplemented with cytokines stem cell factor (SCF, 100 ng/mL), Fms Related Receptor Tyrosine Kinase 3 (FLT-3, 100 ng/mL), and interleukin-6 (IL-6, 20 ng/mL). Cells were maintained for 3 days at 5% CO2, at 37°C, before use. PBMCs were reprogrammed to hiPSCs using the CytoTune-iPS 2.0 Sendai Reprogramming Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions with small modifications. Briefly, 2 x105 PBMCs were transduced with the three CytoTune 2.0 reprogramming vectors per well (24-well plate) in 0.5 mL in complete StemPro-34 SFM. Twenty-four hours post-transduction the PBMCs were pelleted by centrifugation, resuspended in fresh complete StemPro-34 SFM, and plated in one Matrigel-coated well of a 24-wells plate. Three days later, the medium was replaced with StemPro-34 SFM without cytokines and cultured for an additional 3 days. Next, cells were exposed to and maintained in the E8 stem cell medium. Approximately two weeks post-transduction, stem cell-like colonies were manually picked and expanded in E8 stem cell media (Life Technologies) on Matrigel (0.1 mg/mL, BD Biosciences)-coated plates under standard culture conditions (5% CO2, 37ºC). Cells were dissociated with 0.5 mM EDTA-PBS (Invitrogen) with E8 medium supplemented with 2.5 μM Y-27632 (SelleckChem). All hiPSC lines were routinely checked for mycoplasma and characterized as shown in Supplementary Figure 1. Differentiation of hiPSC to CMs. Cardiomyocyte (CM) differentiation was carried out following a small molecule Wnt-activation/inhibition protocol, as previously described.21 Briefly, hiPSCs were first treated with CHIR99021 (8 μM; Tocris) in RPMI+ 2% B27 without insulin (Life Technologies) for 72 hours, and then with Wnt C-59 (2 μM; Selleck Chemicals) for another 48 hours. Media was replaced with RPMI + 2% B27 with insulin (Life Technologies) and refreshed every 2 days. Spontaneously beating cells were typically observed 8-10 days post-differentiation induction. On day 9 of differentiation, hiPSC-CMs were metabolically selected in RPMI-B27 without D-glucose (Life Technologies) supplemented with 2% B27 for 48
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