Tryple select 10x

For cell harvest, cells were washed two times with PBS and incubated with TrypLE™ Select 10X (Life Technologies) in PBS at a 1:5 ratio (v/v) for 10 minutes at 37°C. Following cell detachment, cells were cross-linked by addition of formaldehyde at a final concentration of 1.6% for 10 minutes at room temperature. Cross-linked cells were then centrifuged at 600 × g for 5 minutes at 4°C. After aspirating the supernatant, the cell pellet was re-suspended in -20°C methanol to a suspension of 1 million cells/ml and transferred to −80°C for long-term storage.

Py2T cells were obtained from the laboratory of Gerhard Cristofori, University of Basel, Switzerland [13 (link)]. Cells were tested for mycoplasma contamination upon arrival and regularly during culturing and before being used for experiments. Cells were cultured at 37°C in DMEM (D5671, Sigma Aldrich), supplemented with 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin, at 5% CO_2. For cell passaging, cells were incubated with TrypLE™ Select 10X (Life Technologies) in PBS in a 1:5 ratio (v/v) for 10 minutes at 37°C. For each experiment, cells were seeded at the density of 0.3 million cells per plate (100 mm diameter) and allowed to recover for 36 hours. After reaching 60% confluence, cells were either mock treated or treated with 4ng/ml TGFß (Human recombinant TGFß1, Cell Signaling Technologies) for 2, 3 and 4 days. Cell growth media and 4ng/ml TGFß treatment was renewed every day.

For the generation of iPSC-CFs a protocol developed by Zhang et al. was used^{13 (link)}. Briefly, human iPSCs were dissociated with 1 mL/well 0.5 µM EDTA solution (Invitrogen) at RT for 5 min and seeded on Vitronectin XF (StemCell Technologies) coated 6-well plates at a density of 15.000–30.000 cells/cm² in TeSR-E8 medium (StemCell Technologies) supplemented with 5 μM ROCK inhibitor (Y-27632) (Tocris) for 24 h. Cells were cultured for 6–7 days in TeSR-E8 medium with medium changes every other day until they reached 100% confluency and differentiation started (day 0). At day 0, the medium was changed to 2.5 mL/well RPMI + B27 without insulin (Gibco) and supplemented with 12 µM CHIR99021 (Tocris) for 24 h (day 1). After day 1, the medium was changed to 2.5 mL RPMI + B27 without insulin for 24 h (day 2). Afterwards, the medium was changed to 2.5 mL/well of the CFBM medium (Table S1) supplemented with 75 ng/mL bFGF (StemCell Technologies). Cells were refreshed with 2 mL/well CFBM supplemented with 75 ng/mL bFGF every other day until day 20 when RNA was collected, and cells were dissociated using TrypLE Select (10x) (Thermo Fisher) for 10 min at 37 °C. After dissociation, cells were cultured in DMEM + 10% Fetal bovine serum. For the first two passages, 5 μM ROCK inhibitor was added for 24 h to help cell attachment. Cells between passage 3–6 were used for experiments.

Human iPSC lines were acquired from publicly available cryopreserved stocks in the Stanford Cardiovascular Institute Biobank. Human iPSCs (2 male and 1 female lines) were expanded in monolayer in GIBCO Essential 8 medium (Thermo) on a Matrigel matrix (Corning). Human iPSC differentiation into CM was performed on three individual donor lines using an established small-molecule Wnt-activation/inhibition protocol yielding 95% pure TNNT2+ CM (Burridge et al., 2014 (link); Lee et al., 2019 (link); Kitani et al., 2019 (link)). Briefly, iPSC cultures at ~90% confluence in 6-well-plates were treated with 6 μM CHIR-99021 (SelleckChem) in RPM11640 medium supplemented with B27 supplements (Thermo Fisher Scientific) for 2 days to induce mesoderm specification, allowed to recover 1 day, then treated with 5 μM IWR-1-endo (SelleckChem) for 2 days for cardiac specification. On day 7, the culture medium was changed to RPMI-B27 + insulin, and the cells were glucose-starved on day 10 to day 14. Cells were harvested daily at day 0 to day 14 post-differentiation by dissociation using TrypLE select 10x (Thermo Fisher Scientific) and pelleted by centrifugation (200 × g, ambient temperature, 5 min).

The iPSCs were differentiated into iPSC-CMs using a small molecule-mediated protocol [16 (link)]. Briefly, iPSCs were cultured in RPMI 1640 medium supplemented with 1x B27 minus insulin supplement (Thermo Fisher Scientific) and the small molecule CHIR99021 (6 μM) for 48 hours. The media was then switched to RPMI 1640 medium supplemented with 1x B27 minus insulin supplement and the small molecule IWR-1 (3 μM) for another 48h. Twelve days after cardiac differentiation, iPSC-CMs were enriched with RPMI-1640 without glucose (Life Technologies) supplemented with 1x B27 minus insulin and 5 mM sodium DL-lactate (Sigma) for 96h. Beating iPSC-CMs were maintained in RPMI 1640 medium supplemented with 1x B27 supplement (Thermo Fisher Scientific). Dissociation of iPSC-CMs was performed using pre-warmed TrypLE select 10x (Thermo Fisher Scientific) at 37°C for 10min. After detaching, cells were collected by centrifugation (100g, 5 min), resuspended in RPMI 1640 with 1x B27 media, and plated in Matrigel-coated dishes. All experiments were performed with iPSC-CMs at forty-five to sixty days post differentiation.