Tra 1 60

Alkaline phosphatase activity staining was carried out using an alkaline phosphatase substrate kit from Vector (Newark, CA, USA). In brief, the cells were incubated with the substrate working solution for 30 min and washed with PBS three times. Immunocytochemistry was performed by fixing iPSCs with 4% PFA in PBS for 30 min. Following washing with PBS, the cells were permeabilized with 0.2% Triton X-100 in 0.05% Tween 20/PBS for 30 min. The samples were blocked by incubation in 0.05% Tween 20 in PBS containing 3% BSA and 0.3 M glycine for 1 h. Then, the samples were incubated with a primary antibody (1:100 dilution with blocking buffer) overnight at 4 °C. After incubation with the primary antibody, the samples were washed with PBS three times and incubated with a secondary antibody (1:1000 dilution with blocking buffer) for 1 h at room temperature. Finally, the samples were washed with PBS three times and incubated with Hoechst 33,342 for 10 min at room temperature. Afterward, the samples were washed with PBS three times and preserved with a mounting medium (Bio-Rad, Hercules, CA, USA). This experiment involved the use of primary antibodies against NANOG (Cell Signaling, Danvers, MA, USA) and TRA-1-60 (Millipore, Burlington, MA, USA). Alexa Fluor-conjugated secondary antibodies were acquired from Jackson ImmunoResearch (West Grove, PA, USA).

For 3D cultures, we used 3 hiPSC healthy lines: the SKiPSC-31.3 (Control-1), which was derived from human dermal fibroblasts from a healthy 45-year-old volunteer as previously published (42 (link)), and 2 lines (P11014 [Control-2] and P11007 [Control-3]), which were derived from healthy female and male donors, respectively (24 (link)). We also used a CRISPR/Cas9 generated BRAF T599R hiPSC line. hiPSCs cells were seeded on Matrigel and cultured in mTeSR1 medium (Stemcell Technologies). When hiPSCs reached a confluency of 70%–80%, cells were passaged in clumps by scraping with a pipette tip. A medium change was performed every 24 hours. Cultures were maintained at 37°C in a humidified incubator with 5% CO₂. The hiPSC line used in this study was assessed for pluripotency and routinely tested for mycoplasma.
Total RNA was extracted using NucleoSpin RNA Kit (Macherey-Nagel), and cDNA was synthesized by SuperScript IV VILO kit (Thermo Fisher Scientific). Pluripotency gene expression was assessed by quantitative PCR (qPCR) and normalized to RPL32.
For immunostaining, hiPSCs cells were fixed in 4% paraformaldehyde for 10 minutes at room temperature, permeabilized in blocking solution (PBS 2% BSA, 0.5% Triton) for 1 hour, and stained with primary antibodies: Nanog (4903, Cell Signaling Technology; 1:200) and Tra1.60 (MAB4360, MilliporeSigma; 1:100) overnight at 4°C. Then, cells were washed 3 times with PBS and incubated 1 hour with secondary antibodies and DAPI for nuclear staining, at room temperature. Images were acquired with an EVOS imaging Systems (Thermo Fisher Scientific).
Once confluent, the hiPSC cells were differentiated into CMs using a small molecule–modulated differentiation and glucose starvation (43 (link)). Briefly, mTeSR1 medium (Stemcell Technologies) was changed by RPMI supplemented with B27 without insulin (Thermo Fisher Scientific) and 6 μM CHIR-99021 (Abcam), and it was maintained in a 37°C and 5% CO₂ incubator for 48 hours. The medium was changed to RPMI-B27 without insulin for 24 hours and then to RPMI-B27 without insulin supplemented with 5 μM IWR-1 (MilliporeSigma) for 48 hours. On day 5, the medium was changed back to RPMI-B27 without insulin for 48 hours. From day 7 onward, cells were placed in RPMI-B27 with insulin and media change every 2 days. At day 11, the medium was changed to low-glucose medium for 3 days. CMs were then replated in RPMI-B27 with insulin. At day 15, medium was changed for a second glucose deprivation for 3 more days. Starting from day 18, medium was changed every 2 days with RPMI-B27 with insulin.
To assess the differentiation efficiency, at day 21, cells were strained with an APC anti–cardiac TroponinT (TNNT2) antibody (130-106-689, Miltenyi Biotec; 1:100) or APC isotype control (130-104-615, Miltenyi Biotec; 1:100) and analyzed by flow cytometry.

The primary antibodies used to confirm pluripotency of CERA007c6 iPSCs were OCT4 (5 μg/mL, Santa Cruz Biotechnology, TX, USA) and TRA1-60 (5 μg/mL, Millipore), followed by the appropriate Alexa Fluor-488 secondary antibodies. For in vitro differentiation, CERA007c6 iPSCs were allowed to form EBs for 12 days and subsequently plated down on gelatinized plates to further differentiate for 15 days. Immunocytochemistry is performed using the primary antibodies against alpha-fetoprotein (10 μg/mL AFP, Millipore), alpha smooth muscle actin (10 μg/mL, SMA, R&D Systems, MN, USA), and Nestin (10 μg/mL, Abcam), followed by the Alexa Fluor-488 secondary antibody (10 μg/mL; Invitrogen).
For quantitative assessment of cardiomyocyte differentiation, beating colonies at 14 days after plating were trypsinized into single cell suspension with 0.25% trypsin-EDTA and spun onto coated glass slides (4 min at 900 rpm; Shandon Cytospin 4, Thermo Fisher Scientific, MA, USA). Cells were fixed and permeabilized with 100% ice-cold methanol for 10 min at 4°C. Cells were then incubated with a serum-free blocking solution (Thermo Fisher Scientific) for 10 min prior to incubation with cardiac troponin T antibody (2 μg/mL, mouse monoclonal IgG; Abcam, MA, USA) at 4°C overnight followed by a secondary antibody, Alexa Fluor-488 goat anti-mouse IgG for 60 min at room temperature.
For qualitative analysis, beating colonies at 14-15 days after plating were treated with 10 μM of Y-27632 (Millipore, MA, USA), a Rho-associated kinase (ROCK) inhibitor, for 1-2 hours at 37°C to minimise apoptosis [15 (link)]. Cells were then washed with phosphate buffer saline (PBS) and dissociated with TrypLE Select (Invitrogen) at 37°C for 5 min. Serum-containing media were then added to neutralize the enzymatic reaction of TrypLE. Mixture of single cells and clumps was centrifuged at 400 g for 5 min, resuspended in serum-containing media supplemented with 10 μM of Y-27632, and seeded onto gelatin and fibronectin coated 8-well chamber slides. After 48 h of incubation at 37°C, the media were removed and the cells were fixed with 4% paraformaldehyde for 30 min at room temperature followed by Triton X-100 (0.1% in PBS) for 10 min. Cells were then incubated with a serum-free blocking solution for 10 min prior to incubation with antibodies against cardiac troponin T (4 μg/mL, mouse monoclonal IgG, Abcam), sarcomeric α-actinin (25 μg/mL, mouse monoclonal IgG, Sigma-Aldrich), myosin heavy chain (2 μg/mL, mouse monoclonal IgG, Abcam), and connexin 43 (1.2 μg/mL, rabbit polyclonal IgG, Abcam) at 4°C overnight followed by Alexa Fluor-488 goat anti-mouse IgG (10 μg/mL, Invitrogen) or Cy3 fluorescence-conjugated goat anti-rabbit IgG (7.5 μg/mL, Jackson ImmunoResearch Laboratories, PA, USA) for 60 min at room temperature.
All preparations were counterstained with DAPI (1 μg/mL, Invitrogen) for nuclear staining and mounted with fluorescent mounting medium (Dako, Victoria, Australia). Images were taken using a fluorescence microscope (BX-61, Olympus, Tokyo, Japan). For quantification, at least 100 cells were counted from 10 random fields.