B27 minus insulin

Human iPSCs were differentiated into cardiomyocytes using a new optimized protocol derived from previous publications [13 (link),14 (link),15 (link)] (Figure 1A). In short, a confluent monolayer of human iPSCs was differentiated by adding RPMI media (Thermofisher Scientific) supplemented with B27 minus insulin (Thermofisher Scientific), 50 μg/mL of ascorbic acid (Thermofisher Scientific), 20 ng/mL of BMP4 (R&D Systems, Minneapolis, MN, USA), 20 ng/mL of activinA (StemCell Technologies), and 1.5 μM of CHIR99021 (TOCRIS, Bristol, UK) for 3 days. Next, the medium was changed to an RPMI medium supplemented with B27 minus insulin, 50 μg/mL of ascorbic acid, and 5 μM of XAV939 (TOCRIS) for 3 more days; later, the cells were cultured with an RPMI medium supplemented with B27 minus insulin and 50 μg/mL of ascorbic acid for 2 days. From day 7, the first beating areas appeared and the medium was refreshed with an RPMI medium supplemented with B27 plus insulin (Thermofisher Scientific) and 50 μg/mL of ascorbic acid, with a media change every other day.

iPSCs were cultured in mTeSR1 media (STEMCELL Technologies, Vancouver, Canada; 85851) in 6-cm dishes precoated with Matrigel (Life Technologies, Carlsbad, CA; A1413302) and incubated at 37°C and 5% CO₂. At 85% confluence, iPSCs were disaggregated with ReLeSR (STEMCELL Technologies; 05872), passaged into 24-well plates, and allowed to grow for 4 to 5 days to create a monolayer. The differentiation strategy used has been reported previously.¹¹ For differentiation, the culture medium was changed to RPMI 1640 GlutaMAX plus 25 mM HEPES supplemented with B27-minus insulin (Life Technologies; A18956-01) containing CHIR99021 (Tocris Bioscience, Bristol, United Kingdom; 4423, 6 μM as working concentration) from days 0 to 2. On day 2, medium was changed to RPMI-B27-minus insulin containing IWP2 (Tocris Bioscience; 3533, 5μM as working concentration) and incubated until day 4. On day 4, the medium was changed back to normal RPMI GlutaMAX-B27-minus insulin and cells were maintained in this media until beating cardiomyocytes appeared, typically around day 6 or day 8. After beating was seen, iPSC-CMs were maintained in RPMI GlutaMAX medium with B27 serum-free supplement (Life Technologies; 17504-044).

H1, H9, and PGP1 hPSCs were cultured on growth factor-reduced Matrigel (Corning, 354230)-coated well plates with mTeSR1 media (STEMCELL Technologies, 85850). For CM differentiations, hPSCs were passaged with Accutase (Innovative Cell Technologies, AT104) and treated with small molecular inhibitors as previously described.³² (link) Briefly, hPSCs were grown in mTeSR1 media (STEMCELL Technologies, 85850) until 90% confluence. Cells were then treated with 12 μM CHIR99021 (Tocris Bioscience, 4423) in RPMI 1640 media (Gibco, 11875) containing B27 supplement (Thermo, 17504), defined as day 0 of the differentiation. On day 1, CHIR99021 was removed and cells were cultured in RPMI 1640 media containing B27 minus insulin supplement (Thermo, A18956). On day 3, combined media consisting of 1:1 ratio of used media and fresh RPMI 1640 containing B27 minus insulin media and 5 μM IWP2 (Tocris Bioscience, 3533) were added to cells. Media were changed to RPMI 1640 containing B27 minus insulin on day 5, followed by media changes with RPMI media containing B27 supplement on days 7, 9, and 11. Cardiomyocytes were used for experiments as described on day 12.