Advanced rpmi 1x glutamax 1x b27 supplement

In all, 60,000–120,000 H9 hPSCs were plated per 1.9 cm² and sandwiched, sufficient to produce scattered, isolated spheroid colonies. Forty-eight hours after sandwiching, hPSC spheroids were treated with 12 μM CHIR (Stemgent) for 36 h, then changed to RB (Advanced RPMI+1X Glutamax+1X B27 Supplement, all from Thermo Fisher Scientific) and replaced every 3 days thereafter. Alternatively, spheroids were treated with 12 μM CHIR in RB minus insulin (RBNI) for 24 h, RBNI for 48 h, 5 μM inhibitor of WNT production-2 (IWP2, Tocris) for 48 h, RBNI for 48 h and RB every 3 days thereafter, as described for 2D cardiomyocyte differentiation29 (link).

Work with hPSC was performed under the approval and auspices of the University of Washington Embryonic Stem Cell Research Oversight Committee. Specific cell lines used in this study are described below and are sourced from commercially available hPSC obtained with informed consent. hPSC stocks were maintained in mTeSR1 media with daily media changes and weekly passaging using Accutase or ReLeSR (STEMCELL Technologies, Vancouver). 5,000–20,000 hPSCs per well were placed in each 24-well plate pre-coated with 300 µL of DMEM-F12 containing 0.2 mg/mL Matrigel and sandwiched the following day with 0.2 mg/mL Matrigel in mTeSR1 (STEMCELL Technologies, Vancouver) to produce scattered, isolated spheroid colonies. 48 hrs after sandwiching, hPSC spheroids were treated with 12μM CHIR99021 (Tocris Bioscience) for 36 h, then changed to RB (Advanced RPMI + 1X Glutamax + 1X B27 Supplement, all from Thermo Fisher Scientific) after 48 h, and replaced with fresh RB every 3 days thereafter.