Psc cardiomyocyte differentiation kit

Human embryonic stem cells (RUES) were kindly provided by Dr. Elisa Di Pasquale. The stem cells (passage < 30) were seeded on Matrigel^®-coated well plates in Essential-8 medium (Gibco™, #A1517001) and, once reached 70–80% of confluency within 3 to 4 days, differentiated into cardiomyocytes. The differentiation was performed via the PSC Cardiomyocyte Differentiation Kit (Gibco™, #A2921201). Briefly, the protocol induced differentiation through sequential refreshment of the three different media in the kit every two days (medium A, B and M). Once in medium M, the cells were refreshed every 2 days. On the 26th day of culture, the cells were purified from all non-cardiomyocytes by MACS PSC-Derived Cardiomyocyte Isolation Kit (Miltenyi Biotec, #130-110-188) as the negative fraction to depletion antibodies. A resuspension of 50.000 cells in DMEM (Gibco™, 11960-044 supplemented with glutamine 1:1000) completed with 10% FBS (Microgem, S1860-500) and 1% P/S (Euroclone, ECB3001D) was seeded into round-bottomed ultra-low attachment 96-well plate. After 4 days, the cells started to form small clusters and within a week began to compact into cardioids (Hofbauer et al., 2021). The medium was partially refreshed every 3 days. The cardioids dimensions were measured via Fiji®.

The iPSC lines were maintained in a serum-free and feeder-free system, as previously described [27 (link),28 (link)].Cardiac differentiation was instigated using the PSC Cardiomyocyte Differentiation Kit (Gibco, Waltham, MA, USA) according to the manufacturer’s instructions. In brief, undifferentiated iPSCs were seeded on 12-well plates coated with GelTrex (Gibco, Waltham, MA, USA) and cultured in StemFlex (Gibco, Waltham, MA, USA) media to 90–100% confluence. Afterward, the cells were cultured subsequently in medium A (first 2 days), medium B (next 2 days), and cardiomyocyte maintenance medium (for approximately 7–14 days). Beating human iPSC-CMs were dissociated with collagenase B and subjected to immunostaining using antibodies specific for cardiac troponin T or sarcomeric α-actinin [28 (link)]. To evaluate specific functional properties of interest, iPSC-CMs were treated with different drugs or transfected with siRNAs specific to CAV3 (S2454, Ambion, Waltham, MA, USA) or MeCP2 (AM16708, Ambion, Waltham, MA, USA).

Pluripotent stem cells were generated by reprogramming skin fibroblasts (ID MRIi004-A) of a healthy human donor according to established methods 19 (link). The study participant provided written informed consent and the investigation conformed to the principles outlined in the Declaration of Helsinki. For maintenance culture, hiPSCs were expanded in matrix-coated dishes (Geltrex LDEV-Free, Gibco), using Essential 8 medium (Gibco), which was changed on a daily basis. Cells were split at 85% confluency, using EDTA for dissociation (Versene solution; Gibco). A state-of-the-art differentiation protocol for hiPSC-CMs using small molecules to induce differentiation was adapted from Chen and colleagues 20 (link). Briefly, cardiac differentiation was initiated at about 85% confluence by applying chemically defined factors for 24 h (medium A) and subsequent 48 h (medium B, PSC Cardiomyocyte Differentiation Kit; Gibco). After 72 hours, differentiation of hiPSC-CMs was continued for 12 days with Cardiomyocyte Maintenance Medium (PSC Cardiomyocyte Differentiation Kit; Gibco) that was changed every other day. The workflow is displayed in Figure 1A.

To induce differentiation of myocardial cells, hiPSCs were cultured in six-well plates in StemFit AK03N medium to confluence on a support using a PSC Cardiomyocyte Differentiation Kit, according to the manufacturer’s instructions (Thermo Fisher Scientific K.K.). HCMs (PromoCell, Heidelberg, Germany) were used as a control.