Stemdiff ventricular cardiomyocyte differentiation kit

Commercially available human iPSCs (WTSIi081-A, EBiSC #66540196) were maintained in a humidified CO₂ incubator at 37 °C and 5% CO₂ and cultured in mTeSR™ Plus supplemented media (STEMCELL #100-0276) on Vitronectin XF™ (STEMCELL #07180) coated plates (0.5 µg/cm²). Cells were routinely passaged using Accutase® (Sigma-Aldrich #A6964) for detachment and subsequently seeded in an appropriate density. For differentiation into cardiomyocytes, the STEMdiff™ Ventricular Cardiomyocyte Differentiation Kit (STEMCELL #05010) was used following the manufacturer’s guidelines. The ethics board of the Goethe University, Faculty of Medicine, approved the use of the iPSC line for this study under the approval number 2023-1503-Anfrage.

Cardiomyocytes derived from human induced pluripotent stem cells (iPSCs, ATCC-ACS-1021) were cultured as instructed. For cardiomyocyte differentiation in vitro, a STEMdiff ventricular cardiomyocyte differentiation kit (STEMCELL Technologies, cat# 05010) was used according to the manufacturer’s instructions. Cardiomyocytes were cultured for 15 days in maintenance medium (STEMCELL Technologies, cat# 05020) to promote maturation. To induce hypertrophy, cardiomyocytes were treated with 10 nM of endothelin 1 (ET-1, Sigma-Aldrich, cat#E7764) for 24 h. Human pulmonary artery smooth muscle cells (PASMCs) were purchased (ATCC, cat# PCS-100-023) and cultured in vascular cell basal medium (ATCC, cat# PCS-100-030) supplemented with a vascular smooth muscle cell growth kit (ATCC, cat# PCS-100-042) in 5% CO₂ at 37^°C. Cells from passages 4–7 were used for experiments. Sotagliflozin (Selleckchem, cat# S8103) was used as described for each experiment.

The differentiation of hESCs toward beating cardiomyocyte was performed following STEMdiff™ Ventricular Cardiomyocyte Differentiation Kit (Stem Cell Technologies Catalog #05010) in accordance with the manufacturer instructions. In brief, iPSCs were detached using gentle cell dissociation reagent and seeded at 1.2 × 10⁶ cells/well on Matrigel-coated 12 well plates in presence of mTeSR™ Plus medium and 10 μM Y-27632. Subsequently, the differentiation was initiated by replacing culture medium with Cardiomyocyte Differentiation Medium A for 48 h. at 37 °C, 5% CO₂. Cardiomyocyte Differentiation Medium B was added for another 48 h. Then, Cardiomyocyte Differentiation Medium C was replaced on day 4 and 6. We perform a full-medium change with Cardiomyocyte Maintenance Medium every other day up to 20 days.