Cardiac Differentiation of Murine Embryonic Stem Cells

According to standard protocols W4 murine embryonic stem cells (mESCs), originally isolated from the 129S6 mouse strain, were grown as described previously [9 (link)]. In brief, cells were cultured in DMEM supplemented with 15% FBS Superior (Biochrom AG, Berlin, Germany), 1% Cell Shield® (Minerva Biolabs GmbH, Berlin, Germany), 100 µM non-essential amino acids, 1000 U/mL leukemia inhibitory factor (Phoenix Europe GmbH, Mannheim, Germany) and 100 µM β-mercaptoethanol (Sigma-Aldrich GmbH, Steinheim, Germany) at 37 °C, 5% CO₂, and 20% O₂. We performed cardiovascular differentiation using cardiogenic differentiation medium, containing IBM (Iscove’s Basal Medium, Biochrom AG, Berlin, Germany) supplemented with 10% FBS Superior, 1% Cell Shield®, 100 µM non-essential amino acids, 450 µm 1-thioglycerol (Sigma-Aldrich GmbH, Steinheim, Germany), and 213 µg/mL ascorbic acid (Sigma-Aldrich GmbH, Steinheim, Germany). Cardiovascular differentiation was initiated by hanging-drop culture for two days at 37 °C, 5% CO₂, and 20% O₂. To start formation of embryoid bodies (EB) 400 cells per drop were plated on the cover of a square petri dish and grown for 2 days. Subsequently, EB grew for four more days in suspension culture and were then harvested for transplantation. These cells will be referred to as cardiac induced cells (CiC) [8 (link)].