Dmem with high glucose

We obtained human oesophageal cancer cells (ECA109, TE2, TE3, TE7 and TE8) from the American Type Culture Collection. TE2 and TE7 were cultured in DMEM with high glucose (Invitrogen, CA, USA) containing 10% foetal bovine serum, and these cells were maintained at 37 °C with 5% CO₂.

Primary neonatal rat ventricular myocytes (NRVM) were isolated using the Worthington isolation kit [32 (link)]. Briefly, 2–3 days old pups were sacrificed by decapitation and after opening the thorax, hearts were quickly removed. Cardiomyocytes were isolated by digestion with 0.5 mg/ml Type II collagenase and 0.5% Trypsin. After dissociation, the cells were pre-plated for 45–60 min to allow fibroblasts to attach and then plated on 100-mm cell culture dishes at a density of 3 × 10⁶ cells or onto 6-well dishes at 1 × 10⁶ in DMEM with high glucose (Invitrogen) supplemented with 10% fetal calf serum and antibiotics (100 U/ml penicillin, 10 mg/ml streptomycin solution). Cells were maintained in a humidified CO₂ (5%) chamber at 37 °C for all experiments.

Mouse embryonic fibroblasts and Venus porcine neonatal fibroblasts were cultured in complete DMEM AQ (high‐glucose Dulbecco's modified Eagle's medium [D0819; Sigma–Aldrich, St. Louis, MO] supplemented with 1% penicillin/streptomycin [Pen/Strep] [Sigma–Aldrich] and 10% fetal bovine serum [FBS] [Hyclone]). HEK‐293T cells were cultured in DMEM with high glucose (1965; Invitrogen, Waltham, MA), supplemented with 1× Glutamax (Invitrogen), 1× Pen/Strep, and 10% FBS (In vitro A/S, Denmark). Derived Venus piPSCs were cultured in piPSC medium (DMEM/F12 medium [Sigma–Aldrich] supplemented with 20% Knockout Serum Replacement [Invitrogen]), 1× Pen/Strep [Sigma–Aldrich], 1× non‐essential amino acids [Sigma–Aldrich], and 100 μM β‐mercaptoethanol [Life technologies, Waltham, MA]) supplemented with 10 ng/mL LIF (Millipore, Billerica, MA) or 20 ng/mL human recombinant basic FGF (Prospec, East Brunswick, NJ), 2i (1 μM PD0325901 [Sigma–Aldrich], 3 μM CHIR99021 [Sigma–Aldrich]), and 2 μg/mL doxycycline (Sigma–Aldrich).

EC were seeded at 40,000 cells/cm² in plates coated with 50 ng/mL (mESC) or 10 ng/mL fibronectin (hESC) for the mESC-A3 and hESC-H7, respectively. Once confluent (about 3 days) the cells were incubated with Alexa Fluor 488 acetylated low density lipoprotein (LDL; Invitrogen) diluted 1:100 in DMEM with high glucose (Invitrogen) for 5 hours. The wells were then counterstained with DAPI and fixed with 4% paraformaldehyde.

The brain tissue isolated from a fetus of 15 weeks gestation, was dissociated into single cells by incubation with 0.25% trypsin in phosphate-buffered saline (PBS) for 30 min, as described previously [4] (link), [5] (link). Dissociated cells were suspended in the culture medium, composed of the Dulbecco's modified Eagle medium (DMEM) with high glucose (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS) (Invitrogen) and 20 µg/mL gentamicin (Sigma, St Louis, MO). Dissociated cells were plated at 5×10⁷ cells/T75 flask and were grown in an incubator with 5% CO₂ atmosphere. In primary brain cell cultures, all microglia freely floating in the medium were removed. After replating the cultures for three to five times by treatment with trypsin, most of neurons and oligodendrocytes underwent cell death and detached off, while astrocytes were firmly attached onto the flask surface, resulting in enrichment of highly purified astrocytes.