Linoleic oleic acid

Purified iPSC-CMs were digested as described in “Digestion of iPSC-CMs” section and cultured in maturation medium for 3 to 7 days. The maturation medium contained glucose free DMEM (Thermo Fisher Scientific), 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) HEPES (Thermo Fisher Scientific), 2 mM l-carnitine inner salt (Sigma-Aldrich), 5 mM creatine (Sigma-Aldrich), 5 mM taurine (Sigma-Aldrich), 1 mM nonessential amino acid (Thermo Fisher Scientific), insulin-transferrin-selenium (Thermo Fisher Scientific), linoleic-oleic acid (Sigma-Aldrich), and 1% penicillin/streptomycin (Table 1). iPSC-CMs cultured in maturation medium for 3 to 7 days were used for analysis of bioenergetics and immunostaining, reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and Western blot, respectively, as described below.

TWNT-4, immortalized human stellate cells, kindly provided by Professor Scott Friedman, Icahn School of Medicine at Mount Sinai, NY, USA [29 (link)], and HepG2, human hepatoma cells, were cultured in filtered Dulbecco’s modified eagle’s medium (DMEM) with 2% or 10% fetal bovine serum (Pan-Biotech GmbH, Aidenbach, Germany) and 1% penicillin and streptomycin (Thermo Fisher Scientific, Waltham, MA, USA) at passages 3–8 in 37 °C and 5% CO ${}_2$ in a humidified incubator. For induction of a steatotic phenotype, the cells were treated with 10% linoleic–oleic acid (Sigma-Aldrich, St. Louis, MO, USA) for 24 h. Primary human stellate cells were obtained from Lonza (Basel, Switzerland) and cultured in human stellate cell growth medium (Lonza) according to the manufacturer’s instructions. Serum was taken from female and male patients diagnosed with MAFLD (n = 5, m:f 3:2, ages: 43–77 years, mean age 52.6 years) and controls (n = 5, m:f 3:2, ages 33–56 years, mean age 44.8 years) by venipuncture after informed consent was obtained. The study was approved by the University Hospital RWTH Aachen ethics board. Normal pooled plasma was obtained from healthy volunteers as described [30 (link)]. The antibodies used in this study are directed against human antigens and listed in Table 1.