Essential e8 medium

Human induced pluripotent stem cells (hiPSCs) were generated and characterised as described previously [5 (link),66 (link)]. Next, adherent cultures of hiPSCs were adapted to feeder-free conditions on Matrigel-coated dishes in the presence of a chemically defined medium (E8 Essential Medium, Life Technologies, Bleiswijk, The Netherlands). Subsequently, differentiation of hiPSCs to CMs was performed in 20 days using Wnt signalling modulation by small molecule-based differentiation protocols in a serum-free, chemically defined medium [26 (link),67 (link)]. Finally, we performed a metabolic selection-based enrichment for hiPSC-CMs by applying glucose-depleted culture medium containing 4 mM lactate for six days, thereby removing a large proportion of non-cardiomyocytes [68 (link)]. During the whole process, no serum was applied in the culture medium.
Previous experiments using this differentiation protocol and hiPSC-CM line demonstrated that the vast majority of the cells had a ventricular-like AP morphology [26 (link)]. To increase the amount of hiPSC-CMs with an atrial-like AP morphology, 1 µM all-trans retinoic acid (Sigma-Aldrich, Zwijndrecht, The Netherlands) was added from day 4 to 7 of differentiation, as described recently for hESC-CMs [21 (link)] and hiPSC-CMs [22 (link)].

Human iPS cells were cultured in E8 essential medium (Life Technologies) and expanded according to its protocol. For EB generation, cells were harvested using EDTA and EBs were formed on a shaker as described before12 (link). EB medium consisted of IMDM supplemented with 10% of horse serum and 20% of fetal bovine serum plus penicillin/streptomycin. For myogenic induction, the EBs were induced by a Wnt agonist (3 μM of CHIR99021, Stem Cell Technologies) during the first 4 days and then expanded by basic FGF alone (10 ng/ml). EB day 12 sorted cells were also expanded in EB medium on Matrigel coated plates for a week and were differentiated into myotubes using myotube induction medium (2% horse serum in low glucose DMEM).

Stem cells were maintained in Essential E8 medium (ThermoFisher) on Thermo-Nunc plasticware coated with Geltrex (GIBCO) diluted 1:50 in DMEM/F12 without glutamine. They were passaged by manual dissociation using 0.02% EDTA (GIBCO). MSN differentiation was carried out as described (32) using Activin A to direct ganglionic/striatal fate. Media containing retrovirus encoding shRNA hairpins targeting FAN1 or empty vector was mixed one to one with normal iPSC media and supplemented with polybrene (8 μg/ml). This media was added to iPSC at ∼70% confluence and the cells were incubated for 16 h. Fresh media was added to the cells for a further 48 h prior to selection. For this, the media was supplemented with puromycin (1 μg/ml) and the cells were monitored ensuring regular media changes to minimize the number of dead cells in the culture. Colonies of transduced cells were detected after 2–3 weeks. Untreated cells were cultured alongside the selected cells and used as controls in subsequent experiments.

Stem cells were maintained in Essential E8 medium (ThermoFisher) on Thermo-Nunc plasticware coated with Geltrex (Gibco) diluted 1:50 in DMEM/F12 without glutamine. They were passaged by manual dissociation using 0.02% EDTA (Gibco). MSN differentiation was carried out as described (32 (link)) using Activin A to direct ganglionic/striatal fate. Media containing retrovirus encoding shRNA hairpins targeting FAN1 or empty vector was mixed one to one with normal iPSC media and supplemented with polybrene (8 μg/ml). This media was added to iPSC at ∼70% confluence and the cells were incubated for 16 h. Fresh media was added to the cells for a further 48 h prior to selection. For this, the media was supplemented with puromycin (1 μg/ml) and the cells were monitored ensuring regular media changes to minimize the number of dead cells in the culture. Colonies of transduced cells were detected after 2–3 weeks. Untreated cells were cultured alongside the selected cells and used as controls in subsequent experiments.

Cells (HT-1080, DSMZ-German Collection of Microorganisms and Cell Cultures, Brunswick, Germany, #ACC315; HEK293 c18, ATCC, Manassas, VA, USA, CRL-10852), which do not express endogenous DSC2 (46 (link), 47 (link)) were cultured in Dulbecco's Modified Eagle Medium (DMEM, 4.5 g/L glucose, Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin (Sigma-Aldrich, St. Louis, MO, USA) under standard conditions (37°C, 5% CO₂). The medium for HEK293 c18 was additionally supplemented with 0.25 mg/ml G418 (Thermo Fisher Scientific).
HiPSCs (NP0040-8, UKKi011-A, kindly provided by Dr. Tomo Saric, University of Cologne, Germany) were cultured in Essential E8 Medium (Thermo Fisher Scientific) and differentiated into cardiomyocytes as previously described (25 (link), 48 (link)).
For confocal microscopy the human fibrosarcoma cell line HT-1080 was cultured in 8-well µ-slide cover slides (Ibidi, Gräfelfing, Germany). HiPSC-derived cardiomyocytes were cultured in Matrigel-coated 8-well µ-slide cover slides (Ibidi).
For the secretion assay HEK293 c18 cells containing EBNA1 were cultured in 6-well plates to obtain high and efficient expression of recombinant cadherins (49 (link)).