Biolaminin 521 ln

For RAR pathway modulation, cells were treated for 24 hours either with 0.5 µM RA or with 5 µM BMS493 (a pan‐retinoic acid receptor inverse agonist) or with a combination of RA and BMS493, directly diluted in the culture media. For EBs formation assay, cells were dissociated into single cells using StemPro Accutase (Thermo Fisher Scientific) and cultured for 7 days on poly (2‐hydroxyethyl methacrylate) (Sigma‐Aldrich) – coated dishes in mTeSR1 medium supplemented with 10 µM of the Rho‐kinase inhibitor Y‐27632 (Selleckchem) for the first three days. At day 7, floating EBs were transferred on 5 µg/mL Biolaminin 521LN (Biolamina)‐coated plates and cultured in adhesion for additional thirteen days in medium consisting of DMEM/F12 containing 20% knockout serum replacement (KSR, Thermo Fisher Scientific), 1% Glutamax (Thermo Fisher Scientific), 1% non‐essential Amino Acids (Thermo Fisher Scientific), 100 µM 2‐mercaptoethanol and 0.5% penicillin and streptomycin.

SBAD2 cells, a human induced pluripotent stem cell line that was originally produced for the StemBANCC project [23 (link)], were received from Prof. Marcel Leist (University of Konstanz). The Leibniz-Institute DSMZ (German Collection of Microorganisms and Cell Cultures) validated the cell identity by short tandem repeat profiling.
For the UKN1 test system, cells were cultured in Essential 8^TM (E8) medium (Thermo Fisher Scientific Inc., Waltham, MA, USA) on Biolaminin 521 LN (BioLamina, Sweden) coated culture vessels and in the Cellartis^® DEF-CS^TM 500 Culture System (Takara Bio, Japan), according to the manufacturers’ guidelines. The cells were cultured following a three- or four-day protocol, i.e., the cells were seeded at a density of 20,000 cells/cm² or 12,000 cells/cm², respectively, and cultured (5% CO₂, 37 °C) for three or four days until confluency. The medium was changed daily. For dissociation of the cells during each passaging, the dissociation reagent TrypLE^TM Select (Thermo Fisher Scientific Inc., Waltham, MA, USA) was used. When cells were passaged in Essential 8^TM medium, 10 µM Rho-kinase inhibitor Y-27632 (Cell Guidance Systems, Cambridge, UK) was added to the medium for the first 24 h.