Reagents and chemicals

All reagents for the PluriLum differentiation protocol were identical to those described in Treschow et al. (2024) except for the stem cell growth medium that was changed from mTeSR™1 to an improved formulation, mTeSR™ Plus, obtained from STEMCELL Technologies Inc. (Vancouver, Canada). Corning® Matrigel® hESC-Qualified Matrix and Corning® ITS Premix Universal Culture Supplement were obtained from Corning Inc (NY, USA). TrypLE™, Penicillin–Streptomycin-Glutamine (PSG), KnockOut™ DMEM medium, human fibroblast growth factor-basic (FGF2), activin A, 60 mm cell culture dishes, and 96-well Polystyrene Conical Bottom MicroWell™ plates were supplied by Thermo Fisher Scientific Inc. (Massachusetts, USA). L-Ascorbic acid 2-phosphate trisodium salt (Asc), sodium selenite, human transferrin, all-trans (atRA; CAS number 302-79-4), and 13-cis retinoic acid (13-cisRA; CAS number 4759-48-2) were purchased from Merck KGaA (Darmstadt, Germany). Rho kinase inhibitor was obtained from Abcam Plc (Cambridge, UK). 6-(2-(4-(2,4-Dichlorophenyl)-5-(4-methyl-1H-imidazol-2-yl)-pyrimidin-2-ylamino)ethyl-amino)-nicotinonitrile (CHIR99021) was purchased from Axon Medchem (Groningen, the Netherlands). 4-(2-Methyl-4-pyridinyl)-N-[4-(3-pyridinyl)phenyl]benzeneacetamide (Wnt-C59) and human bone morphogenetic protein 4 (BMP4), and were procured from Bio-Techne (Minnesota, USA).

Cell culture maintenance

The cell line used for the PluriLum assay is the BIONi010-C-NKX2.5-T2A-Nluc-44.37, which was established and quality assessed in collaboration with Bioneer A/S (Hørsholm, Denmark) (Lauschke et al. 2021b). The hiPSCs culture was maintained on Matrigel®-coated cell culture dishes in mTeSR™ Plus medium. Cells were kept at 37 °C and 5% CO2 in a humid environment. Culture medium was changed every day, or second day and cultures were split approximately once a week using 0.02% EDTA in DPBS.

Cardiomyocyte differentiation

The hiPSCs were differentiated into cardiomyocytes as previously described (Lauschke et al. 2020). Near confluent hiPSC cultures were dissociated into single cells by incubation with TrypLE™ for 4 min at 37 °C. Cells were then resuspended in medium and single cells were then seeded into 96-well Polystyrene Conical Bottom MicroWell™ plates in accordance with the desired density in 100 µl medium/well and spun down at 500g for 5 min (Day-1, D-1).

For experiments related to the effects of EB size on differentiation efficiency (Figs. 1 and 2), hiPSCs were seeded at different cell densities (1000, 2000, 3000, 4000, 5000 or 6000 cells/well) on D-1 to generate EBs of varying size.

The seeded plates were then incubated overnight at 37 °C and 5% CO2, and after a 19h (± 2h) overnight incubation (Day 0, D0), EBs had formed in the bottom of the conical wells, and the medium was exchanged into D0 differentiation medium.

After 24 h of incubation (Day 1, D1), medium was changed to TS medium. After an additional 24 h incubation (Day 2, D2), medium was changed into WNT-containing TS medium. After 24 h (Day 3, D3), the medium was changed again to TS medium and left to incubate for extra 72 h until Day 6 (D6). At D6, medium was replaced with fresh TS medium. The differentiation was assessed after an additional 24 h, i.e., on Day 7 (D7), where the experiment was terminated.

The original assay protocol (Lauschke et al. 2021b) runs for a total of 8 days (D-1 to D7). To assess the sensitivity of a shortened protocol, a set of experiments were conducted shortening the original protocol by 24 h, thus terminating on D6.

Chemical exposures

Epoxiconazole, atRA, and 13-cisRA were prepared in a stock solution in DMSO concentrated by a factor of 1000 relative to the highest final exposure concentration (40 mM epoxiconazole, 1 mM atRA and 3 mM 13-cisRA). For exposure experiments, dilutions in DMSO were added in a ratio of 1:1000 to the respective medium on the experimental days D1, D2 and D3, as well as on D6 for experiments terminating on D7, allowing for a constant vehicle (V/V) concentration of 0.1% DMSO in all wells. For each individual experimental condition, 6 EBs were exposed.

Imaging and sizing of embryoid bodies

The imaging of the individual EBs was performed on D0, on a BioTek Cytation 5 Cell Imaging Multimode Reader (Agilent Technologies, Santa Clara, USA), using a 4X objective. Size measurement was done using BioTek Gen5 Image Prime Software, by masking the edge of the EBs and calculating the diameter. Subsequent analog measurements were made to verify the quality of the size measurements using ImageJ (NIH, USA).

Scoring of embryoid body contraction

Upon completion of the assay, the contractility of each individual EB was assessed by visually evaluating the contractility using a light microscope and a 4X objective (Nikon Eclipse Ts2, Tokyo, Japan). Each EB was observed for maximum 15 s, or until beating was established.

The contractility was scored using the criteria: “Full Beat” (whole EB visibly contracting), “Partial Beat” (if contraction was less than whole), or “No Beat” (no visible contraction).

Analysis of NKX2.5 activation by luminescence measurements

Quantification of NKX2.5 activation in the individual EBs was analyzed by luminescence measurements using the Promega Nano-Glo® Luciferase Assay System (Promega, Wisconsin, USA). The EBs were washed by removing 50 µL medium from each well and adding 150 µL DPBS. The EBs were transferred in a volume of 40 µL into flat bottomed white 96-well plates for luminescence measurements. The EBs were incubated with 40 µL papain (Merck, Darmstadt, Germany) at a final activity of 40 U/mL for at least 1.5h. After incubation, the EBs were dissociated into a cell suspension by pipetting up and down 20 times (Fischer et al. 2018). After dissociation, 40 µL cell suspension was transferred into a flat bottomed white 96-well plates containing 40 µL of Nano-Glo® Luciferase Assay Substrate followed by pipetting up and down. To the remaining 40 µL of cell suspensions, 40 µL of CellTiter-Glo® (Promega) were added and plates were shaken on an orbital shaker for 10 min before stabilizing for 20 min. Measurements of both Nano-Glo® and CellTiter-Glo® luminescence were performed on a PerkinElmer EnSpire 2300 Multimode Microplate Reader (PerkinElmer, Inc., Massachusetts, USA).

Data processing and statistical analysis

Statistical analysis on luminescence data from chemical exposures was performed using GraphPad Prism 10 (version 10.0.3). All experiments were performed in three independent experiments using different passages of cells. Each experimental condition contained six technical replicates (EBs) in each experiment.

The value for each experimental condition is the average of the normalized relative luminescence units (RLU) of the technical replicates. Results are presented as mean ± standard deviation.

Statistical analysis was performed using two-way ANOVA, followed by multiple comparisons using the Bonferroni post hoc test.