Mouse embryonic stem cells (SBr line55) were cultured at 370C in 5% CO2 in medium composed of DMEM+Glutamax (#61965-026), 10% ES cell-qualified FBS (#16141-079), 1mM sodium pyruvate (#11360-070), 1x MEM non-essential aminoacids (#11140-035), 0.1mM 2-mercaptoethanol (#31350-010) and 1000u/ml Pen/Strep (#15140-122) supplemented with 3µm GSK3i (#361559), 2µm MEKi (#S1036) and 0.1µg/ml LIF (in house preparation). Cells were routinely passaged every 2-3 days by seeding 8000-9000 cells/cm2 and every 20 passages a fresh vial was thawed. Cells were tested and confirmed free of mycoplasma. Mouse trophoblast stem cells (TS:GFP line45) were cultured at 370C in 5% CO2 in TS medium composed of RPMI 1640+Glutamax (#61870-010), 20% ES cell-qualified FBS (#16141-079), 1mM sodium pyruvate (#11360-070), 0.1mM 2-mercaptoethanol (#31350-010) and 1000u/ml Pen/Strep (#15140-122). TS medium was conditioned on irradiated MEFs for 3 days and stored at -200C. This was repeated three times for one batch of irradiated MEFs. Aliquots of TS conditioned medium (TSCM) were thawed and mixed 3:1 with fresh TS medium before cell passaging. 50ng/ml Fgf4 (#100-31) and 1µg/ml Heparin (#H3149) were added to make final TS medium. TS cells were routinely passaged every 2-3 days by seeding 5000-6000 cells/cm2 and every 20 passages a fresh vial was thawed. MEFs were prepared in house and stocks were prepared at passage 1. MEFs were cultured in TS medium with no additional growth factors. For the co-culture experiments, MEFs from passages 4-7 were used. Cells were tested and confirmed free of mycoplasma.
Preparing EPI and TS differentiation medium
N2B27 medium was prepared by 1:1 mixing of DMEM/F12+Glutamax (#31331-028) and Neurobasal (#21103-049) with the addition of 0.5x N2 supplement (#17502001), 0.5x B27 supplement (#17504001), 0.5x Glutamax (#35050-038), 1mM sodium pyruvate (#11360-070), 1x MEM non-essential aminocacids (#11140-035), 0.1mM 2-mercaptoethanol (#31350-010) and 1000u/ml Pen/Strep (#15140-122). 12ng/ml Fgf2 (#PMG0035), 20ng/ml Activin-A (#338-AC) and 1% KSR (#10828-010) were added to make final EPI differentiation medium (EPIdiff). TS differentiation medium (TSdiff) was prepared by 1:1 mixing of N2B27 and TS medium supplemented with 25ng/ml Fgf4 (#100-31) and 500ng/ml Heparin (#H3149).
Preparing EPI and TSC Aggregates on PEG microwells
Poly(ethylene glycol) (PEG) microwells with 400µm well diameter (121 wells per array) were prepared on 24-well plates as previously described56. Microwells were equilibrated with 50µl of either EPIdiff (for ES cells) or TSdiff (for TS cells) for at least 30 minutes at 370C. Mouse ES and TS cells and were dissociated to single cells with Accutase (#A11105-01) or TrypLE (#12605-028), respectively. Cells were then centrifuged at 1000 rpm for 5 minutes and washed twice with 10 ml PBS at 40C. Cells were resuspended in cold EPIdiff (for ES cells) or TSdiff (for TS cells) and suspension of 484.000 cells/ml was prepared. 35µl of the suspension was added dropwise on microwell arrays to have 100-150 cells/well. Seeding was done at 370C for 15 minutes. Growth factor reduced Matrigel (#356231) was diluted in cold EPIdiff or TSdiff to 3% or 2% (v/v), respectively. The medium was vortexed and 1ml was slowly added from the side of the well, avoiding direct addition from the top of the microwell arrays. Plates were kept at 370C in 5% CO2 for at least 72 hours before further processing.
Forming EpiTS embryoids
At 72-75 hours of culture, aggregates on microwell arrays were flushed out and transferred to non-tissue culture treated 10cm plates in 10ml warm N2B27 medium. Single EPI and TSC aggregates were picked in 10µl and transferred to low adherent U-bottom 96 well plates (#COR-7007). 170µl of N2B27 medium was added on top. At 96 and 120 hours, 150µl of medium was replaced with fresh N2B27 and EpiTS embryoids were kept until 168h. Protein inhibitors Lefty (#746-LF-025), Noggin (in house preparation), Dkk1 (#5897-DK-010) were added at indicated timepoints at 200ng/ml final concentration. Small molecule inhibitors; SB431542 (#S4317), XAV939 (#S1180), LDN193189 (#SML0559) were added at indicated timepoints at 10µm, 10µm and 1µm final concentrations, respectively.