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Skeletal muscle differentiation was performed according to the protocol described in^{46 (link)}, with few adjustments. Briefly, a total of 50,000 cells/cm² were plated on a laminin-521 coated dishes. The next day, differentiation was induced by the use of 10 µM CHIR99021 in myogenic differentiation medium consisting of DMEM-F12, 1 × ITS-X, 100 U/ml Pen/Strep and 1 × Glutamine (all from Thermo Fischer Scientific) for 2 days. Subsequently, the CHIR99021 was replaced by 20 ng/ml FGF2 (Prepotech) for the following 10 days. Medium was refreshed daily.

ETX embryos were generated as described previously^{10 (link)}. Approximately 6000–7000 ES cells, 15,000–19,000 TS cells and 5000–6000 XEN cells were added dropwise into AggreWell plates having 1200 microwells in one well (34411; STEMCELL Technologies). The microwells were treated with rinsing solution (07010; STEMCELL Technologies). Cells were centrifuged at 100g for 3 min. 1.5 ml c-IDG medium containing 7.5 nM ROCK inhibitor (72304; STEMCELL Technologies) was added dropwise to each well. On the following day (day 1), 1 ml medium was removed gently from each well and replaced with 1 ml fresh c-IDG medium without ROCK inhibitor. This step was repeated once to fully remove the ROCK inhibitor. On day 2, 1 ml c-IDG medium was replaced with 1 ml fresh medium. On day 3, the media was replaced with IVC1 medium^{10 (link),24 (link),52 (link)}. IVC1 medium comprises advanced DMEM/F12 (21331-020; Gibco) supplemented with 20% (vol/vol) FBS, 2 mM GlutaMax, 1% vol/vol penicillin–streptomycin, 1× ITS-X (51500-056; Thermo Fisher Scientific), 8 nM β-estradiol, 200 ng ml⁻¹ progesterone and 25 mM N-acetyl-l-cysteine.

To culture embryos through the pre- to post-implantation transition, the zona pellucida was removed at the late blastocyst. Embryos/chimeras were exposed for 30 min to 20% rabbit anti-mouse whole serum (Sigma-Aldrich) in M2 medium at 37 °C. Next, they were incubated for 30 min with 20% guinea pig complement serum (Sigma-Aldrich) in M2 medium at 37 °C. The damaged TE was removed by pipetting the embryos in M2. ICMs were embedded in Matrigel. 20 μl drop of ice-cold growth factor-reduced Matrigel (BD Biosciences) was placed in a well of a μ-Slide 8-well ibiTreat (Ibidi) dish and embryos were mouth pipetted inside the Matrigel drop. The dish was incubated for 5 min at 37 °C to allow the Matrigel to solidify. Then, 300 μl of prewarmed IVC medium was added to the well.
Embryo imaging was performed using a spinning disk confocal at 37 °C and 5% CO₂. The images were captured every 20 min in 50–90 μm stacks of 2.5-μm intervals.
IVC medium constitution: Advanced DMEM F12 (Thermo Fisher Scientific), 20% v/v heat-inactivated fetal bovine serum (FBS) (Stem Cell Institute), GlutaMAX (Thermo Fisher Scientific), 25 U ml⁻¹ penicillin—25 μg ml⁻¹ streptomycin (Thermo Fisher Scientific), 1× ITS-X (Thermo Fisher Scientific), 8 nM β-oestradiol (Sigma-Aldrich), 200 ng ml⁻¹ progesterone (Sigma-Aldrich) and 25 μM N-aceyl-L-cysteine (Sigma-Aldrich).

Stage 7 medium was based on a previous report [7 (link)] with our original modifications. Cells were cultured in the MCDB 131 medium with 1% P/S, 2% fat-free bovine serum albumin (FUJIFILM Wako), 20 mM glucose, 1.5 g/L NaHCO₃, 1% GlutaMAX, 0.5% ITS-X (Thermo Fisher Scientific), 10 μM ALK5iII or the alternative candidates, 1 μM T₃, 10 μM ZnSO₄ (Merck Millipore), 1.4 IU/mL heparin sodium salt (Nacalai Tesque, Kyoto, Japan), 1 mM N-acetyl cysteine (Merck Millipore), 10 μM Trolox (FUJIFILM Wako), 2 μM R428 (Selleck Chemicals, Houston, TX, USA), 1 μM PD-166866, 3 μM TR06141363 (Multi-kinase inhibitor, Takeda Pharmaceutical Company), and 10 μM Y-27632, for 4 days. To generate iPICs for implantation, cells were dissociated and re-sized in an Elplasia 6-well microwell plate (Corning Incorporated, Corning, NY, USA) or a gas-permeable microwell culture bag (Toyo Seikan Group Holdings, Ltd., Yokohama, Japan) [20 (link)] and cultured in the stage 7 medium.