1. Amino acid In vitro recapitulation of the urea cycle using murine embryonic stem cellderived in vitro liver model Miho Tamai, Mami Aoki, Akihito Nishimura, Koji Morishita, and Yoh-ichi Tagawa* *To whom correspondence should be addressed: Yoh-ichiTagawa, Email: ytagawa@bio.titech.ac.jp, Tel.: +81 45-924-5791. Fax: +81 45-924-5815. Department of Biomolecular Engineering, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology 4.5 3.0 Urea (mM) 1.5 0 [day] 1 4 7 1 4 7 On collagen On EHS gel Supplementary Fig. S1 Day of Culture(day) B A Isolated Hepatocyte Day of Culture (day) Adult Liver On collagen On EHS gel 1 4 7 1 4 7 [day] cps1 otc arg1 ornt1 hprt

2. Supplementary Fig. S2 150 100 50 B A 0 * * Released Enzymes (IU/L) Cell Viability (%) - - - - + + L-Ornithine L-Ornithine - - + + + + Ethanol Ethanol C D * * 5.0 NH4+ (mM) 2.5 Urea (mM) 0 - - - - + + L-Ornithine L-Ornithine - - + + + + Ethanol Ethanol