Nicotinamide

Cells were cultured in iMEM containing 1% P/S, 0.5× B27, 1% Pluronic® F-68, 100 ng/mL KGF, 50 ng/mL epidermal growth factor (EGF, R&D Systems), 10 mM nicotinamide (STEMCELL Technologies, Vancouver, Canada), 0.1 μM TR05991851 (ROCK inhibitor, Takeda Pharmaceutical Company, Osaka, Japan), 0.5 μM PDBu (Merck Millipore, Billerica, MA, USA), and 5 ng/mL activin A for 4 days.
In the following stages 5–7, we used 10 μM ALK5iII and their alternatives (ALK5 inhibitors, CDK8/19 inhibitors, and their combination such as 3 μM SB 431542 and 0.3 μM senexin B, depending on the experimental purposes as mentioned above.

For pancreatic differentiation, we followed Yuya Kunisada’s protocol with slight modification^{19 (link)}. Briefly stem cells were passaged on Matrigel-coated 24-well plates at a density of 10 × 10⁴ cells per well. Stem cells were cultured with mTeSR1 including Y27632 for 24 h and then 3 days with mTeSR1 to nearly reach confluence. For differentiation, the cells were cultured in RPMI1640 (Gibco) containing B27 insulin (Gibco), 100 ng/mL activin A (Pepro Tech) and 3 μM CHIR99021 (Stemgent) for 24 h and for 48 h in RPMI 1640 containing B27 minus insulin and 100 ng/mL activin A. Subsequently, the media was changed with DMEM/F12 containing B27 (Gibco), 1 μM dorsomorphin (Calbiochem), 10 μM SB431542 (Sigma) and 2 μM retinoic acid (Sigma) for 7 days and medium was replaced every other day. For insulin-producing cell differentiation, the medium was changed to DMEM/F12 containing B27, 10 μM forskolin (Stemgent), 10 μM dexamethasone (Enzo Life Sciences), 5 μM Alk5 inhibitor II (Calbiochem) and 10 mM nicotinamide (Stemcell) for 12 days and provided with fresh medium every other day.

Mouse HPs were generated as previously described [31 (link)]. Briefly, primary adult mice hepatocytes were isolated from 8 to 12 weeks old female BALB/c mice by two-step perfusion method. After perfusion with Ca⁺² free Hank’s/EGTA solution and digestion with collagenase solution (Millipore Sigma), the digested livers were filtered and the suspension was collected via centrifugation at 50 g at 4 °C. Dead cells were removed, the remaining cells were washed twice and cultured with small hepatocytes basal medium (SHM) [DMEM/F12 (high glucose, Hyclone) supplemented with 5 mM HEPES (Sigma), 10 ng epidermal growth factor (PeproTech), 1% ITS (Gibco), 30 mg/L L-proline (Sigma), 0.05% BSA (Gibco), 10–7 M dexamethasone (Dex) (Selleck), 1 mM ascorbic acid, 10 mM nicotinamide (Stem Cell), and 1% penicillin and streptomycin solution (Life Technologies)] supplemented with 10% FBS (Gibco). Purified mouse hepatocytes were then seeded on collagen-coated plates in SHM with or without the combination of the small molecule inhibitors, Y-27632 (Selleck), 0.5 mM A-83–01 (Selleck) and CHIR99021 (Selleck). One day after seeding, the medium was changed and every other day thereafter. We confirmed all procedures for hepatic and biliary functions described for mouse hepatic progenitors as mentioned in [31 (link)].