Activin a

46C mESCs (3×10⁵) were seeded in Fibronectin (16.7 μL/mL, F1141-5MG, Sigma) coated plates and cultured in N2B27 medium supplemented with 20 ng/mL Activin A (C678, Novoprotein, China), 12 ng/mL bFGF (C044, Novoprotein, China) and 1% KSR (10828028, Invitrogen) to induce epiblast like stem cells (EpiLCs). After two days, 2×10⁵ EpiLCs were inoculated into GK15 medium for suspension culture for 4 days to induce PGCLCs in the presence of the inductive cytokines BMP4 (500 ng/mL, 315-27-10, Peprotech), LIF (1000 U/ml, Millipore), SCF (100 ng/mL, AF-250-03, Peprotech) and EGF (50 ng/mL, AF-100-15, Peprotech). GK15 consists of GMEM (11710035, Gibco), 15% KSR, 1×MEM nonessential amino acids, 0.1 mM β-mercaptoethanol and 1 mM sodium pyruvate (SP0100, Solarbio, China). After dissociation, the PGCLCs were resuspended in 1×PBS and analyzed on flow cytometers (FACS Calibur, BD Biosciences).

Hepatocytes were differentiated as described,⁵³ (link) with some modifications. The differentiation process was divided into three stages: pretreatment step, differentiation step and maturation step. Passage 2-Passage 8 UC-MSCs were seeded at 4×10³ cells/cm² in six-well plates in serum-free medium. Culture medium was switched 24 h later to pretreatment medium based on the serum-free medium supplemented with 20 ng/ml epidermal growth factor (Peprotech), 100 ng/ml activin A (Novoprotein) and 10 ng/mL fibroblast growth factor 4 (Peprotech) for 3 days. Thereafter, differentiation was induced by treating UC-MSCs with serum-free medium containing 20 ng/ml hepatocyte growth factor (Peprotech), 10 ng/mL fibroblast growth factor 4, 0.61 g/L nicotinamide (Sigma), 1% dimethyl sulfoxide (MP Biomedicals), and 1% insulin-transferrin-selenium (Gibco) for 10 days. Then cells were incubated with maturation medium containing 20 ng/mL oncostatin M (Peprotech), 1 μmol/L dexamethasone (Sigma), 1% dimethyl sulfoxide and 1% insulin-transferrin-selenium for 15 days. Medium was changed every 3 days.