1).16 They were differentiated in vitro to hepatocyte-like cells at passages 4 to 10 (Fig. 1A), according to a well-established multistep protocol.15, 16 Quality of differentiation was proven by an increased gene expression of cytochrome P450 3A4 (CYP3A4; P = 0.016), hepatocyte nuclear factor 4-α (HNF4α; P = 0.031), and albumin (P = 0.016), and the exhibition of functions typical of mature hepatocytes,

such as CYP3A4 activity (P = 0.031; Fig. 1B,C). Variability in differentiation mTOR inhibitor quality among different donors is shown in Supporting Fig. 1E. To study the effect of differentiation state on HBV-cell interactions, we first assessed viral attachment to the cell membranes. At a temperature below 18°C, endocytosis is inhibited (Supporting Fig. 4A), whereas binding of viral particles to membrane receptors remains active.26 We incubated PHHs, UD-UCMSCs, and D-UCMSCs with HBV at an MOI of 1.0 ± find more 0.8 × 105, for 2 hours at 4°C. Under these conditions, endocytosis was totally inhibited while cellular viability was not affected (Supporting Fig. 4B,C). After extensive washing (Supporting Fig. 4D), the amount of membrane-bound HBV DNA was similar for PHHs and D-UCMSCs, but lower for UD-UCMSCs (P = 0.052; Fig. 2A). To prove that binding of viral particles on cell membrane was receptor-mediated, after incubation with HBV at 4°C and extensive washing, cells were treated

with trypsin before DNA extraction. Protease detached 95% of the viral particles, without any difference between cell types (Fig. 2B), indicating that proteinaceous structures were involved in HBV binding. To assess whether viral particles attached to membrane receptors could be internalized, after the 2-hour incubation at 4°C and extensive washing cells were moved to a 37°C environment. They were cultured under standard conditions and DNA was extracted after 1, 4, and 3-mercaptopyruvate sulfurtransferase 24 hours. To make sure to extract only intracellular DNA,

trypsin was applied before DNA extraction, in order to detach all particles still bound to the cell membrane. After 1 hour at 37°C, PHHs, UD-UCMSCs, and D-UCMSCs were able to internalize 4.9 ± 0.7%, 6.3 ± 1.5%, and 5.5 ± 1.3% of membrane-bound HBV, respectively (P = ns; Fig. 2C). The proportion of viral uptake increased at 4 and 24 hours for PHHs (P = ns) and D-UCMSCs (P = 0.016), but remained stable for UD-UCMSCs. HBV uptake after 24 hours was significantly greater in D-UCMSCs than in UD-UCMSCs (P = 0.004). The amount of virus taken up by D-UCMSCs at 24 hours increased with the increase of MOI (Fig. 2D). Little increase was seen for MOI >103, suggesting saturation of the receptor(s). Viral entry after 24 hours at 37°C was confirmed by immunofluorescence. Both PHHs and D-UCMSCs, but not UD-UCMSCs, showed a positive staining for intracellular HBcAg (Fig. 2E).