3A,B). In this regard, D-UCMSCs resembled PHHs serving as positive controls. ASGPR has been suggested to play a role in HBV binding and uptake.8-10 HBV uptake by D-UCMSCs was directly correlated with ASGPR mRNA expression see more (R2 = 0.924, P < 0.01; Fig. 3C). In Fig. 3D we show that HBV binding

to D-UCMSCs may be partially inhibited by Ca2+ chelation (21.1 ± 2.5% inhibition) and thyroglobulin addition (77.8 ± 1%), the latter being one known ASGPR-specific ligand. Suramin, which is known to block HBV attachment,22, 23 inhibited 87.4 ± 1% of HBV binding to D-UCMSCs. The addition of increasing concentrations of D-galactose (0.03-100 μM), before and during inoculation of D-UCMSCs, resulted in a dose-dependent inhibition of HBV uptake (up to 79.3 ± 0.7%, P < 0.0001; Fig. 3E). The median effective concentration (EC50) = 0.2 μM (95% confidence interval [CI], 0.17-0.23) was calculated for D-galactose by dose-response analysis (Supporting Fig. 4F). Total HBV DNA was quantified BMN 673 clinical trial by qPCR at 3 and

7 days postinfection and compared to the amount of viral DNA present inside the cells at 24 hours postincubation (Fig. 4A). Intracellular HBV DNA levels decreased along time in UD-UCMSCs (P < 0.0001), whereas they increased in PHHs (P = ns) and D-UCMSCs (P = 0.016) at day 3 and 7, suggesting productive viral replication. At 7 days postinfection, intracellular HBV DNA levels did not differ between D-UCMSCs and PHHs, but were significantly higher in D-UCMSCs than in UD-UCMSCs (P < 0.01). A further increase of HBV DNA levels was seen at 10 days postinfection in D-UCMSCs (P = 0.029; Fig. 4B). PHHs were not tested beyond 7 days postinfection to avoid biases due to dedifferentiation.11 In D-UCMSCs, an MOI of at least 100 was needed to yield detectable viral replication (Fig. 4C). Increase of HBV DNA replication intermediates along time was confirmed at Southern blotting

on the same samples used for qPCR (Supporting Fig. 5C). We subsequently measured cccDNA by qPCR (Fig. 4D). In D-UCMSCs, cccDNA levels increased along time up to 0.03 ± 0.04 copies/cell at 7 days postinfection, corresponding Bcl-w to approximately every 30th cell containing at least one copy of cccDNA. By contrast, 0.7 ± 0.8 copies/per cell were found in PHHs, and no cccDNA was detected in UD-UCMSCs. Controls to prove specificity of cccDNA detection by qPCR are shown in the Supporting Data. To further confirm the effect of differentiation on cellular susceptibility to viral replication, we infected UD-UCMSCs as described above and induced differentiation after 24 hours. For this postinfection differentiation we used a serum-free medium corresponding to the final step of the differentiation protocol described above. HBV DNA levels in UCMSCs undergoing postinfection differentiation increased along time as compared to UD-UCMSCs (P = 0.018), suggesting replication of the small quantity of HBV (0.22 ± 0.32 vge/cell) internalized by undifferentiated cells (Fig. 4E).