Figure 5

Differentiation of hepatoblasts into hepatocyte-like cells free of vector integration. (A) Fluorescence microscopy (left panel) 18 days after sorting and fluorescence-activated cell sorting (FACS) analysis of green fluorescent protein (GFP) expression from (left) non-transduced progenitors (left FACS panel), and transduced progenitors (middle) 3 days after transduction and (right) and 18 days after sorting. The numbers indicate the percentages of GFP-positive cells in the analyzed P15 gate. (B) Representative Southern blots of integrated and non-integrated forms (1-long terminal repeat (LTR) and 2-LTR circles) of the lentivector at 3 days and 14 days after transduction (days 16 and 27, respectively, of differentiation) in the presence or absence of raltegravir. (C) Total copy number of lentivectors was analyzed in the same cells as in (B) by quantitative-(q)PC). (D) Representative phase-contrast image of differentiating purified hepatic progenitors on day 14 after sorting. (E, F) Uptake and excretion of indocyanin green. (G) Albumin secretion of undifferentiated H9 cells, non-purified hepatic cells, purified hepatic cells and Hepa-RG hepatoma cell line in the culture medium was evaluated by ELISA. (H) Representative transcript levels of hepatocyte-specific clotting Factor IX (FIX) on days 16, 25, and 35 of differentiation. (I) Glycogen storage of the purified cells was assessed by periodic acid-schiff (PAS) staining, 14 days after sorting (day 30 of differentiation).