Autonomous hepatic differentiation of hiHSCs in Essential 6 medium.

<p>Self-renewing hiHSCs (clone AFB1-1) differentiate into hepatocyte-like cells in defined Essential 6 medium that consists simply of insulin, transferrin, selenium, and L-ascorbic acid in DMEM/F12 medium. (<b>A–G</b>) Gene expression was analyzed by quantitative RT-PCR at day 12. Relative expression is shown in the histogram. The expression is normalized to 1 in the self-renewing hiHSCs (mTeSR1/MEF) and compared to that of hepatocyte-like cells (E6). Gene symbols are shown for (<b>A</b>) serum hepatic proteins, (<b>B</b>) cytochrome P450 enzymes, (<b>C</b>) conjugating enzymes and transporters, (<b>D</b>) hepatic transcription factors, (<b>E</b>) endodermal transcription factors, (<b>F</b>) ESC transcription factors, and (<b>G</b>) urea cycle-related enzymes. Data are presented as mean+SEM and represent a minimum of three independent samples with at least two technical duplicates. (<b>H</b>) Urea production was measured by urea nitrogen detection kits on the supernatants of cells at day 12. E6 fresh medium was also measured as a negative control. Data are presented as mean+SEM and represent a minimum of three independent samples. (<b>I</b>) Immunofluorescent staining of hepatocyte-like cells for FABP1, ALB, CK8, and ASGPR1. Scale bar represents 100 μm. (<b>J</b>) Release of ALB was measured by ELISA at three time points. Data are presented as mean±SEM and represent a minimum of three independent samples with at least two technical duplicates. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123193#pone.0123193.t001" target="_blank">Table 1</a>. (<b>K</b>) Phase contrast micrographs showing the morphology of differentiating cells. Scale bar represents 100 μm. (<b>L</b>) At day 12, hepatocyte-like cells are shown to store glycogen by Periodic Acid—Schiff (PAS) staining. Otherwise, the cells were stained to uptake indocyanine green (ICG) for 5 hr and BODIPY FL–labeled low density lipoprotein (LDL) for 48 hr. Scale bar represents 100 μm.