Figure 1.
Schematic protocol for hepatic differentiation of hiPS cells.
Human iPS (hiPS) cells were differentiated into endodermal cells using Roswell Park Memorial Institute (RPMI) + GlutaMAX medium containing 100 ng/mL of activin A for 5 days, and then into hepatic progenitor cells (HPCs) using KnockOut Dulbecco's modified Eagle medium (KnockOut DMEM) containing 1% dimethyl sulfoxide (DMSO) for 7 days. Thereafter, HPCs were matured using Cosmedium 004 (Cosmedium) containing 10 ng/mL of hepatocyte growth factor (HGF), 20 ng/mL of oncostatin M (OSM), and 100 nM of dexamethasone (DEX) for 10 days. Finally, the cells were cultured in only Cosmedium for 3 days. Valproic acid (VPA) was added to the culture medium for 72 h from day 18, 168 h from day 12, or 312 h from day 12 at a final concentration of 2 mM.
Table 1.
Sequences of primers for real-time RT-PCR analysis.
Table 2.
Probe substrates of drug-metabolizing enzyme and the metabolites.
Figure 2.
Effects of VPA on hepatic marker gene expression and induction of the CYP3A4 mRNA by RIF.
Human iPS (hiPS) cells (Windy) were differentiated into hepatocytes. Valproic acid (VPA) was added to the medium for 72 h from day 18 (72 h), 168 h from day 12 (168 h), or 312 h from day 12 (312 h). (A) Cryopreserved human primary hepatocytes (HPHs) were cultured for 0 (just after thawing) and 48 h. Each bar represents the mean ± standard deviation (n = 3). The graph represents gene expression relative to that detected in HPHs cultured for 48 h. Levels of statistical significance compared with VPA-untreated hepatocyte-like cells [control (Ctrl)]: *P<0.05 and **P<0.01; and (B) hiPS cell-derived hepatocyte-like cells were treated with 40-µM rifampicin (RIF) for the last 48 h of culture. Each bar represents the mean ± standard deviation (n = 2–3). The graph represents gene expression relative to that detected in VPA-untreated hepatocyte-like cells without RIF. Levels of statistical significance compared with Ctrl in the RIF-untreated group (†), and RIF-treated group compared with each RIF-untreated group (*), respectively: †P<0.05 and *P<0.05. The abbreviations used are: AFP, α-fetoprotein; ALB, albumin; TAT, tyrosine aminotransferase; PXR, pregnane X receptor; CYP, cytochrome P450.
Figure 3.
Morphological changes observed in hiPS cells during hepatic differentiation.
The images show morphological changes in human iPS (hiPS) cells (Windy). (A) Undifferentiated hiPS cells; (B) hepatic progenitor cell-like cells after 12 days of differentiation; (C, D) hepatocyte-like cells after 25 days of differentiation in the absence of valproic acid (VPA); and (E, F) hepatocyte-like cells after 25 days of differentiation in the presence of 2-mM VPA for 168 h from day 12. Arrows indicate binuclear cells. Scale bar, 100 µm.
Figure 4.
Immunofluorescence staining of ALB and effects of VPA on mRNAs encoding drug-metabolizing enzymes.
Human iPS (hiPS) cells (Windy) were differentiated into hepatocytes. Valproic acid (VPA) was added to the medium for 168 h from day 12. (A, B) hiPS cell-derived hepatocyte-like cells in the absence of VPA (A) and hiPS cell-derived hepatocyte-like cells treated with VPA (B) were stained for ALB (red). Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI; blue); (C) Cryopreserved human primary hepatocytes (HPHs) were cultured for 0 (just after thawing) and 48 h. Each bar represents the mean ± standard deviation (n = 3). The graph represents gene expression relative to that detected in human hepatocytes cultured for 48 h. Levels of statistical significance compared with VPA-untreated hepatocyte-like cells [control (Ctrl)]: **P<0.01. CYP, cytochrome P450; UGT, UDP-glucuronosyltransferase; N.D., not detected.
Figure 5.
Drug-metabolizing enzyme activities in hepatocyte-like cells differentiated from hiPS cells using VPA.
Human iPS (hiPS) cells (Windy) were differentiated into hepatocytes. Valproic acid (VPA) was added to medium for 168 h from day 12. Acetaminophen, hydroxybupropion, 4′-hydroxydiclofenac, 4′-hydroxymephenytoin, 1′-hydroxybufuralol, 1′-hydroxymidazolam, 7-hydroxycoumarin glucuronide, and 7-hydroxycoumarin sulfate were biotransformed from phenacetin, bupropion, diclofenac, (S)-mephenytoin, bufuralol, midazolam, 7-hydroxycoumarin, and 7-hydroxycoumarin by cytochrome P450 (CYP) 1A1/2, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP3A4/5, UDP-glucuronosyltransferase (UGT), and sulfotransferase (SULT), respectively. Each bar represents the mean ± standard deviation (n = 3). Levels of statistical significance compared with VPA-untreated hepatocyte-like cells [control (Ctrl)]: *P<0.05 and **P<0.01.
Figure 6.
Effects of small-molecule compounds on hepatic differentiation from hiPS cells.
All compounds were added to the medium for 168(A) The albumin (ALB) mRNA expression level was analyzed in hepatocyte-like cells differentiated from hiPS cells (Windy). Each bar represents the mean ± standard deviation (n = 3). The graph represents gene expression relative to that detected in compound-untreated hepatocyte-like cells [control (Ctrl)]. Levels of statistical significance compared with Ctrl: *P<0.05 and **P<0.01. (B) Time-dependent changes in HDAC activity in differentiating human iPS (hiPS) cells (Windy). Symbols represent the mean ± standard deviation (n = 4). Levels of statistical significance compared with Ctrl: **P<0.01. (C) The ALB mRNA expression level was analyzed in hepatocyte-like cells differentiated from three hiPS cell lines (Windy, Dotcom, and Fetch). Each bar represents the mean ± standard deviation (n = 3). The graph represents gene expression relative to that detected in VPA-untreated hepatocyte-like cells differentiated from Windy. Levels of statistical significance in each cell line compared with each Ctrl, respectively: **P<0.01. The abbreviations used are: NaB, sodium butyrate; TSA, trichostatin A.