One of the critical functions of the liver is the ability to metabolize drugs, which requires functional drug-processing machinery—cytochrome P450 (CYP) enzymes, phase II enzymes, and transporters. In the field of preclinical drug discovery research, current in vitro hepatic models fail to recapitulate these functions and maintain them over a long period of time. Animal models fall short in that drug metabolism and pharmacokinetics vary across species, and primary hepatocytes are fundamentally limited by donor availability.

Hepatocytes derived from human induced pluripotent stem cells have the potential to serve as a predictive in vitro model system for toxicity testing and preclinical drug development studies. Using an optimized differentiation protocol and an improved maintenance medium, we have developed hepatocytes from human induced pluripotent stem (hiPS) cells that display mature adult hepatic characteristics. Cellartis enhanced hiPS-HEP cells are available from three different hiPSC lines: ChiPSC12, ChiPSC18, and ChiPSC22 (abbreviated as C12, C18, and C22). Each line is included in a complete kit containing a thawing/plating kit and Cellartis Long-Term Maintenance Medium.

Here, we show that the enhanced hiPS-HEP cells maintain the following characteristics for two weeks post-thawing:

• Stable and substantial CYP expression and activities
• Phase II enzyme expression and activities
• Transporter expression
• Increasing sensitivity to hepatotoxic compounds over repeated dosing

Sustained cytochrome P450 expression and activity

Several methods were used to measure CYP enzyme activities and expression in the enhanced hiPS-HEP cells maintained with the v2 kit. First, CYP expression was analyzed by qRT-PCR (Figure 1). CYP mRNA levels were stable or slightly increasing between Days 4 and 20 post-thawing (Figure 1, Panel A). A comparison to cryoplateable primary hepatocytes (hphep cells) showed that multiple CYP enzymes were expressed at similar levels in the enhanced hiPS-HEP cells on Day 20 post-thawing versus hphep cells at 24 hr post-thawing (Figure 1, Panel B). Gene expression of adult liver enzymes (CYP1A2, CYP2C9, and CYP3A4) and a fetal enzyme (CYP3A7) were expressed at similar levels in the enhanced hiPS-HEP cells and hphep cells, indicating that the enhanced hiPS-HEP cells show mature hepatocyte features.

In addition to qRT-PCR analyses, immunostaining for CYP1A2, CYP2C9, and CYP3A4 was performed on the enhanced hiPS-HEP cells from C12, C18, and C22 (Figure 2), revealing expression of the CYP enzymes in a subpopulation of the enhanced hiPS-HEP cells. These data are reminiscent of metabolic zonation of the liver lobe, wherein differential expression of CYP enzymes occurs in distinct periportal and perivenous hepatocyte phenotypes (Jungermann and Kietzmann 2000).

Next, the activities of the CYP enzymes listed in Figure 3, Panel A were analyzed using liquid chromatography-mass spectrometry (LC/MS). The enhanced hiPS-HEP cells showed similar CYP1A, CYP3A, and CYP2C9 activities to hphep cells cultured for 20 hr post-thawing (Figure 3, Panel B). CYP2B6, CYP2D6, and CYP2C19 activities were present in the enhanced hiPS-HEP cells but at lower levels than in hphep cells cultured for 20 hr post-thawing. Importantly, CYP activities in the enhanced hiPS-HEP cells vary little between different batches derived from the same cell line (as shown by the small error bars in Figure 3, Panel B), which indicates a robust, stable differentiation procedure. In contrast to the rapidly decreasing CYP activities seen in hphep cells in 2D cultures (Richert et al. 2006), the enhanced hiPS-HEP cells displayed stable CYP activities between Days 4 and 21 post-thawing (Figure 3, Panel C). Taken together, the low batch-to-batch variation and the stable or increasing CYP activities over time provide users with a continuous, unlimited source of cells that have a consistent, stable phenotype.

CYP	Drug substrate	Metabolite
CYP3A4/5	Midazolam	α-OH-midazolam
CYP2D6	Bufuralol	1'-OH-bufuralol
CYP2B6	Bupropion	(2S,3R)-OH-bupropion
CYP2C9	Diclofenac	4'-OH-diclofenac
CYP1A	Phenacetin	Paracetamol
CYP2C19	Mephenytoin	4'-OH-mephenytoin

Figure 3. CYP activities in enhanced hiPS-HEP cells and hphep cells. Panel A. A list of the CYP substrates and their respective metabolites used for assessment of CYP activities. The CYP enzymes are ranked by how much they contribute to drug metabolism (i.e., percentage of drugs metabolized by the enzyme; Hewitt et al. 2007). Panel B. Enhanced hiPS-HEP cells from C12, C18, and C22 were cultured for 4, 12, and 19 days post-thawing before performing CYP activity assays. CYP activity assays were performed on two batches per cell line. The small error bars indicate low batch-to-batch variation. Hphep cells were cultured for 20 hr post-thawing, including plating, culturing, and CYP activity assay (n = 4 donors). Data are presented as mean values ± standard deviation. Panel C. Enhanced hiPS-HEP cells from C12, C18, and C22 were cultured for 4, 8, 12, 15, 19, or 21 days post-thawing before CYP activity assays were performed. CYP activity levels were stable for all six CYP enzymes analyzed during this 21-day period. Per cell line, one batch was analyzed, and for each data point, samples were pooled from two wells.

Interestingly, the enhanced hiPS-HEP cells derived from different hiPS cell lines display different CYP activity profiles, reflecting well-characterized CYP variation between individuals. (Note the large error bars for hphep cells in Figure 3, Panel B.) For example, CYP3A activity (Figure 3, Panel B) is higher in C18-derived enhanced hiPS-HEP cells than in C12- or C22-derived cells. Since CYP3A isoenzymes (CYP3A4, CYP3A5, and CYP3A7) cannot be distinguished by the activity assay, qRT-PCR analysis (Figure 1) was used to reveal the variation in expression between the isoforms and across cell lines. In Figure 1, C18-derived enhanced hiPS-HEP cells have substantially higher expression of the polymorphic gene CYP3A5 than C12- or C22-derived cells, whereas CYP3A4 levels are similar across the lines.

Effective expression and activity of phase II enzymes over an extended culture time

Phase II enzymes continue the drug metabolizing process. The modifications made by phase II enzymes like sulfotransferases (SULT) and uridine diphosphate glucuronosyltransferase (UGT) increase the solubility of most compounds, generally leading to their renal excretion. Phase II enzyme activity and expression were measured in the enhanced hiPS-HEP cells using several methods. First, qRT-PCR analysis showed stable or slightly increasing UGT1A1 and UGT2B7 mRNA expression levels between Days 4 and 20 post-thawing (Figure 4, Panel A). These mRNA levels were similar to those in hphep (dashed line; Figure 4, Panel B). SULT and UGT enzyme activities were analyzed using LC/MS (Figure 4, Panel C), revealing high levels of activity in the enhanced hiPS-HEP cells derived from all three hiPS cell lines, similar to those in hphep cells. The enhanced hiPS-HEP cells displayed stable or slightly increasing SULT and UGT activities between Days 4 and 19 post-thawing. 

Hepatic uptake and efflux transporter expression

In addition to phase I and II enzymes, expression of the uptake transporters (NTCP, OCT1, OATP1B1, and OATP1B3) and the efflux transporters (MRP2, MDR1, and BSEP) was analyzed in the enhanced hiPS-HEP cells and compared to hphep cells. qRT-PCR analyses showed that transporter expression was stable or slightly increasing over time in enhanced hiPS-HEP cells (Figure 5, Panels A and B), similar to the findings on phase I and II enzyme expression (Figures 1, 2, and 4). In Figure 5, Panel C, a comparison of levels in the enhanced hiPS-HEP cells to hphep cells (represented as a dashed line) shows that most of the efflux transporters as well as uptake transporter NTCP were expressed at similar levels, whereas OATP1B1, OATP1B3, and OCT1 were expressed at lower levels in the enhanced hiPS-HEP cells. mRNA expression of OATP1B3 was not observed in enhanced hiPS-HEP cells derived from ChiPSC18.

Proof-of-concept chronic toxicity study

Basic characterization revealed the expression and activity of important drug metabolizing enzymes in the enhanced hiPS-HEP cells. Next, the enhanced hiPS-HEP cells were tested for a correct response to known hepatotoxic drugs in a chronic toxicity study. Other commonly used hepatic cell models were also tested: hphep cells grown as 3D spheroids and an immortalized hepatic cell line (HepaRG).

In Figure 6, we studied the effect of aflatoxin on viability of hphep cells (Panel A), HepaRG cells (Panel B), enhanced hiPS-HEP cells from C18 (Panel C) and C22 (Panel D). Dosing was started on enhanced hiPS-HEP cells on Day 4 post-thawing, and viability was assessed following exposure for 2, 7, or 14 days. Dose-response curves were plotted as percentage viability of vehicle control. EC₅₀ values (Table I) dropped for all four compounds after repeated dosing at 7 or 14 days, as seen by a leftward shift in the dose response curves from Figure 6. This indicates, as expected, an increased sensitivity after prolonged exposure to the selected compounds in all cell types tested.

Human iPS cell-derived hepatocytes differentiated with our robust differentiation protocol and cultured using a novel maintenance medium encompass a functional human hepatocyte model for applications that demand hepatic functionality, high reproducibility, and an unlimited supply of hepatocytes from one or several donors. 

Enhanced hiPS-HEP cells are available from three different iPSC lines derived from healthy donors. This enables the repeated production of enhanced hiPS-HEP cells with high batch-to-batch consistency, unlike highly variable human primary hepatocytes. Additionally, these enhanced hiPS-HEP cells meet or exceed the performance of human primary hepatocytes in their long-term functionality, with CYP enzymes, phase II enzymes, and hepatic uptake and efflux transporters maintained for up to 20 days post-thawing. With full cellular functionality obtainable on Day 5, the v2 kits can achieve a >14-day assay window, typically from Day 5 through at least Day 19. Furthermore, the enhanced hiPS-HEP cells display variation in CYP activity profiles between cell lines, which reflects well-characterized CYP variation between individuals.

Overall, the enhanced hiPS-HEP cells can be considered an inexhaustible and consistent supply of functional hepatocytes for drug discovery, safety toxicology, and other long-term applications. We can also provide customized lines through our hepatocyte differentiation service.

Cell culture

Cryopreserved Cellartis enhanced hiPS-HEP cells derived from ChiPSC12, ChiPSC18, and ChiPSC22 were thawed, plated, and maintained in long-term maintenance medium according to the Cellartis Enhanced hiPS-HEP v2 Kits User Manual. Cells were maintained for up to 21 days post-thawing with media changes every second or third day. Cryoplateable human primary hepatocytes (hphep cells; BioreclamationIVT) were thawed and plated according to the manufacturer's recommendations. For Figures 1–5, hphep cells were grown in 2D conditions. For Figures 6 and 7, hphep cells were grown as 3D spheroids. HepaRG cells (Thermo Fisher Scientific) were grown in 2D for all experiments, according to the manufacturer's instructions. 

CYP activity assay

The CYP activities of the enhanced hiPS-HEP cells were analyzed at multiple time-points after thawing. LC/MS was used to measure the formation of specific metabolites: α-OH-midazolam (CYP3A), 1'-OH-bufuralol (CYP2D6), (2S,3R)-OH-bupropion (CYP2B6), 4'-OH-diclofenac (CYP2C9), paracetamol (CYP1A), and 4'-OH-mephenytoin (CYP2C19). LC/MS analysis was performed by Pharmacelsus GmbH.

The cells were carefully washed twice with prewarmed William medium E (+0.1% PEST). Then, the activity assay was started by adding 110 μl per cm² culture area of prewarmed William medium E containing 0.1% PEST, 25 mM HEPES, 2 mM L-glutamine, and the probe substrate cocktail (see Table II, below). After 2 hr at 37°C, 100 μl of the supernatant was collected and kept at –80°C until LC/MS analysis. The metabolite concentrations measured by LC/MS were normalized to the amount of protein per well (determined using the Pierce BCA Protein Assay Kit) and the assay duration (120 min). 

Phase II enzyme activity assay

The phase II enzyme activities of the enhanced hiPS-HEP cells were analyzed at multiple time-points after thawing. LC/MS was used to measure the formation of 7-OH-coumarin sulfate and 7-OH-coumarin glucuronide, specific metabolites for sulfotransferases and UDP-glucuronosyltransferases, respectively. LC/MS analysis was performed by Pharmacelsus GmbH.

The cells were carefully washed twice with prewarmed William medium E (+0.1% PEST). Then, the activity assay was started by adding 110 μl per cm² culture area of prewarmed William medium E containing 0.1% PEST, 25 mM HEPES, 2 mM L-glutamine, and 200 μM 7-OH-coumarin. After 2 hr at 37°C, 100 μl of the supernatant was collected and kept at –80°C until LC/MS analysis. The metabolite concentrations measured by LC/MS were normalized to the amount of protein per well (determined using the Pierce BCA Protein Assay Kit) and the assay duration (120 min).  

qRT-PCR

Total RNA from the enhanced hiPS-HEP cells was extracted using the MagMAX-96 Total RNA Isolation Kit (Life Technologies) according to the manufacturer's instructions. cDNA was synthesized, and qRT-PCR amplification reactions were performed using the ABI 7500 Real-Time PCR System (Life Technologies). Gene expression was analyzed using TaqMan Gene Expression Assays (Life Technologies) according to the manufacturer's recommendations. Each sample was analyzed in duplicate.

The following assays were used (Life Technologies): CYP1A2 (Hs01070374_m1), CYP2B6 (Hs04183483_g1), CYP2C9 (Hs004260376_m1), CYP2C19 (Hs00426380_m1), CYP2D6 (Hs00164385_m1), CYP3A4 (Hs00604506_m1), CYP3A5 (Hs00241417_m1), CYP3A7 (Hs00426361_m1), UGT1A1 (Hs02511055_s1), UGT2B7 (Hs00426592_m1), OCT1 (Hs00427552_m1), OATP1B1 (SLCO1B1; Hs00272374_m1), OATP1B3 (SLCO1B3; Hs00251986_m1), NTCP (Hs00161820_m1), BSEP (Hs00184824_m1), MRP2 (Hs00960494_m1), and MDR1 (Hs00184500_m1). Expression levels were calculated using the ΔΔCt method and normalized to a calibrator mix consisting of cDNA from hiPSCs, hiPSC-derived embryoid bodies, hiPSC-derived DE, hiPSC-derived cardiomyocytes, hphep cells, HepG2 cells, and HEK293 cells. Expression was normalized to CEBPα expression as a reference gene and presented as relative quantification. ΔΔCt was transformed into fold change by the formula: fold change = 2^–ΔΔCt.

Immunocytochemistry

Cells were stained as described previously (Ulvestad et al. 2013). The enhanced hiPS-HEP cells were fixed on Day 6 post-thawing for 15 min with 4% formaldehyde, and then stained with the following primary and secondary antibodies: rabbit anti-CYP1A2 (1:100), rabbit anti-CYP2C9 (1:3,000), rabbit anti-CYP3A4 (1:200), and donkey anti-rabbit Alexa Fluor 594 IgG (1:1,000).

Chronic toxicity study

The four compounds selected for the study were aflatoxin B1, amiodarone, chlorpromazine, and troglitazone. Compounds were first dissolved in DMSO and diluted in long-term maintenance medium to a final DMSO concentration of 0.4%. Dosing was started on the enhanced hiPS-HEP cells on Day 4 post-thawing and continued every 2–3 days thereafter. Viability was determined following a single-dose exposure for 2 days and repeated dose exposures for 7 days (three repeated doses) and 14 days (six repeated doses), using the CellTiter-Glo Luminescent Cell Viability Assay (Promega, Sweden). Luminescence was measured, and the samples were blank-corrected and normalized to the DMSO vehicle control. Dose-response curves were plotted as percentage of the vehicle control using GraphPad Prism 4 (GraphPad Software Inc., La Jolla, CA). Effective concentrations at 50% mortality (EC₅₀) were generated using nonlinear sigmoidal dose-response regression.

Acknowledgements  

The chronic toxicity experiments on the enhanced hiPS-HEP cells, HepaRG cells, and 3D hphep cell spheroids were performed by Gustav Holmgren (Skövde University, Sweden), Tommy B. Andersson's group (AstraZeneca Mölndal, Sweden), and Magnus Ingelman-Sundberg's group (Karolinska Institute, Stockholm, Sweden), respectively. These studies were performed within the ScrTox EU project (FP7 EC funded network; Grant Agreement No. 266753).

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