Anti-EGFR antibodies (sheep polyclonal, Upstate^®, Merck, Darmstadt, DE), phospho-EGFR (Y1068) [mouse monoclonal (m), Abcam, Cambridge, UK], GAPDH (m, MBL, Nagoya, Japan), human albumin (goat polyclonal, Bethyl Laboratories, Montgomery, TX), α-fetoprotein [rabbit monoclonal (r, mAb), Abcam, Cambridge, UK], and hepatocyte nuclear factor 4 alpha (HNF4α) (r, mAb, Abcam, Cambridge, UK) were used for our experiments.

iPSCs were cultured in a feeder-free system. Briefly, 6-well plates were pre-incubated in 1.5 ml/wells of Stem Fit AK03N (Ajinomoto, Tokyo, Japan) containing 0.25% iMatrix 511 silk (TaKaRa, Shiga, Japan) and 10 µM Y-27632, a rho-associated coiled-coil kinase (ROCK) inhibitor (FUJIFILM Wako Pure Chemical Corp., Osaka, Japan), in a CO₂ incubator. iPSCs (1–1.4×10⁴ cells), suspended in 500 µl of Stem Fit AK03N (Ajinomoto, Tokyo, Japan) and then plated onto a pre-incubated 6-well plates. The following day, the medium was replaced with Stem Fit AK03N (Ajinomoto, Tokyo, Japan) alone and changed every other day.

At confluency, iPSCs were washed twice with PBS (FUJIFILM Wako Pure Chemical Corp., Osaka, Japan) and incubated with Accutase^™ (Innovative Cell Technologies, Inc., San Diego, CA) for approximately 5 min at 37°C, after which the supernatant was removed. The cells were washed with PBS, suspended in Stem Fit AK03N (Ajinomoto, Tokyo, Japan), and counted using an automated cell counter (TC20, Bio-Rad, Hercules, CA, USA). The cells were cultured as described above.

iPSCs were plated at 60–80% of confluence with 1 ml/well of Stem Fit AK03N (Ajinomoto, Tokyo, Japan) containing 0.25% iMatrix 511 silk (TaKaRa, Shiga, Japan) and 10 µM Y-27632 in a 12-well plate. Hepatic differentiation was performed using 4-step protocol. (STAGE 1) The next day, the medium was changed to RPMI1640 (Thermo Fisher Scientific, Waltham, MA) with 2% B27 (Thermo Fisher Scientific, Amarillo, TX) with insulin, 75 ng/ml Activin A (FUJIFILM Wako Pure Chemical Corp., Osaka, Japan), 3 µM CHIR99021 (FUJIFILM Wako Pure Chemical Corp., Osaka, Japan), GSK3β inhibitor, and 10 µM LY294002 (FUJIFILM Wako Pure Chemical Corp., Osaka, Japan), PI3K inhibitor. The cells were then incubated for 5 days and the medium changed daily. (STAGE 2) The medium was replaced with RPMI1640 (Thermo Fisher Scientific, Waltham, MA) containing 2% B27 (Thermo Fisher Scientific, Amarillo, TX) with insulin, 10 ng/ml BMP4 (FUJIFILM Wako Pure Chemical Corp., Osaka, Japan), and 10 ng/ml FGF2 (FUJIFILM Wako Pure Chemical Corp., Osaka, Japan). The cells were then incubated for 5 days and the medium changed daily. (STAGE 3) At day 11, the cells were incubated with RPMI1640 (Thermo Fisher Scientific, Waltham, MA) containing 2% B27 (Thermo Fisher Scientific, Amarillo, TX) with insulin, and 20 ng/ml HGF (FUJIFILM Wako Pure Chemical Corp., Osaka, Japan) for 5 days with daily medium change. (STAGE 4). On day 16, the medium was again replaced with RPMI1640 (Thermo Fisher Scientific, Waltham, MA) containing 2% B27 (Thermo Fisher Scientific, Amarillo, TX) with insulin, 20 ng/ml Oncostatin M (FUJIFILM Wako Pure Chemical Corp., Osaka, Japan) for a further 5 days with daily medium change, after which the iPSC-derived hepatocytes (iPSC-heps) were characterized.

HaCaT cells, an immortalized human keratinocyte cell line, obtained from COSMO BIO CO., Ltd. (300493-ACADEMIC) were cultured in MCDB 153 medium containing 5% FBS and 10 µg epidermal growth factor (FUJIFILM Wako Pure Chemical Corp., Osaka, Japan). To collect the cell lysates, the cells were grown until they reached 80–100% confluence.

The samples were prepared with RIPA buffer and quantified using a Pierce^™ BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA). The quantified samples were electrophoresed and electroblotted using a semi-dry system (Trans-Blot SD Semi-Dry Transfer cell, Bio-Rad, Hercules, CA, USA) onto a PVDF membrane (Merck Millipore, Burlington, MA). The membrane was then incubated with the appropriate primary antibodies followed by HRP-conjugated anti-mouse or -rabbit secondary antibodies (SouthernBiotech, Birmingham, AL) and developed using Luminata^™ Crescendo (Merck Millipore, Burlington, MA). The images were captured using a FUSION SYSTEM FX7 (VILBER LOURMAT, Marne-la-Vallée Cedex 3, France).

Total RNA was prepared using the RNAzol^®RT Reagent (Molecular Research Center, Inc., Cincinnati, OH, USA) and then transcribed into cDNA with a PrimeScript^™ RT reagent kit (Perfect Real Time) (TaKaRa, Shiga, Japan), 1 µg of total RNA. TaqMan^® probes for AFP (Hs01040598_m1), ALB (Hs00609411_m1), NANOG (Hs02387400_g1), and POU5F1 (Hs04260367_gH) were obtained from Thermo Fisher Scientific (Waltham, MA), and qPCR was performed using the StepOnePlus real-time PCR system (Thermo Fisher Scientific, Waltham, MA). The data were analyzed using the ΔΔCq method and expressed as relative quantities (RQ) according to the manufacturer's instructions.

iPSCs were plated in Stem Fit AK03N (Ajinomoto, Tokyo, Japan) containing 0.25% iMatrix 511 silk (TaKaRa, Shiga, Japan) and 10 µM Y-27632 (FUJIFILM Wako Pure Chemical Corp., Osaka, Japan) in a 12-well plate. as described above. The following day, the medium was replaced with Stem Fit AK03N (Ajinomoto, Tokyo, Japan) containing gefitinib (0, 3, 6, and 10 µM). The medium was collected on days 0, 1, and 2. The medium was not replaced during gefitinib treatment. For iPSC-heps, gefitinib (0, 3, 6, and 10 µM) was added on day 2 of STAGE 4, and the conditioned medium and iPSCs were collected. LDH was measured using the LDH-Glo^™ Cytotoxicity Assay combined with the GloMax Multi/Luminescence System (Promega, Madison, WI, USA) according to the manufacturer's instructions.

All analyses were performed using ‘MEPHAS’ (http://www.gen-info.osaka-u.ac.jp/testdocs/tomocom/). One-way ANOVA with Dunnett's test or Tukey's test, and Student's-t test were employed for statistical analyses. P<0.05 was considered to indicate a statistically significant difference.

To prevent dose reduction or cessation of gefitinib treatment caused by hepatotoxicity, it is important to establish a cell-based assay system that can predict gefitinib-induced toxicity before clinical use. We hypothesized that iPSC-heps may predict gefitinib-induced hepatotoxicity in vitro and thus generated iPSCs from two groups of gefitinib-treated patients, those with grade 3 or greater hepatotoxicity (T group), and those who had grade 1 or less hepatotoxicity (N group) in the CTCAE v5.0. iPSCs were generated from 6 patients, 3 each from T and N groups. The patient characteristics are displayed in Table SI. We established 3 clones from a patient and got 18 clones in total. To confirm the pluripotency, we examined the mRNA expression levels of POU5F1 and NANOG, stem cell markers, showing that the clones were in a pluripotent state (Fig. 1A). In addition, the other stem cell markers, alkaline phosphatase (ALP) and SOX2, were highly expressed in the clones, whereas they were barely detected in the differentiated cells, HaCaT cells, in western blot analyses (Fig. S1). Since all clones were in a pluripotent state, we randomly chose 1 clone from each of the patients (6 clones), and was subjected to further analysis including the cyto/hepatotoxicity assay. We then differentiated iPSCs into iPSC-heps. A qPCR analysis showed that the expression levels of AFP and ALB, hepatocyte-specific markers, in iPSC-heps were much higher than those in iPSCs (Fig. 1B). In contrast, NANOG and POU5F1 were not expressed in iPSC-heps (Fig. 1A). A western blot analysis also showed that HNF4α, ALBUMIN (ALB), and AFP, hepatocyte-specific markers, were comparably expressed in iPSC-heps (Fig. 1C). These data suggest that iPSCs had differentiated into cells in a hepatocyte lineage. The four-step protocol we used for differentiation of iPSCs into iPSC-heps was depicted in Fig. 2. Collectively, we concluded that the iPSC clones were successfully differentiated into the iPSC-heps.

To evaluate the hepatotoxicity of gefitinib, we employed a chemiluminescence-based LDH release assay using iPSC-heps. iPSCs (N-1, N-3, T-1, and T-3) were simultaneously differentiated into iPSC-heps. Gefitinib (0, 3, 6, and 10 µM) was administered on day 2 of STAGE 4 (Fig. 2). We confirmed that AFP and ALB, hepatocyte-specific markers, were already expressed at the beginning of STAGE 4 (data not shown). As hepatic maturation proceeded, evidence of naturally dying cells appeared. To differentiate natural cell death from gefitinib-related cytotoxicity as much as possible, day 2 of STAGE 4 was selected as the starting point. Each iPSC-heps were treated with 0, 2, 6, and 10 µM gefitinib, and conditioned medium was collected on days 0, 1, and 2 after administration. However, we could not find dose- or group (N and T)-dependency (Fig. 1D-G). These data suggest that our experimental design may need to be improved to be a good platform for gefitinib-induced hepatotoxicity assays.

Since gefitinib is an EGFR-TKI, we examined the expression level of EGFR and the phosphorylation following gefitinib treatment in the clones of iPSCs and iPSC-heps from the N or T group. Western blot analyses showed that they were expressed similarly among the clones of iPSCs (Fig. 3A and C) and iPSC-heps (Fig. 3B and D) from each group, irrespective of whether those were from the N or T group. EGFR phosphorylation status (the ratio of pEGFR to EGFR) were also similar among the clones of iPSCs from the two groups following gefitinib treatment (Fig. 3E and F). Although treatment significantly suppressed EGFR phosphorylation in a dose-dependent manner, we did not find any differences in phosphorylation status between the two groups following gefitinib treatment. Fig. 3E and F shows that gefitinib treatment inhibited the EGFR signaling pathway equally in each iPSCs. Taken together, an LDH-release assay using iPSCs would potentially be a good predictor of gefitinib-induced hepatotoxicity.

While we evaluated the gefitinib-induced hepatotoxicity with the iPSC-heps, we used iPSCs as a control in the assays. We found that iPSCs in T group released LDH more than those in N group after gefitinib treatment, and the increase was dose dependent (Fig. 4A-F). Because the difference between the groups at day 1 after gefitinib treatment seemed to be the largest among the time points analyzed, we focused on the toxicity at day 1. T-2 was significantly more sensitive to gefitinib than N-1 after 6 µM gefitinib treatment (Fig. 4H). There were no significant differences between the T and N groups in any other concentrations or combinations except that T-2 was significantly more sensitive to gefitinib than N-1 after 6 µM gefitinib treatment (Fig. 4G-I). However, iPSCs in T group tended to have the cytotoxicity more than those in N group. Therefore, we combined the data of each group and compared the 2 groups (Fig. 4J-L). The result showed that iPSCs in T group had higher cytotoxicity than those in N group after 6 or 10 µM gefitinib treatment (Fig. 4K and L), whereas there were no significant differences between N and T groups after 3 µM treatment (Fig. 4J). These results were consistent with the data of the cell viability assay (Fig. S2) and the cell morphology (Fig. S3). The cell viability in T group at day 2 after gefitinib treatment was significantly aggravated, except for T-2 and T-3 at 3 µM and T-2 at 6 µM, whereas no significant damages were observed in N group, except for N-1 and N-3 at 10 µM (Fig. S2). In the cell morphology at day 2 after gefitinib treatment, gefitinib decreased the number and size of attached iPSCs in the T group in a dose-dependent manner, whereas no significant damages were observed in N group, except for N-2 that seemed to be slightly damaged at 10 µM treatment (Fig. S3).