Hepatocytes with a phenotype of substantial CYP3A4 induction generated from drug-associated fulminant hepatitis-derived induced pluripotent stem cells

Center for Regenerative Medicine, National Center for Child Health and Development Research Institute, Tokyo, 157-8535, Japan Graduate school of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, 113-0034, Japan Research team for Geriatric Medicine (Vascular Medicine), Tokyo Metropolitan Institute of Gerontology, Tokyo, 173-0015, Japan Department of Applied Biological Science, Tokyo University of Science, Tokyo, 162-8601, Japan Department of Developmental and Regenerative Biology, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, 113-0034, Japan Department of BioSciences, Kitasato University School of Science, Kanagawa, 252-0373, Japan Organ Transplantation Center, National Center for Child Health and Development, Tokyo, 157-8535, Japan


INTRODUCTION
Preclinical testing of low molecular weight drugs for hepatotoxicity has traditionally been carried out in animal models such as rats and mice (Food and Drug Administration, 2012).
Primary human hepatocytes can also be used for such pharmaceutical testing, but their drawbacks in drug screening include limited supply of cell lots and large variations between the lots due to genetic and environmental backgrounds. To solve the problem of lot variation, HepG2 cells have also been used to examine hepatotoxicity, because of their clonal nature.
HepaRG, another hepatocyte-like clonal line established from a hepatoblastoma has the advantage of high inducibility of cytochrome p450 genes. We hypothesized that the technology to create induced pluripotent stem cells (iPSCs) would enable us to obtain hepatocytes from patients with drug-induced liver injury and establish a new and superior cell type for preclinical testing.
iPSCs have impacted numerous medical fields including clinical therapy development, drug discovery, research on inherited diseases and studies on reprogramming of differentiated cells (De Assuncao et al., 2015;Hankowski et al., 2011;Santostefano et al., 2015;Takahashi and Yamanaka, 2006). Human iPSCs also prove valuable for toxicology testing. For example, iPSC-derived hepatocytes have been shown to serve as an in vitro tool in drug metabolism and toxicology (Gripon et al., 2002;Takayama et al., 2012). iPSC-derived hepatocytes or hepatocyte-like cells can be obtained from the same origin repeatedly due to immortality of iPSCs (Holmgren et al., 2014;Lu et al., 2015;Sirenko et al., 2014). Although it is expected that hepatocytes differentiated from iPSCs can be utilized in drug toxicity testing, the actual applicability of iPSC-derived hepatocytes in this context has not yet been thoroughly examined. In this study, we generated iPSCs from a pediatric patient with hepatic failure, presumably drug-induced fulminant hepatitis, and investigated in vitro hepatotoxicity of various drugs. To generate mature hepatocytes that would be applicable for drug toxicity testing, we manufactured HepaKI from drug-induced hepatitis-derived iPSCs by improvement and optimization of a previously used differentiation method. HepaKI induced cyp3A4 57.2-fold upon exposure to rifampicin, suggesting that HepaKI can serve as a useful source for in vitro hepatic toxicology testing.
To investigate multipotency in vitro, iPSCs were differentiated into ectodermal, mesodermal, and endodermal lineages. The differentiation of iPSC-K was confirmed by immunostaining using antibodies against TUJ1, α -SMA, and AFP as ectodermal, mesodermal, and endodermal markers, respectively ( Figure 1G, H). To address whether the iPSC-K have the competence to differentiate into specific tissues in vivo, teratomas were formed by implantation of iPSC-K in the subcutaneous tissue of immunodeficient NOD/SCID mice.
iPSC-K produced teratomas within 6-10 weeks after implantation. Histological analysis of paraffin-embedded sections demonstrated that the three primary germ layers were generated as shown by the presence of ectodermal, mesodermal, and endodermal tissues in the teratoma ( Figure 1I), implying iPSC-K has potential for multilineage differentiation in vitro and in vivo.
Among iPSC-K clones, #25, #66, and #100 generated larger area of liver-like tissues in the teratomas, while the other clones did not.

Hepatic differentiation
We investigated the efficiency of hepatic differentiation of iPSC-K by two different methods Protocol H and Protocol S (described in detail in Figure 2A, B, C). iPSC-K exhibited hepatocyte-like morphology, i.e. a polygonal and/or cuboidal shape that had tight cell-cell contact when generated by either method ( Figure 2D, E) and the expression of the hepatocyte-associated genes were comparable. We thus employed the Saeko method hereafter for the iPSC-K experiments. Quantitative analysis revealed that iPSC-K expressed the genes for AFP, ALB and AAT 21 days after the start of induction ( Figure 2F, G, H).
Among the iPSC-K clones, iPSC-K#25 exhibited morphology that most resembled primary hepatocytes, and high expression of hepatocyte-associated genes, and was therefore used for subsequent experiments. Time-course analysis revealed that similar expression levels of liver-associated genes were observed at 21, 28 and 35 days after the start of hepatic induction ( Figure 2I, J).

Verification of the manufacturing process
To ensure the consistent production of hepatocyte-like cells from iPSC-K, we established a master cell bank and used a working cell bank (WCB) for starting material. We then developed a standard operating procedure for hepatic differentiation ( Figure 3A). HepaKI developed into a cell type that was positive for both AFP and ALB ( Figure 3B). Karyotypic analysis showed that the same chromosomes, without any aberration, were present in the WCB stock as parental fibroblastic cells from the patient ( Figure 3C, D). Exome analysis revealed that iPSC-K had no significant single nucleotide alterations in a homozygous manner. To verify the procedure, we repeatedly manufactured and characterized hepatocytelike cells from the WCB. Immunocytochemistry confirmed that these hepatocyte-like cells were positive for CK7, CK8/18 (AE1/3) and Hep1, but negative for CD31 and CD34 ( Figure   3E).

Induction of the genes for cyp1A2, 2B6, and 3A4
To investigate whether HepaKI exhibits CYP induction, we exposed HepaKI to omeprazole, phenobarbital, and rifampicin for 24, 48, and 48 h, respectively ( Figure 4). Expression of the genes for AFP and ALB was unchanged with exposure to these drugs. Cyp1A2 was increased after exposure to omeprazole and phenobarbital, while Cyp2B6 remained unchanged ( Figure   4C, D). Interestingly, Cyp3A4 was up-regulated 57.2-fold, on average, upon exposure to rifampicin ( Figure 4E).

DISCUSSION
In this study, we succeeded in promoting hepatocytic differentiation from fulminant hepatitis iPSCs. We also were able to reduce the culture period to less than 30 days through EB formation, and extended the window for hepatic differentiation to more than 30 days. The reduction of the culture time and the increase in the window for differentiation could decrease manufacturing costs, which is an important requirement for efficient generation of HepaKI.
Moreover, we found that HepaKI showed marked drug-mediated CYP3A4 induction, much higher than previously reported hepatocyte-related cells such as HepG2, HepaRG and pluripotent stem cell-derived hepatocytes.

Advantages and limitations of HepaKI
Because of its extremely high CYP3A4 induction, HepaKI is a superior cell system for toxicology testing. However, further maturation into hepatocytes is required because their hepatic characteristics, such as drug metabolism capacity, are lower than those of primary hepatocytes; the CYP2B6 and CYP3A4 genes are expressed at about one-tenth of the level seen in HepaKI. The major advantage of the manufacturing process of HepaKI is the long window of time available forcytochrome p450 induction tests. After approximately 30 days of differentiation, HepaKI cells remained stable as measured by hepatotoxicity assays, suggesting that HepaKI provides a better cost/performance balance.

Modeling of drug-mediated CYP3A4 induction by HepaKI
Because CYP3A4 contributes to the first-pass metabolism of many commercial drugs, it is important to investigate the CYP3A4-mediated hepatic metabolism in order to estimate hepatotoxicity. It is known that CYP3A4 expression in hepatocytes can be induced by various drugs, such as dexamethasone, PB, RIF, and 1α,25-dihydroxyvitamin D3. The induction of CYP3A4 expression in hepatocytes by such drugs might affect the pharmacokinetics of concomitant drugs administered orally. Therefore, a HepaKI model that could evaluate drugmediated CYP3A4 induction in hepatocytes would be useful for drug discovery.
The mechanism by which the high induction of cyp3A4 (57.2-fold, on average) is achieved in HepaKI is unknown. The most likely explanation is the cell origin; iPSC-K was generated from a patient with drug-induced hepatitis, and the patient may have undergone a specific genetic alteration/mutation causing the high induction. Exome analysis revealed that HepaKI had a number of single-nucleotide alterations, both homozygous and heterozygous, but we failed to find significant null alterations related to drug-induced hepatic injury or high CYP3A4 induction. In addition, continuous generation of iPSCs from other patients with drug-induced fulminant hepatitis is necessary to produce a variety of cell sources with different genetic backgrounds for in vitro CYP3A4 induction testing and in vitro hepatotoxicity tests.

Ethical statement
Human cells in this study were obtained in full compliance with the Ethical Guidelines for IBL CO., Ltd, Gunma, Japan), and re-plated at a density of 5 x 10 5 cells in a 100-mm dish.
Medium changes were carried out twice a week thereafter.
Elimination of Sendai virus was confirmed by RT-PCR. Cells just after infection served as a positive control. Sequences of the primer sets for the Sendai virus are shown in Table 1.

Immunocytochemical analysis
Cells were fixed with 4% paraformaldehyde in PBS for 10 min at 4°C. After washing with PBS and treatment with 0.2% Triton X-100 in PBS for 10 min, cells were pre-incubated with blocking buffer (10% goat serum in PBS) for 30 min at room temperature, and then exposed to primary antibodies in blocking buffer overnight at 4°C. Following washing with 0.2% PBST, cells were incubated with secondary antibodies; either anti-rabbit or anti-mouse IgG conjugated with Alexa 488 or 546 (1:300) (Invitrogen) in blocking buffer for 30 min at room temperature. Then, the cells were counterstained with DAPI and mounted.

Karyotypic analysis
Karyotypic analysis was contracted out to Nihon Gene Research Laboratories Inc. (Sendai, Japan). Metaphase spreads were prepared from cells treated with 100 ng/mL of Colcemid

Statistical analysis
Statistical analysis was performed using the unpaired two-tailed Student's t test.