Transgenic HepaRG cells expressing CYP2D6 as an improved model of primary human hepatocytes

Abstract CYP2D6 and CYP3A4, which are members of the cytochrome P450 superfamily of metabolic enzymes, play major roles in the metabolism of commonly available drugs. CYP3A4 is involved in the metabolism of 50% of drugs on the market, whereas CYP2D6 is involved in the metabolism of 25% of them. CYP2D6 exhibits a high degree of polymorphic nature in the human population, causing individual differences in CYP2D6 expression and enzymatic activity. Therefore, accurate prediction of drug metabolism and toxicity require a human adult hepatocyte cell model that mimics individual responses in the average population. HepaRG cells, a human hepatocellular carcinoma cell line, is the only cell line that can differentiate into hepatocyte‐like cells with high expression of CYP3A4 but poor expression of CYP2D6. To solve this problem, we developed transgenic HepaRG cell clones expressing either full‐length or spliced CYP2D6 at various levels with an easy monitoring system for CYP2D6 expression in living cells under a fluorescent microscope. As CYP2D6 mRNA, protein, and fluorescence intensity were closely correlated among transgenic HepaRG clones, fluorescence levels will provide a simple tool for quality assurance of CYP2D6‐expressing HepaRG cells. Thus, the package of transgenic HepaRG cell clones expressing CYP2D6 at various levels will provide an improved hepatocyte model that reflects the average or individual reactions in the human population for in vitro studies of drug metabolism and toxicity involving CYP2D6 and CYP3A4.

gree of polymorphic nature in the human population, causing individual differences in CYP2D6 expression and enzymatic activity. Therefore, accurate prediction of drug metabolism and toxicity require a human adult hepatocyte cell model that mimics individual responses in the average population. HepaRG cells, a human hepatocellular carcinoma cell line, is the only cell line that can differentiate into hepatocyte-like cells with high expression of CYP3A4 but poor expression of CYP2D6. To solve this problem, we developed transgenic HepaRG cell clones expressing either full-length or spliced CYP2D6 at various levels with an easy monitoring system for CYP2D6 expression in living cells under a fluorescent microscope. As CYP2D6 mRNA, protein, and fluorescence intensity were closely correlated among transgenic HepaRG clones, fluorescence levels will provide a simple tool for quality assurance of CYP2D6expressing HepaRG cells. Thus, the package of transgenic HepaRG cell clones expressing CYP2D6 at various levels will provide an improved hepatocyte model that reflects the average or individual reactions in the human population for in vitro studies of drug metabolism and toxicity involving CYP2D6 and CYP3A4.

K E Y W O R D S
CYP2D6, CYP3A4, HepaRG, live-cell imaging, polymorphism, transgenics development. In contrast, CYP2D6 is rarely transcriptional inducible, but it is polymorphic (showing mutations, copy number variation, and alternative splicing), resulting in individual differences in response to drugs. 1 Therefore, there is a need for model cells that mimic primary human adult hepatocytes, reflecting real human metabolism and the polymorphic nature in the human population.
HepaRG cells are widely used as model cells for adult human hepatocytes because HepaRG cells are the only cultured cells that can be differentiated into adult human hepatocyte-like cells (HLCs) highly expressing CYP3A4 to the levels of normal adult human hepatocytes. 2 However, CYP2D6 mRNA expression is limited in HepaRG cells to 1/35 to 50 of that in adult human hepatocytes, which constrains the use of HepaRG as model hepatocyte cells. 3 Therefore, in this study, we developed transgenic HepaRG cells to act as HLCs that co-express CYP3A4 and CYP2D6. We also tried to introduce the polymorphic nature of CYP2D6 in the transgenic HepaRG cells.
CYP2D6 metabolizes analgesics, antidepressants, antihypertensives, and anticancer drugs. In particular, the anticancer agent tamoxifen has therapeutic effects as a prodrug, and the metabolites of CYP2D6, 4-OH-tamoxifen and endoxifen, are highly effective in breast cancer treatment. 4 Differences in the metabolic activity of CYP2D6 can seriously affect therapeutic efficiency and prognosis. 5 CYP2D6 shows differences in protein structure due to selective splicing of exon 3 or exon 6. 6 However, it remains unclear whether these splicing variants affect the metabolic function of CYP2D6.
In this study, therefore, we generated not only HepaRG cell clones expressing various levels of CY2D6, but also HepaRG cells expressing the full-length or splicing variant of CYP2D6 to provide a package of HLCs that reflect both individual and population differences. In these living cells, the expression level of CYP2D6 can be easily monitored under conventional fluorescence microscopy.

| RNA extraction from cryopreserved primary human hepatocytes
Total RNA was extracted from human cryopreserved hepatocytes

Promega. The HaloTag ORF Clone FHC02139 ubiquitously expresses
HaloTag-CYP2D6 under the CMV promoter. By adding a fluorescent ligand that specifically and covalently binds HaloTag to the culture medium, 7 CYP2D6 protein in living cells was visualized by adding TMR ligands (Promega) into cell culture medium for 15 min followed by extensive washing.

Wild-type (WT) HepaRG cells were purchased from Biopredic
International. The cell seeding density and differentiation conditions have been described in detail previously 8 and SI METHODS.

| Fluorescence image analysis and PCR analysis
Fluorescence microscopic images were captured using a BZ-9000 fluorescence microscope (Keyence) and an A1 confocal microscope (Nikon). Reagents, software, and methods used in this study have

The CYP2D6-iGFP expression vector allows ubiquitous expression
of CYP2D6-IRES-GFP mRNA under the CMV promoter and encodes two independent proteins, CYP2D6 and GFP, in relatively comparable amounts. Therefore, we planned to use it as a live-cell monitoring system to predict the amount of CYP2D6 by green fluorescence intensity.
In contrast, we planned to use the HaloTag-CYP2D6 expression vector for quality assurance of the amount of CYP2D6 protein in the cell population just prior to its use in metabolic studies ( Figure 1B). We isolated many transgenic HepaRG clones for each expression vector with different expression levels of CYP2D6 variants ( Figure 1C), as shown in Table 1. These transgenic clones generated HLCs similar to the origi-

nal WT HepaRG cells and continuously expressed green fluorescence
and HaloTag-CYP2D6 before and after cell differentiation ( Figure 1D).

HaloTag-CYP2D6 accumulated in endoplasmic reticulum (ER)-rich
perinuclear regions ( Figure 1E), as CYP2D6 protein is an integral membrane protein and localizes to the ER. 10 HepaRG clones of CYP2D6iGFP showed various levels of green fluorescence, of which three clones, classified as higher, intermediate, and lower according to their expression levels, were shown in Figure 2A.  Figure 2C. RNA expression levels varied in transgenic HepaRG cell clones and were lower than in adult human liver but much higher than in WT HepaRG cells ( Figure 2D).
Clonal differences in red fluorescent intensity were consistent with differences in CYP2D6 mRNA expression levels, with one exception ( Figure 2E).

| DISCUSS IONS
HepaRG is the only cell line that can provide an unlimited number of HLCs expressing high levels of CYP3A4. This is our second report of the creation of transgenic HepaRG cells. Previously, we generated transgenic HepaRG cells that can monitor CYP3A7-expressing hepatoblast-like cells and CYP3A4-expressing HLCs with red and green fluorescence intensities, respectively. 11,12 In those HLCs, transcriptional induction of CYP3A4 can be detected as an increase in green fluorescence levels. In this study, we generated many HepaRG clones expressing various levels of CYP2D6. We found an approximate correlation between RNA expression and GFP fluorescence intensity in the CYP2D6-iGFP expressing HepaRG clones. In the future, we plan to measure the metabolic activity of

(E)
Relative levels (C1=100) S1 L30 L 22 L35 L37 enhanced hepatocyte model cells that reflect individual differences in CYP2D6 expression. The widespread use of these cells will help solve many current issues in drug development and toxicity testing.

ACK N OWLED G EM ENTS
We sincerely thank Dr C. Guguen-Guillouzo, Dr A. Jamin, and Dr C. Chesne (Biopredic International, France) for expert advice on HepaRG cells and primary human hepatocytes.

D I SCLOS U R E
The authors declare that there is no conflict of interest, no ethical issue, and no use of clinical materials and materials from other sources.

AUTH O R CO NTR I B UTI O N S
MT: research design, corresponding author, final proofreading before submission, collected data, and contributing to data analysis.

E TH I C S S TATEM ENT
The authors declare that this study was performed in accordance with the research policy of Toho University.

This article has earned Open Data, Open Materials and Preregistered
Research Design badges. Data, materials and the preregistered design and analysis plan are available in the article.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data discussed in this publication are included in this manuscript.
Further information and requests for data and reagents should be requested to the corresponding author, Masako Tada.