Enrichment of high-functioning human iPS cell-derived hepatocyte-like cells for pharmaceutical research
Introduction
Human induced pluripotent stem (iPS) cell-derived hepatocyte-like cells are expected to be utilized in pharmaceutical development, such as drug screening and drug metabolism testing. Many research groups, including ours, have been engaged in developing an efficient hepatocyte differentiation protocol from human iPS cells [[1], [2], [3], [4], [5]]. Hepatocyte-like cells have the capacity to secrete albumin (ALB), urea, and bile acid and also have drug metabolism capacity. However, several issues must be overcome before hepatocyte-like cells can be used in pharmaceutical applications. It has been reported that the hepatocyte-like cells are similar to fetal hepatocytes rather than adult hepatocytes [6,7]. In addition, although almost all of the hepatocyte-like cells are positive for early hepatic markers (such as ALB), some of the hepatocyte-like cells are negative for late hepatic markers (such as cytochrome P450 (CYP)) [8]. These facts suggest that it is still difficult to generate mature hepatocyte-like cells at high purity.
To obtain mature hepatocyte-like cells at high purity, some groups have sorted mature hepatocyte-like cells by using hepatocyte-specific cell surface markers [8,9]. Basma et al. have reported that mature hepatocyte-like cells could be concentrated by sorting asialoglycoprotein receptor 1 (ASGR1)-positive cells [9]. Mallanna et al. have also reported that mature hepatocyte-like cells could be concentrated by sorting sodium taurocholate cotransporting polypeptide (NTCP)-positive cells [8]. However, although cell suspensions of mature hepatocyte-like cells can be obtained by cell sorting, it is difficult to replace the sorted mature hepatocyte-like cells, because hepatocyte-like cells easily lose their hepatic functions during the cell reattachment process. In the drug screening and CYP induction experiments, attached hepatocytes, not suspended hepatocytes, are highly required. Thus, the cell sorting technology might not be suitable for preparing attached mature hepatocyte-like cells at high purity. Therefore, we decided to purify mature hepatocyte-like cells by drug-selection.
In this study, we attempted to establish a CYP3A4-neomycin resistance gene (NeoR)-enhanced green fluorescent protein (EGFP) transgenic reporter human iPS cell line (CYP3A4-NeoR-EGFP iPS cells) by using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 (Cas9) technology. Although it is known that the genome editing efficiency of human ES/iPS cell clones is quite low at the heterochromatin locus [10,11], we have recently found that RAD51 recombinase (RAD51) overexpression and valproic acid treatment enhanced genome targeting efficiency in human ES/iPS cells regardless of the transcriptional activity of the targeted locus [12]. Therefore, we targeted the CYP3A4 locus by using our genome editing technology. After the hepatocyte differentiation, we tried to concentrate the hepatocyte-like cells that strongly expressed CYP3A4 by treating the CYP3A4-NeoR-EGFP iPS cell-derived hepatocyte-like cells with neomycin. In addition, we examined the pharmaceutical value of CYP3A4-positive high-functioning hepatocyte-like cells.
Section snippets
Human iPS cells
The human iPS cell line, Tic [13,14] (provided by Dr. Akihiro Umezawa at the National Center for Child Health and Development), was maintained on 1 μg/cm2 recombinant human laminin 511 E8 fragments (iMatrix-511, Nippi) with StemFit AK02N medium (Ajinomoto) [15]. Human iPS cell lines, YOW-iPS cells and FCL-iPS cells [16], generated from primary human hepatocytes was maintained on 1 μg/cm2 iMatrix-511 with StemFit AK02N medium. To passage human iPS cells, near-confluent human iPS cell colonies
Generation of CYP3A4-NeoR-EGFP iPS cells
In this study, we attempted to generate high-purity and high-functioning human iPS cell-derived hepatocyte-like cells for pharmaceutical research. It is known that CYP3A4 is the representative drug metabolizing enzyme in the liver [18]. Therefore, we integrated a NeoR-EGFP cassette into the CYP3A4 locus to concentrate CYP3A4-expressing hepatocyte-like cells. We tried to establish a CYP3A4-NeoR-EGFP transgenic reporter human iPS cell line (CYP3A4-NeoR-EGFP iPS cells) by using a CRISPR/Cas9
Discussion
In this study, we succeeded in obtaining human iPS cell-derived hepatocyte-like cells that strongly express CYP3A4 at high purity. By using our cells and technology, high-functioning hepatocyte-like cells could be purified only by drug-selection, without the need for cell sorting. Because expensive materials or complicated procedures are not required in our protocol, the high-functioning hepatocyte-like cells generated by this study might be suitable cell source for drug screening. In addition,
Conclusions
In this study, we succeeded in obtaining human iPS cell-derived hepatocyte-like cells that highly express CYP3A4 at high purity. We believe that the neomycin-treated CYP3A4-NeoR-EGFP-iPS cell-derived hepatocyte-like cells could be utilized in various aspects of pharmaceutical research, including drug screening and pharmacokinetic research.
Conflicts of interests
K. Sekiguchi owns stock in Matrixome, Inc. Other authors have declared that no conflict of interest exists.
Acknowledgements
We thank Ms. Ayaka Sakamoto and Ms. Natsumi Mimura for her excellent technical support. We also thank Dr. Kazuo Harada (Osaka University) for his excellent technical support in LC-MS/MS analysis.
This research was supported by Japan Society for the Promotion of Science (JSPS) KAKENHI Grant Number 10759509, and the Keihanshin Consortium for Fostering the Next Generation of Global Leaders in Research (K-CONNEX), established by Human Resource Development Program for Science and Technology, MEXT.
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