Stem Cells International
Volume 2019 (2019), Article ID 5129526, 12 pages
https://doi.org/10.1155/2019/5129526

Review Article

Potential for Isolation of Immortalized Hepatocyte Cell Lines by Liver-Directed In Vivo Gene Delivery of Transposons in Mice

Masahiro Sato,¹ Issei Saitoh,² Emi Inada,³ Shingo Nakamura,⁴ and Satoshi Watanabe⁵

¹Section of Gene Expression Regulation, Frontier Science Research Center, Kagoshima University, Kagoshima 890-8544, Japan
²Division of Pediatric Dentistry, Graduate School of Medical and Dental Science, Niigata University, Niigata 951-8514, Japan
³Department of Pediatric Dentistry, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima 890-8544, Japan
⁴Division of Biomedical Engineering, National Defense Medical College Research Institute, Saitama 359-8513, Japan
⁵Animal Genome Unit, Institute of Livestock and Grassland Science, National Agriculture and Food Research Organization (NARO), Tsukuba, Ibaraki 305-0901, Japan

Correspondence should be addressed to Masahiro Sato; masasato@m.kufm.kagoshima-u.ac.jp

Received 9 March 2019; Accepted 6 May 2019; Published 2 June 2019

Guest Editor: Luis E. Gomez-Quiroz

Copyright © 2019 Masahiro Sato et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract.

Isolation of hepatocytes and their culture in vitro represent important avenues to explore the function of such cells. However, these studies are often difficult to perform because of the inability of hepatocytes to proliferate in vitro. Immortalization of isolated hepatocytes is thus an important step toward continuous in vitro culture. For cellular immortalization, integration of relevant genes into the host chromosomes is a prerequisite. Transposons, which are mobile genetic elements, are known to facilitate integration of genes of interest (GOI) into chromosomes in vitro and in vivo. Here, we proposed that a combination of transposon- and liver-directed introduction of nucleic acids may confer acquisition of unlimited cellular proliferative potential on hepatocytes, enabling the possible isolation of immortalized hepatocyte cell lines, which has often failed using more traditional immortalization methods.

1. Introduction

It is well-known that primary cells can only undergo a limited number of cell divisions in culture, although the number varies according to species, cell type, and culture conditions [1]. Cells at a state where they can no longer divide are referred to as being in “replicative senescence,” which is characterized by changes in cellular morphology, as exemplified by enlarged cell size and formation of multiple nuclei [2, 3]. Replicative senescence is also associated with the activation of tumor suppressor genes, such as p53, retinoblastoma (RB), and p16. However, this replicative senescence can be overcome by overexpression of viral genes such as simian virus 40 large T antigen (SV40T) [4] or telomerase reverse transcriptase protein (TERT), the latter being responsible for elongation of telomeres [5, 6], through in vitro transfection of primary cells. It is known that expression of SV40T causes inactivation of p53 [7]. As a result, cells acquiring continuous cell division capacity can retain many of the original and relevant characteristics of the source tissue. These types of cells are generally called “immortalized cells.”

Immortalized cells can also be acquired from transgenic (Tg) mice that harbor a temperature-sensitive SV40T gene called tsA58 [8, 9]. Mutant SV40T is inactive at 39°C; therefore, the gene functions normally in Tg mice. However, at 33°C, which is below the normal physiological temperature, the gene becomes active. Therefore, when researchers dissect cells from the target organs/tissues of this Tg line for in vitro primary culture, these primary cells must always be cultured under low-temperature conditions to ensure that the temperature-sensitive SV40T gene is activated, allowing unlimited cell proliferation. According to Obinata’s review [10], numerous different cell lines have been established using this Tg system, including bone marrow stromal cell lines (TBR series), a stromal cell-dependent hematopoietic stem-like cell line (THS-119), a dendritic cell line (SVDC), a hepatocyte cell line (TLR), a Leydig cell line (TTE1), and a Sertoli cell line (TTE3). One of the drawbacks associated with this system is the initial establishment of the Tg line, which requires considerable time and cost. Additionally, if they are subjected to in-house breeding, animal maintenance and genotyping are required, which are laborious and expensive. Similarly, fibroblasts derived from p53 knockout (KO) mice proliferate continuously without showing aging or crisis. However, cardiac muscle cells or hepatocytes isolated from such p53 KO mice fail to proliferate indefinitely [11].

Generally, acquiring hepatocyte cell lines from normal liver has been considered difficult, since cells tend to lose hepatocyte-specific functions soon after in vitro cultivation [12, 13]. Only HepaRG cells, derived from liver tumors, are known to retain hepatic functions with respect to having the ability to produce albumin and being susceptible to infection by hepatitis B virus (HBV) [14]. In earlier stages of attempts to acquire immortalized hepatocytes, many researchers employed viral infection approaches involving primary cultured hepatic cells obtained soon after isolation from liver tissue after perfusion with collagenase (Figure 1(a)). These viruses include adenovirus and SV40 virus containing oncogenic factors such as E1A/E1B (adenovirus) and transforming genes (SV40) [15–17]. Plasmid vectors containing an expression cassette for the expression of oncogenic factors (such as SV40T) have also been used for immortalization of hepatic cells [18]. In this case, electroporation- (EP-) [19] or gene delivery-related reagent-based transfection such as calcium phosphate [20–23] and liposomes [24–27] has been employed, as shown in Figure 1(a). In terms of oncogenic factors involved, DNA coding for SV40T is most frequently used. E6 and E7 genes from human papilloma virus (HPV) have also been employed for hepatocyte immortalization. The E6 gene, derived from HPV16, has the ability to promote the degradation of p53 cell cycle-regulating proteins similar to SV40T [28]. On the other hand, E7 induces the degradation of the retinoblastoma protein RB, another type of cell cycle regulator [29]. Moreover, human-derived TERT (hTERT) gene has also been used for inducing immortalized hepatocytes [30]. In addition to the function of hTERT to maintain telomere length, it is reported to bind to transcription factors such as p65 or β-catenin and to regulate gene expression related to tumorigenesis [31].

Figure 1: Methods for establishing hepatocyte cell lines using conventional approaches (a), HGD-based gene delivery (b) and EP-based gene delivery (c). In (a), the liver is first perfused with collagenase to isolate single hepatocytes, to which in vitro gene delivery using EP, liposomes, or virus is applied. In (b), HGD is performed with transposon vectors, and 2 days later, perfusion with collagenase is performed to isolate single hepatocytes. In (c), transposon vectors are directly introduced into the parenchyma of the livers of anesthetized mice, and then, the injected portion is immediately subjected to in vivo EP using tweezer-type electrodes and a square-pulse generator. The treated mice are kept for 7 days prior to collagenase perfusion. These resulting collagenase-dissociated hepatocytes cells are then cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 20% fetal bovine serum (FBS), 1 μg/mL of insulin, and 4 μg/mL dexamethasone on a collagen-coated dish (d). One day after hepatocyte isolation, puromycin (0.3 μg/mL) is added to the medium to eliminate untransfected hepatocytes and then kept for 7-10 days for generation of viable colonies.

At later stages of acquisition of immortalized hepatocytes, retroviral [32–44] and lentiviral [45–48] vectors are frequently used. These approaches are completely different from those reported earlier. For example, retroviral vectors can insert a single copy of a transgene into a cellular chromosome only at the periods when cells exhibit active cell division [49]. On the other hand, lentiviral vectors are active independent of cell cycle [50]. Since these vectors have high efficiency in terms of gene transfer and low cytotoxicity, they are thought to be suitable for gene transfer into cells at the interfuse stage after terminal differentiation [51]. However, construction of these vectors strictly requires cells dedicated to packaging transgenes into virus particles. Moreover, experimental equipment for containing viral particles is needed for prevention of possible contamination. These procedures are laborious and time-consuming. In contrast, gene transfer of nonviral DNA using chemical reagents or EP is much more simplified and cost-effective, although gene transfer efficiency appears to be lower than that involving viruses. Furthermore, the frequency of chromosomal integration of transgenes appears to be lower compared to viral systems. Therefore, employment of new gene delivery systems enabling effective chromosomal integration of genes of interest (GOI) into hepatocytes is required.

More importantly, many of the immortalized hepatocyte lines established by the above-mentioned technologies appear to lose hepatocyte-specific functions, as exemplified by reduced production of albumin, urea, and cytochromes, compared to the living liver. Almost all of these cells lose infectivity by HBV except the HuS-E/2 human hepatocyte cell line [52]. One reason for this failure appears to be due to in vitro immortalization of in vitro cultured hepatocytes.

2. Transposons as Useful Tools to Obtain Chromosomal Integration of GOI

In mammalian cells, transposon-mediated gene transposition is often performed to achieve chromosomal integration of GOI [53]. The mobility of transposons can be controlled by conditionally providing the transposase that mediates the transposition reaction. Thus, a GOI (i.e., a fluorescent marker, a small hairpin (sh)RNA expression cassette, or a therapeutic gene construct) cloned between the inverted terminal repeat sequences (called ITRs) of transposon-based vectors can be inserted into host chromosomes in a highly efficient manner.

Sleeping Beauty (SB) was the first transposon shown to be capable of gene transfer in vertebrate cells, and recent studies have shown that SB supports the full spectrum of genetic engineering techniques, including transgenesis, insertional mutagenesis, and therapeutic somatic gene transfer, both ex vivo and in vivo [54–56]. PiggyBac (PB) represents an alternative transposon technique, allowing efficient integration of exogenous DNA into host chromosomes in several organisms, including humans [57–59], bovines [60], goats [61], pigs [62, 63], rats [64], mice [65], fish [66], insects [67, 68], malaria parasites [69], yeast [70], and plants [71]. This system is now widely used in gene discovery via insertional mutagenesis [72], generation of induced pluripotent stem (iPS)/embryonic stem (ES) cells [73–75], production of Tg animals [76], introduction of large transposons (>100 kb) [77], generation of stable cell lines with multiple constructs [78], and generation of genome-edited cells [79, 80]. The PB-based gene delivery system is very simple: creation of a PB transposase expression vector and transposons carrying GOI flanked by the two ITR sequences. When they are transfected into a cell, the transposase binds to the ITR to allow the GOI alone to be integrated into host chromosomal sites that contain the TTAA sequence, which is duplicated on the two flanks of the integrated fragment [81, 82]. In Figure 2, the mechanism for PB-based integration of GOI is shown schematically. Furthermore, integrated transposons can be removed by transient retransfection with the PB transposase expression vector [83, 84]. This excision is very precise, as evidenced by the typical absence of “footprint” mutations at the site of transposon excision [85].

Figure 2: Schematic illustration of the mechanism of piggyBac-based gene delivery, based on the website https://www.funakoshi.co.jp/contents/5301. Abbreviations: ITR: inverted terminal repeat; P: promoter; p(A): poly(A) sites; PB: piggyBac.

3. Transposons Confer Efficient Integration of GOI In Vivo

As described above, previous approaches to establish immortalized hepatocytes adopted primary hepatocytes cultured as a source for gene engineering-based immortalization. In general, under the culture conditions used, isolated hepatocytes are known to show reduced viability and dramatic alterations to their gene expression profiles, probably because of drastic alterations involving cell-to-cell contact or cell-to-extracellular matrix contact [86]. This suggests that immortalization of primary cultured hepatocytes may not be the best choice for acquiring immortalized hepatocyte lines. Instead, immortalization of hepatocytes under in vivo condition would be the best because hepatocyte function in these in situ immortalized hepatocytes appears to be retained in the in vivo environment. We therefore considered that transfection of hepatocytes in vivo through liver-directed gene delivery of PB transposons may fit the above concept. As mentioned previously, the PB transposon system is useful for efficient integration of GOI into host chromosomes in cultured cells and for efficient transgenesis in mice [87]. However, little is known about whether this system is also effective in vivo. Recently, a number of studies have described the effectiveness of this system in vivo. For example, Saridey et al. [88] demonstrated that a single injection of plasmid-based PB transposons via the tail vein confers long-term (approximately 300 days after gene delivery) expression of a GOI (coding for luciferase) in the liver and lungs of mice, suggesting chromosomal integration of the GOI. Similar results were also provided by other groups who used repeated intravenous injections of PB transposons [89] or intravenous injections of hybrid PB/viral vectors [90]. We recently performed intraparenchymal injection of exogenous plasmid DNA containing a PB transposase expression vector and PB transposons and subsequent in vivo EP using tweezer-type electrodes to stably transfect murine pancreatic cells. This approach was originally developed to transfect pancreatic cells with naked plasmid DNA and was termed “intrapancreatic parenchymal injection for gene transfer (IPPIGT)” [91]. We found that expression of a GOI (coding for red fluorescent protein) continued for at least 1.5 months after IPPIGT (our unpublished results).

4. New Approaches for Generating Immortalized Hepatocyte Cell Lines Based on In Vivo Transfection of Hepatocyte with Transposons Carrying Immortalization Genes

Hydrodynamic (HGD) injection is a useful method for gene delivery to the liver, involving the rapid injection of a large volume of vector-containing solution into the tail vein [92, 93]. When this approach was employed for transfection with nonviral DNA in mice, the right median lobe of the liver was found to be preferentially transfected (Figure 1(b)) [94]. We recently tested whether HGD-based gene delivery using a DNA solution containing the PB transposon and a PB transposase expression construct could be used to establish prolonged GOI expression in hepatocytes of the right median lobe [94]. Coinjection of a PB transposon containing an enhanced green fluorescent protein expression unit (pT-EGFP; Figure 3(a)) and a PB transposase expression construct (pTrans; Figure 3(a)) together with the nontransposon vector, ptdTomato (conferring expression of tdTomato; [95]), resulted in EGFP expression, even after 56 days postgene delivery, while no appreciable tdTomato expression was observed in the liver sampled 28 days or more after gene delivery [95]. The result of this experiment suggests that the in vivo PB-based gene delivery system confers stable GOI integration in hepatocytes, indicating that HGD-based delivery of PB transposons carrying immortalizing genes may be a useful in vivo approach for the acquisition of immortalized hepatocyte lines. In Figure 4(a), we show an example-of-principle using HGD-based intravenous delivery of two fluorescent marker-containing transposons (pT-EGFP and ptdTomato; Figure 3(a), unpublished data). Two days after gene delivery, liver tissue was dissected for analysis of fluorescence, and two constructs were shown to have been simultaneously introduced into hepatocytes (Figure 4(a), G, H, and I). This suggests that multigene constructs can be delivered simultaneously into hepatocytes, which will be beneficial for chromosomal integration of the transposons with the aid of transposase, a product derived from the pTrans construct delivered concomitantly.

(a)

(b)

(c)

Figure 3: Establishing hepatocyte cell lines by in vivo gene transfer of PB transposons in murine liver. (a) Schematic illustrating the vectors used. pT-LT, pT-E6/E7, pT-Liv#11, pT-EGFP, pT-tdTomato, and pT-ALB/pac are transposon vectors. pTrans is a vector conferring expression of PB transposase. Upon in vivo gene delivery, pT-EGFP, pT-E6/E7, and pT-Liv#11 (or pT-LT and pT-ALB/pac) are cotransfected with pTrans to obtain the 567 cell line (carrying EGFP, pac, E6, E7, and hTERT genes) or LT line (carrying EGFP, pac, and SV40T genes). Arrows under each vector show the orientation of transcription in each expression unit. Abbreviation: PB: PB ITRs; CAG: chicken β-actin-based promoter; p(A): poly(A) signal; E6: HPV18-derived E6 protein gene; E7: HPV18-derived E7 protein gene; SV40T: SV40 T antigen gene; EGFP: enhanced green fluorescent protein gene; hTERT: hemagglutinin-tagged human telomerase reverse transcriptase gene; CMVp: cytomegalovirus promoter; ALBp: human albumin promoter; pac: puromycin acetyltransferase gene; Transposase: PB transposase; pA: poly(A) sites. (b) Cell colonies 10 days after puromycin selection. Both LT (A-D) and 567 (E-H) lines are viable, showing bright EGFP-derived fluorescence. However, there are no viable cells in the control group (data not shown). From these colonies, we obtained clonal lines called LT1-1 and LT1-2, LT2-1 and LT2-2, 5671-1 and 5671-2, and 5672-1 and 5672-2. . (c) RT-PCR analysis of hepatocyte marker gene expression in the isolated clones. The primer sets used for albumin, HNF4α, and C/EBPα were 5-ctcaggtgtcaaccccaa-3 and 5-tccacacaaggcagtctc-3, 5-tgccaacctcaattcatcca-3 and 5-gctcgaggctccgtagtgtt-3, and 5-aagaagtcggtggacaagaacag-3 and 5-gttgcgttgtttggctttatctc-3, respectively. These primer sets yielded 103 bp (for albumin), 94 bp (for HNF4α), and 100 bp (for C/EBPα) PCR products. Notably, almost all of the clones tested still exhibited expression of albumin. F9 cells: murine embryonal carcinoma cells used as negative control; intact liver: adult murine liver used as positive control. M: 100 bp ladder size marker.

(a)

(b)

Figure 4: Analysis of gene expression in murine liver and hepatocytes after HGD-based gene delivery (a) and EP-based gene delivery (b). In (a), the liver is dissected 2 days after HGD with pT-EGFP, ptdTomato, and pTrans and inspected for fluorescence under a fluorescence microscope. Both green and red fluorescence are seen in the DNA-introduced experimental group (D-F) but not in the DNA-noninjected control group (A-C). Higher magnification of images in (D-F) reveals colocalization of both fluorescence colors in hepatocytes (G-I). In (b), the liver is dissected 7 days after EP with pT-EGFP, pT-Liv#11, and pTrans and perfusion with collagenase. Inspection for fluorescence in the dissected liver reveals bright green fluorescence in the electroporated region (A, B). When the fluorescent region is dissociated into single cells and subjected to culture in the presence of puromycin for 7 days, all of the surviving cells are found to exhibit green fluorescence (C, D), suggesting chromosomal integration of introduced transposons. (for a) and 20 μm (for b).

Another approach for in vivo immortalization of hepatocytes involves the direct introduction of foreign DNA into the liver and subsequent in vivo EP in combination with the use of a transposon-based gene delivery system like IPPIGT [91] (Figure 1(c)). This option is always accompanied by surgical procedures, in which the liver is exposed outside the skin. Although this procedure is often laborious, site-specific gene delivery is possible and easy because researchers can control this under observation using a dissecting microscope. Direct introduction of transposon-based vectors carrying immortalizing genes (i.e., SV40 T and hTERT) into animal livers would result in the in vivo establishment of immortalized hepatocyte cell lines. In Figure 4(b), we show an example-of-principle (unpublished data) for the possible isolation of immortalized hepatocyte cell lines using this novel approach. First, a small volume (2–3 μL) of solution containing two PB transposons, pT-EGFP and pT-Liv#11, as well as pTrans, each at a concentration of 100 ng/μL, is introduced into the internal area of the murine liver by inserting a glass micropipette under observation using a dissecting microscope and subsequently injecting the solution using the procedure described by Sato et al. [91] (Figure 1(c)). A small amount of India ink is added to the solution to visualize the location of injection sites. pT-Liv#11 is a transposon vector carrying hTERT- and HVP18-derived E7 expression units, together with a puromycin acetyltransferase gene (pac) expression unit (under the control of an albumin promoter construct) (Figure 3(a)). Simultaneous delivery of these three vectors into a cell should result in EGFP-derived green fluorescence and resistance against puromycin in cells of the hepatocyte lineage. For transfection with pT-E6E7 or pT-LT (Figure 3(a)) together with pTrans and pT-ALB/pac (Figure 3(a)), a plasmid carrying the pac gene under the control of the albumin promoter is cotransfected in the liver. The injection site is then subjected to in vivo EP using tweezer-type electrodes (Figure 1(c)). Eight square electric pulses of 50 V with a constant time of 50 milliseconds (ms) are applied using a pulse generator. With this treatment, some hepatocytes receiving the foreign DNA may be stably transfected. Seven days after gene delivery, the liver is dissected after perfusion with Hanks’ balanced salt solution (HBSS) without Ca²⁺ or Mg²⁺ but containing 1 mg/mL collagenase (Figure 1(c)). The injected portion is easily recognizable by the expression of a fluorescent marker visible under a fluorescent dissecting microscope. In Figure 4(b), A and B, bright fluorescence is easily discernible in the electroporated area. The dissected liver can be dissociated into single cells by teasing apart in HBSS containing collagenase, followed by further incubation at 37°C for more than 1 h to further dissociate the cells prior to culturing in hepatocyte culture medium containing hepatocyte growth factor (HGF) and dexamethasone. To obtain immortalized hepatocytes, the dissociated cells obtained by collagenase perfusion are seeded () into a 6 cm collagen-coated cell culture dish with hepatocyte culture medium. Puromycin is then added to the medium at a concentration of 2 μg/mL, and cells are cultured for 7 days to obtain recombinant cells. The emerging surviving cells (colonies) are picked and seeded onto a collagen-coated 24-well plate and cultured until subconfluency and are then propagated by seeding cells onto a 35 mm collagen-coated dish. The medium is changed every 2 days until subconfluency. The surviving cells should contain transposons in their genomes, conferring resistance against the selective drug and driving cell proliferation due to the expression of the exogenous immortalizing genes. Fluorescence in the surviving hepatocytes (7 days after puromycin treatment) is shown in Figure 4(b), C and D. Notably, almost all the cells are fluorescent, suggesting stable transfection with both the pT-EGFP and pT-Liv#11 transposons. For further propagation, these engineered hepatocytes must be cultured in medium containing factors (i.e., insulin and dexamethasone) that support hepatocyte growth (Figure 1(d)). Several hepatocyte lines (called LT1-1 to LT1-2, LT2-1 to LT2-2, 5671-1 to 5671-2, and 5672-1 to 5672-2) were eventually obtained, all of which survived after puromycin treatment and exhibited EGFP fluorescence (Figure 3(b)). LT1-1 to LT1-2 (Figure 3(b), A, B, C, and D) and LT2-1 to LT2-2 were derived from liver tissue transfected with pT-EGFP, pT-LT, pT-ALB/pac, and pTrans. 5671-1 to 5671-2 (Figure 3(b), E, F, G, and H) and 5672-1 to 5672-2 were derived from liver cells transfected with pT-EGFP, pT-Liv#11, pT-E6E7, and pTrans. Analysis of these established lines by RT-PCR demonstrated that almost all the lines expressed hepatocyte marker genes, such as albumin (Figure 3(c)). Since albumin expression is an important marker of hepatocyte function, the resulting lines are considered to retain the properties of functional hepatocytes. Our future efforts to characterize these established lines would involve examination of the expression of other hepatocyte-specific proteins and urea synthesis. Performing RNA sequencing (RNA-Seq) analysis would also be useful to examine whether our lines indeed resemble hepatocytes at the transcriptional level in vivo.

5. Perspective for Translational Medicine

There is an increasing demand for human hepatocytes differentiated from pluripotent stem cells (as exemplified by ES/iPS cells) for translational medicine [96]. Since the first report by D’Amour et al. [97], who demonstrated the ability of activin A to induce efficient differentiation of human ES cells to definitive endoderm, extensive studies have been carried out on the induction of ES/iPS cell differentiation towards an endodermal lineage. For example, generation of hepatocyte precursors from endodermal cells is achieved by combined treatment with fibroblast growth factor 4 (FGF4) and bone morphogenetic protein 2 (BMP2) [98] or FGF1/2/4 and BMP2/4 [99]. Differentiation of hepatocyte precursors into functional hepatocyte-like cells (HLCs) has typically been achieved by treatment with factors such as HGF, oncostatin M, and dexamethasone [100, 101]. The resulting HLCs have the potential to cure a patient with liver failure through hepatocyte transplantation. Nagamoto et al. [102] demonstrated that transplantation of human iPS cell-derived HLC sheets (created by culturing iPS cells in a temperature-responsive culture dish) into the liver of model mice that show acute liver failure resulted in increased survival rates. However, the use of iPS cells for therapeutic purposes still retains immunogenic and tumorigenic potential [103], and several groups have tried to apply immortalized human hepatocytes for clinical use to bypass the concerns related to the nature of iPS cells. However, ethical issues still remain. For example, there is concern regarding potential tumor generation after transplantation of immortalized hepatocytes, although subcutaneous injection of immortalized hepatocytes into severe combined immunodeficiency (SCID) model mice did not induce tumor development [104]. However, despite the report, the potential for tumorigenesis cannot be completely excluded since the genomes of the immortalized hepatocytes still retain immortalizing genes. Urschitz and Moisyadi [105] suggested that these genes chromosomally integrated through PB-mediated gene delivery could be completely removed before cell transplantation by transient retransfection with a transposase expression vector. Totsugawa et al. [106] used tamoxifen for the Cre/loxP-mediated removal of a floxed immortalizing gene from immortalized hepatocytes after transfection with a gene coding for tamoxifen-dependent Cre recombinase.

Notably, the present technology appears to be confined to smaller experimental animals such as mice and rats (shown in Figures 3 and 4). However, we think that it is also theoretically applicable to larger animals (such as the pig and cow), as well as humans, particularly since the development of in vivo liver-targeting gene delivery methods for gene therapy. Interestingly, some reports have described HGD-based gene delivery in the pig [107, 108]. These experiments were aimed at developing techniques related to gene therapy as basic research but hold potential for the acquisition of functional immortalized porcine hepatocytes. In this context, pigs may be a useful resource to examine whether our present strategy will work well. Indeed, we successfully obtained immortalized hepatocytes from dissected porcine liver using our vector system (shown in Figure 3(a)), although the efficiency was very low (data not shown).

6. Conclusion

To date, an enormous number of immortalized cell lines have been generated. Most of these are derived from transfection of primary cells with vectors carrying genes for immortalizing factors or by primary culture of tissues/organs dissected from Tg mice carrying immortalizing genes. Our present idea, based on site-directed introduction of chromosomal-integrating transposons into living animals, appears to be unique and simple and will provide an additional, easy way to establish novel, immortalized cell lines (including immortalized hepatocytes), which are often refractory to in vitro transfection with vectors carrying immortalizing genes.

Ethical Approval

Experiments related to in vivo gene delivery described in our study were performed according to the “Guide for the Care and Use of Laboratory Animals” of the National Academy of Sciences, USA, and approved by our committee addressing the ethics of animal testing. For example, the experiments shown in Figures 3 and 4 were performed in agreement with the guidelines of the Institute of Livestock and Grassland Science Committee on Recombinant DNA Security and approved by the Animal Care and Experimentation Committee of the Institute of Livestock and Grassland Science (no. 1811B031 dated 26 April 2015; no. 908527-B dated 22 January 2016).

Conflicts of Interest

There are no potential conflicts of interest to disclose.

Authors’ Contributions

Masahiro Sato drafted and revised the manuscript; Issei Saitoh and Emi Inada critically revised the manuscript; Shingo Nakamura was involved in providing experimental ideas; Satoshi Watanabe conceived the study and performed experiments.

Acknowledgments

The authors thank Dr. Masato Ohtsuka (Division of Basic Molecular Science and Molecular Medicine, School of Medicine, Tokai University) for providing some of the transposon vectors. This study was partly supported by a grant (no. 16H05049 for S.N., no. 18K09839 for E.I., and no. 24580411 for M.S.) from the Ministry of Education, Science, Sports, and Culture of the Japanese Government.

References

J. R. W. Masters, “Human cancer cell lines: fact and fantasy,” Nature Reviews Molecular Cell Biology, vol. 1, no. 3, pp. 233–236, 2000.

S. A. Stewart and R. A. Weinberg, “Senescence: does it all happen at the ends?” Oncogene, vol. 21, no. 4, pp. 627–630, 2002.

S. A. Stewart and R. A. Weinberg, “Telomeres: cancer to human aging,” Annual Review of Cell and Developmental Biology, vol. 22, no. 1, pp. 531–557, 2006.

S. H. Ali and J. A. DeCaprio, “Cellular transformation by SV40 large T antigen: interaction with host proteins,” Seminars in Cancer Biology, vol. 11, no. 1, pp. 15–23, 2001.

A. G. Bodnar, M. Ouellette, M. Frolkis et al., “Extension of life-span by introduction of telomerase into normal human cells,” Science, vol. 279, no. 5349, pp. 349–352, 1998.

J. Zhu, H. Wang, J. M. Bishop, and E. H. Blackburn, “Telomerase extends the lifespan of virus-transformed human cells without net telomere lengthening,” Proceedings of the National Academy of Sciences of the United States of America, vol. 96, no. 7, pp. 3723–3728, 1999.

A. S. Lundberg, W. C. Hahn, P. Gupta, and R. A. Weinberg, “Genes involved in senescence and immortalization,” Current Opinion in Cell Biology, vol. 12, no. 6, pp. 705–709, 2000.

N. Yanai, T. Satoh, S. Kyo, K. Abe, M. Suzuki, and M. Masuo, “A tubule cell line established from transgenic mice harboring temperature-sensitive simian virus 40 large T-antigen gene,” Japanese Journal of Cancer Research, vol. 82, no. 12, pp. 1344–1348, 1991.

N. Yanai, M. Suzuki, and M. Obinata, “Hepatocyte cell lines established from transgenic mice harboring temperature-sensitive simian virus 40 large T-antigen gene,” Experimental Cell Research, vol. 197, no. 1, pp. 50–56, 1991.

M. Obinata, “The immortalized cell lines with differentiation potentials: their establishment and possible application,” Cancer Science, vol. 98, no. 3, pp. 275–283, 2007.

T. Tsukada, Y. Tomooka, S. Takai et al., “Enhanced proliferative potential in culture of cells from p53-deficient mice,” Oncogene, vol. 8, pp. 3313–3322, 1993.

H. J. Moshage, H. J. W. de Haard, H. M. G. Princen, and S. H. Yap, “The influence of glucocorticoid on albumin synthesis and its messenger RNA in rat in vivo and in hepatocyte suspension culture,” Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression, vol. 824, no. 1, pp. 27–33, 1985.

C. R. W. Padgham, C. C. Boyle, X. J. Wang, S. M. Raleigh, M. C. Wright, and A. J. Paine, “Alteration of transcription factor mRNAs during the isolation and culture of rat hepatocytes suggests the activation of a proliferative mode underlies their dedifferentiation,” Biochemical and Biophysical Research Communications, vol. 197, no. 2, pp. 599–605, 1993.

P. Gripon, S. Rumin, S. Urban et al., “Infection of a human hepatoma cell line by hepatitis B virus,” Proceedings of the National Academy of Sciences of the United States of America, vol. 99, no. 24, pp. 15655–15660, 2002.

W. W. Colby and T. Shenk, “Fragments of the simian virus 40 transforming gene facilitate transformation of rat embryo cells,” Proceedings of the National Academy of Sciences of the United States of America, vol. 79, no. 17, pp. 5189–5193, 1982.

C. D. Woodworth and H. C. Isom, “Transformation of differentiated rat hepatocytes with adenovirus and adenovirus DNA,” Journal of Virology, vol. 61, no. 11, pp. 3570–3579, 1987.

D. Paul, M. Höhne, and B. Hoffmann, “Immortalization and malignant transformation of hepatocytes by transforming genes of polyoma virus and of SV40 virus in vitro and in vivo,” Wiener klinische Wochenschrift, vol. 66, pp. 134–139, 1988.

S. Chen and E. Paucha, “Identification of a region of simian virus 40 large T antigen required for cell transformation,” Journal of Virology, vol. 64, no. 7, pp. 3350–3357, 1990.

N. Watanabe, H. Odagiri, E. Totsuka, and M. Sasaki, “A new method to immortalize primary cultured rat hepatocytes,” Transplantation Proceedings, vol. 36, no. 8, pp. 2457–2461, 2004.

C. Woodworth, T. Secott, and H. C. Isom, “Transformation of rat hepatocytes by transfection with simian virus 40 DNA to yield proliferating differentiated cells,” Cancer Research, vol. 46, no. 8, pp. 4018–4026, 1986.

C. D. Woodworth, J. W. Kreider, L. Mengel, T. Miller, Y. L. Meng, and H. C. Isom, “Tumorigenicity of simian virus 40-hepatocyte cell lines: effect of in vitro and in vivo passage on expression of liver-specific genes and oncogenes,” Molecular and Cellular Biology, vol. 8, no. 10, pp. 4492–4501, 1988.

C. D. Woodworth and H. C. Isom, “Immortalized hepatocytes as in vitro model systems for toxicity testing: the comparative toxicity of menadione in immortalized cells, primary cultures of hepatocytes and HTC hepatoma cells,” Toxicology in Vitro, vol. 10, no. 6, pp. 721–727, 1996.

C. Macdonald and B. Willett, “The immortalisation of rat hepatocytes by transfection with SV40 sequences,” Cytotechnology, vol. 23, no. 1/3, pp. 161–170, 1997.

A. Werner, S. Duvar, J. Müthing et al., “Cultivation and characterization of a new immortalized human hepatocyte cell line, HepZ, for use in an artificial liver support system,” Annals of the New York Academy of Sciences, vol. 875, no. 1, pp. 364–368, 1999.

K. Fukaya, S. Asahi, S. Nagamori et al., “Establishment of a human hepatocyte line (OUMS-29) having CYP 1A1 and 1A2 activities from fetal liver tissue by transfection of SV40 LT,” In Vitro Cellular & Developmental Biology - Animal, vol. 37, no. 5, pp. 266–269, 2001.

N. Kobayashi, H. Noguchi, T. Watanabe et al., “Role of immortalized hepatocyte transplantation in acute liver failure,” Transplantation Proceedings, vol. 33, no. 1-2, pp. 645-646, 2001.

J. Li, L. J. Li, H. C. Cao et al., “Establishment of highly differentiated immortalized human hepatocyte line with simian virus 40 large tumor antigen for liver based cell therapy,” ASAIO Journal, vol. 51, no. 3, pp. 262–268, 2005.

B. Werness, A. Levine, and P. Howley, “Association of human papillomavirus types 16 and 18 E6 proteins with p53,” Science, vol. 248, no. 4951, pp. 76–79, 1990.

K. Münger, B. A. Werness, N. Dyson, W. C. Phelps, E. Harlow, and P. M. Howley, “Complex formation of human papillomavirus E7 proteins with the retinoblastoma tumor suppressor gene product,” The EMBO Journal, vol. 8, no. 13, pp. 4099–4105, 1989.

C. P. Morales, S. E. Holt, M. Ouellette et al., “Absence of cancer–associated changes in human fibroblasts immortalized with telomerase,” Nature Genetics, vol. 21, no. 1, pp. 115–118, 1999.

J. Zhou, D. Ding, M. Wang, and Y. S. Cong, “Telomerase reverse transcriptase in the regulation of gene expression,” BMB Reports, vol. 47, no. 1, pp. 8–14, 2014.

A. M. Pfeifer, K. E. Cole, D. T. Smoot et al., “Simian virus 40 large tumor antigen-immortalized normal human liver epithelial cells express hepatocyte characteristics and metabolize chemical carcinogens,” Proceedings of the National Academy of Sciences of the United States of America, vol. 90, no. 11, pp. 5123–5127, 1993.

B. H. Kim, S. R. Sung, E. H. Choi et al., “Dedifferentiation of conditionally immortalized hepatocytes with long-term in vitro passage,” Experimental & Molecular Medicine, vol. 32, no. 1, pp. 29–37, 2000.

N. Kobayashi, T. Fujiwara, K. A. Westerman et al., “Prevention of acute liver failure in rats with reversibly immortalized human hepatocytes,” Science, vol. 287, no. 5456, pp. 1258–1262, 2000.

N. Kobayashi, H. Noguchi, T. Fujiwara, K. A. Westerman, P. Leboulch, and N. Tanaka, “Establishment of a highly differentiated immortalized adult human hepatocyte cell line by retroviral gene transfer,” Transplantation Proceedings, vol. 32, no. 7, pp. 2368-2369, 2000.

M. Smalley, K. Leiper, R. Tootle, P. Mccloskey, M. J. O'Hare, and H. Hodgson, “Immortalization of human hepatocytes by temperature-sensitive SV40 large-T antigen,” In Vitro Cellular & Developmental Biology - Animal, vol. 37, no. 3, pp. 166–168, 2001.

N. Kobayashi, T. Kunieda, M. Sakaguchi et al., “Active expression of p21 facilitates differentiation of immortalized human hepatocytes,” Transplantation Proceedings, vol. 35, no. 1, pp. 433-434, 2003.

H. Wege, H. T. le, M. S. Chui et al., “Telomerase reconstitution immortalizes human fetal hepatocytes without disrupting their differentiation potential,” Gastroenterology, vol. 124, no. 2, pp. 432–444, 2003.

R. A. F. M. Chamuleau, T. Deurholt, and R. Hoekstra, “Which are the right cells to be used in a bioartificial liver?” Metabolic Brain Disease, vol. 20, no. 4, pp. 327–335, 2005.

B. Haker, S. Fuchs, J. Dierlamm, T. H. Brümmendorf, and H. Wege, “Absence of oncogenic transformation despite acquisition of cytogenetic aberrations in long-term cultured telomerase-immortalized human fetal hepatocytes,” Cancer Letters, vol. 256, no. 1, pp. 120–127, 2007.

Y. Reid, J. P. Gaddipati, D. Yadav, and J. Kantor, “Establishment of a human neonatal hepatocyte cell line,” In Vitro Cellular & Developmental Biology - Animal, vol. 45, no. 9, pp. 535–542, 2009.

Y. Chen, J. Li, X. Liu, W. Zhao, Y. Wang, and X. Wang, “Transplantation of immortalized human fetal hepatocytes prevents acute liver failure in 90% hepatectomized mice,” Transplantation Proceedings, vol. 42, no. 5, pp. 1907–1914, 2010.

X. Pan, J. Z. Li, W. B. du et al., “Establishment and characterization of immortalized human hepatocyte cell line for applications in bioartificial livers,” Biotechnology Letters, vol. 34, no. 12, pp. 2183–2190, 2012.

F. Y. Meng, L. Liu, F. H. Yang, C. Y. Li, J. Liu, and P. Zhou, “Reversible immortalization of human hepatocytes mediated by retroviral transfer and site-specific recombination,” World Journal of Gastroenterology, vol. 20, no. 36, pp. 13119–13126, 2014.

P. Salmon, J. Oberholzer, T. Occhiodoro, P. Morel, J. Lou, and D. Trono, “Reversible immortalization of human primary cells by lentivector-mediated transfer of specific genes,” Molecular Therapy, vol. 2, no. 4, pp. 404–414, 2000.

T. H. Nguyen, G. Mai, P. Villiger et al., “Treatment of acetaminophen-induced acute liver failure in the mouse with conditionally immortalized human hepatocytes,” Journal of Hepatology, vol. 43, no. 6, pp. 1031–1037, 2005.

Y. Tsuruga, T. Kiyono, M. Matsushita et al., “Effect of intrasplenic transplantation of immortalized human hepatocytes in the treatment of acetaminophen-induced acute liver failure SCID mice,” Transplantation Proceedings, vol. 40, no. 2, pp. 617–619, 2008.

T. Deurholt, N. P. van Til, A. A. Chhatta et al., “Novel immortalized human fetal liver cell line, cBAL111, has the potential to differentiate into functional hepatocytes,” BMC Biotechnology, vol. 9, no. 1, p. 89, 2009.

G. M. Cooper and S. Okenquist, “Mechanism of transfection of chicken embryo fibroblasts by Rous sarcoma virus DNA,” Journal of Virology, vol. 28, no. 1, pp. 45–52, 1978.

P. F. Lewis and M. Emerman, “Passage through mitosis is required for oncoretroviruses but not for the human immunodeficiency virus,” Journal of Virology, vol. 68, no. 1, pp. 510–516, 1994.

L. Naldini, U. Blomer, P. Gallay et al., “In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector,” Science, vol. 272, no. 5259, pp. 263–267, 1996.

H. C. Huang, C. C. Chen, W. C. Chang, M. H. Tao, and C. Huang, “Entry of hepatitis B virus into immortalized human primary hepatocytes by clathrin-dependent endocytosis,” Journal of Virology, vol. 86, no. 17, pp. 9443–9453, 2012.

Z. Ivics and Z. Izsvák, “The expanding universe of transposon technologies for gene and cell engineering,” Mobile DNA, vol. 1, no. 1, p. 25, 2010.

Z. Ivics, P. B. Hackett, R. H. Plasterk, and Z. Izsvák, “Molecular reconstruction of Sleeping Beauty, a Tc1-like transposon from fish, and its transposition in human cells,” Cell, vol. 91, no. 4, pp. 501–510, 1997.

Z. Izsvák, E. E. Stüwe, D. Fiedler, A. Katzer, P. A. Jeggo, and Z. Ivics, “Healing the wounds inflicted by sleeping beauty transposition by double-strand break repair in mammalian somatic cells,” Molecular Cell, vol. 13, no. 2, pp. 279–290, 2004.

Z. Izsvák and Z. Ivics, “Sleeping beauty transposition: biology and applications for molecular therapy,” Molecular Therapy, vol. 9, no. 2, pp. 147–156, 2004.

M. H. Wilson, C. J. Coates, and A. L. George Jr., “PiggyBac transposon-mediated gene transfer in human cells,” Molecular Therapy, vol. 15, no. 1, pp. 139–145, 2007.

C. Kettlun, D. L. Galvan, A. L. George Jr., A. Kaja, and M. H. Wilson, “Manipulating piggyBac transposon chromosomal integration site selection in human cells,” Molecular Therapy, vol. 19, no. 9, pp. 1636–1644, 2011.

E. Inada, I. Saitoh, S. Watanabe et al., “PiggyBac transposon-mediated gene delivery efficiently generates stable transfectants derived from cultured primary human deciduous tooth dental pulp cells (HDDPCs) and HDDPC-derived iPS cells,” International Journal of Oral Science, vol. 7, no. 3, pp. 144–154, 2015.

S. Kim, I. M. Saadeldin, W. J. Choi et al., “Production of transgenic bovine cloned embryos using piggybac transposition,” Journal of Veterinary Medical Science, vol. 73, no. 11, pp. 1453–1457, 2011.

D. P. Bai, M. M. Yang, and Y. L. Chen, “PiggyBac transposon-mediated gene transfer in Cashmere goat fetal fibroblast cells,” Bioscience, Biotechnology and Biochemistry, vol. 76, no. 5, pp. 933–937, 2012.

Z. Wu, Z. Xu, X. Zou et al., “Pig transgenesis by piggyBac transposition in combination with somatic cell nuclear transfer,” Transgenic Research, vol. 22, no. 6, pp. 1107–1118, 2013.

Z. Li, F. Zeng, F. Meng et al., “Generation of transgenic pigs by cytoplasmic injection of piggyBac transposase-based pmGENIE-3 plasmids,” Biology of Reproduction, vol. 90, no. 5, p. 93, 2014.

W. Li, X. Li, T. Li et al., “Genetic modification and screening in rat using haploid embryonic stem cells,” Cell Stem Cell, vol. 14, no. 3, pp. 404–414, 2014.

H. Miura, H. Inoko, I. Inoue et al., “piggyBac-mediated generation of stable transfectants with surface human leukocyte antigen expression from a small number of cells,” Analytical Biochemistry, vol. 437, no. 1, pp. 29–31, 2013.

S. C.-Y. Wu, Y. J. J. Meir, C. J. Coates et al., “piggyBac is a flexible and highly active transposon as compared to sleeping beauty, Tol2, and Mos1 in mammalian cells,” Proceedings of the National Academy of Sciences of the United States of America, vol. 103, no. 41, pp. 15008–15013, 2006.

D. A. O'Brochta and P. W. Atkinson, “Transposable elements and gene transformation in non-drosophilid insects,” Insect Biochemistry and Molecular Biology, vol. 26, no. 8-9, pp. 739–753, 1996.

A. M. Handler, “Use of the piggyBac transposon for germ-line transformation of insects,” Insect Biochemistry and Molecular Biology, vol. 32, no. 10, pp. 1211–1220, 2002.

B. S. Crabb, T. F. de Koning-Ward, and P. R. Gilson, “Toward forward genetic screens in malaria-causing parasites using the piggyBac transposon,” BMC Biology, vol. 9, no. 1, p. 21, 2011.

J. Li, J. M. Zhang, X. Li et al., “A piggyBac transposon-based mutagenesis system for the fission yeast Schizosaccharomyces pombe,” Nucleic Acids Research, vol. 39, no. 6, p. e40, 2011.

A. Nishizawa-Yokoi, M. Endo, N. Ohtsuki, H. Saika, and S. Toki, “Precision genome editing in plants via gene targeting and piggyBac-mediated marker excision,” The Plant Journal, vol. 81, no. 1, pp. 160–168, 2015.

R. Rad, L. Rad, W. Wang et al., “PiggyBac transposon mutagenesis: a tool for cancer gene discovery in mice,” Science, vol. 330, no. 6007, pp. 1104–1107, 2010.

K. Woltjen, I. P. Michael, P. Mohseni et al., “piggyBac transposition reprograms fibroblasts to induced pluripotent stem cells,” Nature, vol. 458, no. 7239, pp. 766–770, 2009.

K. Kaji, K. Norrby, A. Paca, M. Mileikovsky, P. Mohseni, and K. Woltjen, “Virus-free induction of pluripotency and subsequent excision of reprogramming factors,” Nature, vol. 458, no. 7239, pp. 771–775, 2009.

K. Yusa, R. Rad, J. Takeda, and A. Bradley, “Generation of transgene-free induced pluripotent mouse stem cells by the piggyBac transposon,” Nature Methods, vol. 6, no. 5, pp. 363–369, 2009.

S. Ding, X. Wu, G. Li, M. Han, Y. Zhuang, and T. Xu, “Efficient transposition of the piggyBac (PB) transposon in mammalian cells and mice,” Cell, vol. 122, no. 3, pp. 473–483, 2005.

M. A. Li, D. J. Turner, Z. Ning et al., “Mobilization of giant piggyBac transposons in the mouse genome,” Nucleic Acids Research, vol. 39, no. 22, article e148, 2011.

K. M. Kahlig, S. K. Saridey, A. Kaja, M. A. Daniels, A. L. George, and M. H. Wilson, “Multiplexed transposon-mediated stable gene transfer in human cells,” Proceedings of the National Academy of Sciences of the United States of America, vol. 107, no. 4, pp. 1343–1348, 2010.

F. Xie, L. Ye, J. C. Chang et al., “Seamless gene correction of β-thalassemia mutations in patient-specific iPSCs using CRISPR/Cas9 and piggyBac,” Genome Research, vol. 24, no. 9, pp. 1526–1533, 2014.

F. Chen, J. Rosiene, A. Che, A. Becker, and J. LoTurco, “Tracking and transforming neocortical progenitors by CRISPR/Cas9 gene targeting and piggyBac transposase lineage labeling,” Development, vol. 142, no. 20, pp. 3601–3611, 2015.

M. J. Fraser, T. Clszczon, T. Elick, and C. Bauser, “Precise excision of TTAA-specific lepidopteran transposons piggyBac (IFP2) and tagalong (TFP3) from the baculovirus genome in cell lines from two species of Lepidoptera,” Insect Molecular Biology, vol. 5, no. 2, pp. 141–151, 1996.

C. A. Bauser, T. A. Elick, and M. J. Fraser, “Proteins from nuclear extracts of two lepidopteran cell lines recognize the ends of TTAA-specific transposons piggyBac and tagalong,” Insect Molecular Biology, vol. 8, no. 2, pp. 223–230, 1999.

J. Cadiñanos and A. Bradley, “Generation of an inducible and optimized piggyBac transposon system,” Nucleic Acids Research, vol. 35, no. 12, article e87, 2007.

W. Wang, C. Lin, D. Lu et al., “Chromosomal transposition of PiggyBac in mouse embryonic stem cells,” Proceedings of the National Academy of Sciences of the United States of America, vol. 105, no. 27, pp. 9290–9295, 2008.

R. Mitra, J. Fain-Thornton, and N. L. Craig, “piggyBac can bypass DNA synthesis during cut and paste transposition,” The EMBO Journal, vol. 27, no. 7, pp. 1097–1109, 2008.

K. Otsu, K. Ito, T. Kuzumaki, and Y. Iuchi, “Differential regulation of liver-specific and ubiquitously-expressed genes in primary rat hepatocytes by the extracellular matrix,” Cellular Physiology and Biochemistry, vol. 11, no. 1, pp. 33–40, 2001.

L. E. Woodard and M. H. Wilson, “piggyBac-ing models and new therapeutic strategies,” Trends in Biotechnology, vol. 33, no. 9, pp. 525–533, 2015.

S. K. Saridey, L. Liu, J. E. Doherty et al., “PiggyBac transposon-based inducible gene expression in vivo after somatic cell gene transfer,” Molecular Therapy, vol. 17, no. 12, pp. 2115–2120, 2009.

H. Nakanishi, Y. Higuchi, S. Kawakami, F. Yamashita, and M. Hashida, “piggyBac transposon-mediated long-term gene expression in mice,” Molecular Therapy, vol. 18, no. 4, pp. 707–714, 2010.

A. L. Cooney, B. K. Singh, and P. L. Sinn, “Hybrid nonviral/viral vector systems for improved piggyBac DNA transposon in vivo delivery,” Molecular Therapy, vol. 23, no. 4, pp. 667–674, 2015.

M. Sato, E. Inada, I. Saitoh et al., “Site-targeted non-viral gene delivery by direct DNA injection into the pancreatic parenchyma and subsequent in vivo electroporation in mice,” Biotechnology Journal, vol. 8, no. 11, pp. 1355–1361, 2013.

F. Liu, Y. K. Song, and D. Liu, “Hydrodynamics-based transfection in animals by systemic administration of plasmid DNA,” Gene Therapy, vol. 6, no. 7, pp. 1258–1266, 1999.

G. Zhang, V. Budker, and J. A. Wolff, “High levels of foreign gene expression in hepatocytes after tail vein injections of naked plasmid DNA,” Human Gene Therapy, vol. 10, no. 10, pp. 1735–1737, 1999.

S. Nakamura, T. Maehara, S. Watanabe, M. Ishihara, and M. Sato, “Liver lobe and strain difference in gene expression after hydrodynamics-based gene delivery in mice,” Animal Biotechnology, vol. 26, no. 1, pp. 51–57, 2015.

S. Nakamura, M. Ishihara, S. Watanabe, N. Ando, M. Ohtsuka, and M. Sato, “Intravenous delivery of piggyBac transposons as a useful tool for liver-specific gene-switching,” International Journal of Molecular Sciences, vol. 19, no. 11, p. 3452, 2018.

D. Szkolnicka and D. C. Hay, “Concise review: advances in generating hepatocytes from pluripotent stem cells for translational medicine,” Stem Cells, vol. 34, no. 6, pp. 1421–1426, 2016.

K. A. D'Amour, A. D. Agulnick, S. Eliazer, O. G. Kelly, E. Kroon, and E. E. Baetge, “Efficient differentiation of human embryonic stem cells to definitive endoderm,” Nature Biotechnology, vol. 23, no. 12, pp. 1534–1541, 2005.

J. Cai, Y. Zhao, Y. Liu et al., “Directed differentiation of human embryonic stem cells into functional hepatic cells,” Hepatology, vol. 45, no. 5, pp. 1229–1239, 2007.

G. Brolén, L. Sivertsson, P. Björquist et al., “Hepatocyte-like cells derived from human embryonic stem cells specifically via definitive endoderm and a progenitor stage,” Journal of Biotechnology, vol. 145, no. 3, pp. 284–294, 2010.

S. Snykers, J. De Kock, V. Rogiers, and T. Vanhaecke, “In vitro differentiation of embryonic and adult stem cells into hepatocytes: state of the art,” Stem Cells, vol. 27, no. 3, pp. 577–605, 2009.

K. Si-Tayeb, F. P. Lemaigre, and S. A. Duncan, “Organogenesis and development of the liver,” Developmental Cell, vol. 18, no. 2, pp. 175–189, 2010.

Y. Nagamoto, K. Takayama, K. Ohashi et al., “Transplantation of a human iPSC-derived hepatocyte sheet increases survival in mice with acute liver failure,” Journal of Hepatology, vol. 64, no. 5, pp. 1068–1075, 2016.

S. Diecke, S. M. Jung, J. Lee, and J. H. Ju, “Recent technological updates and clinical applications of induced pluripotent stem cells,” The Korean Journal of Internal Medicine, vol. 29, no. 5, pp. 547–557, 2014.

Y. Tsuruga, T. Kiyono, M. Matsushita et al., “Establishment of immortalized human hepatocytes by introduction of HPV16 E6/E7 and hTERT as cell sources for liver cell-based therapy,” Cell Transplantation, vol. 17, no. 9, pp. 1083–1094, 2008.

J. Urschitz and S. Moisyadi, “Transpositional transgenesis with piggyBac,” Mobile Genetic Elements, vol. 3, no. 3, article e25167, 2013.

T. Totsugawa, C. Yong, J. D. Rivas-Carrillo et al., “Survival of liver failure pigs by transplantation of reversibly immortalized human hepatocytes with Tamoxifen-mediated self-recombination,” Journal of Hepatology, vol. 47, no. 1, pp. 74–82, 2007.

H. Yoshino, K. Hashizume, and E. Kobayashi, “Naked plasmid DNA transfer to the porcine liver using rapid injection with large volume,” Gene Therapy, vol. 13, no. 24, pp. 1696–1702, 2006.

J. W. Fabre, A. Grehan, M. Whitehorne et al., “Hydrodynamic gene delivery to the pig liver via an isolated segment of the inferior vena cava,” Gene Therapy, vol. 15, no. 6, pp. 452–462, 2008.