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Induction of Hepatocyte Differentiation in Human Pluripotent Stem Cells

Minoru Tomizawa, Fuminobu Shinozaki, Yasufumi Motoyoshi, Takao Sugiyama, Shigenori Yamamoto, Naoki Ishige

Minoru Tomizawa, Department of Gastroenterology, National Hospital Organization, Shimoshizu Hospital, 934-5 Shikawatashi, Yotsukaido City, Chiba 284-0003, Japan
Fuminobu Shinozaki, Department of Radiology, National Hospital Organization, Shimoshizu Hospital, 934-5 Shikawatashi, Yotsukaido City, Chiba 284-0003, Japan
Yasufumi Motoyoshi, Department of Neurology, National Hospital Organization, Shimoshizu Hospital, 934-5 Shikawatashi, Yotsukaido City, Chiba 284-0003, Japan
Takao Sugiyama, Department of Rheumatology, National Hospital Organization, Shimoshizu Hospital, 934-5 Shikawatashi, Yotsukaido City, Chiba 284-0003, Japan
Shigenori Yamamoto, Department of Pediatrics, National Hospital Organization, Shimoshizu Hospital, 934-5 Shikawatashi, Yotsukaido City, Chiba 284-0003, Japan
Naoki Ishige, Department of Neurosurgery, National Hospital Organization, Shimoshizu Hospital, 934-5 Shikawatashi, Yotsukaido City, Chiba 284-0003, Japan

Correspondence to: Minoru Tomizawa, Department of Gastroenterology, National Hospital Organization Shimoshizu Hospital, 934-5 Shikawatashi, Yotsukaido City, Chiba 284-0003, Japan
Email: nihminor-cib@umin.ac.jp
Telephone: +81-43-422-2511
Fax: +81-43-421-3007
Received: January 11, 2015
Revised: February 14, 2015
Accepted: February 16, 2015
Published online: June 21, 2015

ABSTRACT

Culturing of hepatocytes is used in both experimental and clinical procedures. Human induced pluripotent stem (hiPS) cells can be established from the somatic cells of individual patients. The In vitro production of hepatocytes from these hiPS cells would be advantageous in that it could resolve the ethical problems associated with stem cell research and could eliminate the need for administration of immunosuppresants in hepatocyte transplantation. To establish a method for the production of hepatocytes from hiPS cells, it is necessary to understand both the mechanism of hepatocyte differentiation as well as the current techniques for culturing of primary hepatocytes. Meanwhile, hepatocytes can also be differentiated from endodermal cells upon stimulation with growth factors that induce expression of specific transcriptional regulators. Current protocols for the generation of hepatocytes from hiPS cells include the sequential application of growth factors to mimic the environment of fetal liver. Initially, cells are treated with activin and bone morphogenetic proteins, followed by the application of hepatocyte growth factor and, at the final differentiation stage, Oncostatin M. Expression of transcription factors is necessary for hepatocyte differentiation. However, because the efficiency at which vectors expressing these factors can be transfected into hiPS cells is disappointingly low, adenoviral vectors have been used, which have a transduction efficiency of 100%. This method is used to sequentially introduce sex-determining region Y (Sry) HMG box (Sox) 17, hematopoietically expressed homeobox, and hepatocyte nuclear factor-4α into hiPS cells. These factors, in concert with the stimulation with growth factors, promote differentiation of these cells to hepatocytes. Despite the development of these procedures, however, the resulting cells remain immature and only perform certain hepatocyte functions. In this review, we describe each of the above points, and then discuss novel approaches and future directions for the In vitro production of hepatocytes.

Key words:Transcription factor; Growth factor; Drug metabolism; Transplantation

© 2015 The Authors. Published by ACT Publishing Group Ltd.

Tomizawa M, Shinozaki F, Motoyoshi Y, Sugiyama T, Yamamoto S, Ishige N. Induction of Hepatocyte Differentiation in Human Pluripotent Stem Cells. Journal of Gastroenterology and Hepatology Research 2015; 4(6): 1627-1639 Available from: URL: http://www.ghrnet.org/index.php/joghr/article/view/1257

Introduction

The liver is a single large organ, and hepatocytes account for approximately 70-80% of its cells. These liver-specific cells play a major role in protein synthesis, glucose metabolism, and detoxification. Notably, even when a portion of the hepatocytes are lost, the remaining cells maintain the ability to proliferate[1]. Indeed, one of the ideal methods for the treatment of loss of liver tissue is transplantation of hepatocytes[2].

Human induced pluripotent stem (hiPS) cells are generated with cellular reprogramming factors[3], and have the potential to differentiate into a variety of cell types. Because hiPS cells can be generated from each patient individually, the use of these cells for treating liver ailments could avoid potential ethical complications as well as the development of graft-versus-host disease. Therefore, hiPS cells may comprise an ideal cell source for patients needing transplantation of liver tissues. As a result, the development of methods for producing hepatocytes from hiPS cells is currently underway.

In this review, we begin by discussing current methods for the culturing of primary hepatocytes. This information is also applicable to the maintenance of hepatocytes differentiated from hiPS cells. Subsequently, we discuss the applications for cultured hepatocytes, as these applications could provide potential uses for the hiPS cells. The mechanism governing hepatocyte differentiation has been applied to the In vitro production of hepatocytes from hiPS cells. This cellular mechanism as well as the current protocols for In vitro differentiation of hiPS cells into hepatocytes will therefore be summarized. Despite the development of these protocols, however, the production of functioning hepatocytes from hiPS cells remains technically challenging. As such, the limitations of these differentiation methods will be discussed. Lastly, we present novel approaches for In vitro hepatocyte production.

Culturing of primary hepatocytes

The culturing of hepatocytes can be applied to drug development, cell-mediated therapies, and In vitro disease models. Indeed, primary hepatocyte culturing is an ideal In vitro model for investigating drug metabolism and toxicology, and primary hepatocytes can be transplanted into patients experiencing liver failure[4]; however, the culturing of these cells remains technically difficult. Hepatocytes are typically isolated from a fragment of resected donor liver using a 2-step collagenase perfusion technique[4]. Fetal hepatocytes (107 cells) have been transplanted into patients with hepatic encephalopathy[2]. While there was improvement in liver function there was no observed increase in patient survival times. While the reason for this was unclear, it was speculated that the number of cells transplanted was insufficient to enhance patient survival[5]. There are several disadvantages to utilizing primary hepatocytes, however. Once isolated, these cells are prone to apoptosis and damage[6], and exhibit low levels of proliferation in culture[7]. The use of hiPS cells, however, could overcome several of the difficulties associated with the In vitro culturing of primary hepatocyte.

Other sources of hepatocytes

There are three cell types that can be utilized to generate transplantable hepatocytes: bone marrow stem cells, mesenchymal stem cells, and adipose stem cells. Bone marrow stem cells have the capacity for self-renewal, differentiation, and homing. Indeed, bone marrow stem cells harvested from independent donors have been shown to home to recipient bone marrow tissues[8]. Surprisingly, a previous study demonstrated that mouse bone marrow stem cells, which express CD34 and SCA-1, successfully differentiated into liver epithelial cells In vitro[9]. Likewise, autopsy analyses demonstrated that transplanted bone marrow cells differentiated into hepatocytes in humans[10]. Notably, these transplanted bone marrow cells reduced liver fibrosis in mice[11]. Hepatocyte-like cells have also been produced from human umbilical mesenchymal stem cells through the introduction of hepatocyte nuclear factor-4α[12,13]. Meanwhile, adipose stem cells can be purified, cultured, and subjected to sequential stimulation with growth factors, thereby mimicking the in vivo hepatocyte differentiation process[14]. However, none of these three cell sources is considered feasible for clinical use. This is largely due to the fact that so called hepatocyte from these stem cells are far from the functional hepatocytes because these stem cell populations do not exhibit sufficient pluripotency to differentiate into hepatocytes. As such, hiPS cells are considered an ideal source for the In vitro production of hepatocytes as they are pluripotent.

Putative applications of hepatocytes differentiated from hiPS cells

Successful differentiation of hepatocytes from hiPS cells would be beneficial for both medical practice and biological analyses. Potential applications would be as follows: (1) Transplantation into patients with hepatic insufficiency; (2) A method for providing support to patients with hepatic insufficiency; for use in bioartificial liver devices; (3). Toxicology analyses of drugs; (4) In vitro models of liver disease; (5) In vitro model of hepatocyte infection with hepatitis viruses; (6) In vitro model of hepatocyte differentiation. Each of these applications will be presented in detail.

1. Transplantation into patients with hepatic insufficiency

One of the most important applications of hepatocytes generated from hiPS cells would be transplantation into patients with hepatic insufficiency caused by fulminant hepatitis. Because this disease is the result of a significant loss of functional hepatocytes, transplantation of healthy cells could be an ideal therapeutic option. Hepatic progenitor cells have the potential to differentiate into both mature hepatocytes and bile duct epithelial cells. Furthermore, hepatic progenitor cells could be expected to construct normal liver structures, including hepatic lobules and bile ducts. Hepatic progenitor cells derived from mouse embryonic stem (ES) cells were previously shown to engraft in host liver tissues and to differentiate into hepatocytes when transplanted into partially hepatectomized mice[15]. Likewise, hepatocytes engrafted in mice suffering from acute liver failure caused by carbon tetrachloride intoxication[16]. This promising finding suggests that hepatocytes from pluripotent cells are transplantable. Indeed, hepatocytes have been differentiated from human ES cells and then successfully transplanted[17]. While a major disadvantage associated with the use of human ES cells is the possibility of inducing graft-versus-host disease, the ability to treat patients with hepatocytes derived from their own tissues would eradicate this limitation. Therefore, this approach would allow for successful treatment of patients suffering from acute liver failure. Hepatocyte-like cells derived from mouse iPS cells have been shown to improve acute liver failure caused by carbon tetrachloride[18]. These cells were transplanted through peritoneal injection, which led to significant reduction in the extent of liver necrosis. The authors concluded that the observed hepatoprotective effects were due to antioxidant activities of the transplanted cells. Meanwhile, transplanted hepatocytes generated from human iPS cells maintain their proliferative capacity, as evidenced by immunostaining with Ki67-specific antibodies[19]. This report is promising in that it indicates that transplanted cells can proliferate and compensate for lost liver tissue in patients experiencing acute liver failure.

2. A method for providing support to patients with hepatic insufficiency

Hepatocytes are utilized in bioartificial liver systems of patients suffering from liver failure. While these systems currently employ either primary hepatocytes or hepatocellular carcinoma cells[20], which can be rejected by the host, Iwamuro et al recently tested the use of hepatocytes derived from mouse iPS cells in bioartificial liver systems[21]. In these experiments, the mouse cells produced albumin and expressed enzymes involved in the urea cycle, consistent with hepatocyte-like cells. It is expected that similar results could be obtained using hiPS cells.

3. Drug screening for toxicology

The liver is the primary organ affected by drug toxicity[22]. Primary hepatocytes and hepatocellular cancer cells have been used to assess drug-induced liver injury[23-25]. Again, the major limitations of using primary hepatocytes are the technical difficulties associated with obtaining and maintaining these cells. In addition, the innate limitation of hepatocellular carcinoma cells is that they are distinct from hepatocytes. Hepatocytes derived from hiPS cells are expected to overcome these limitations. Indeed, hepatocyte-like cells were recently used for analyzing the toxicity of particular compounds to the liver[26]. In this study, the authors used cell viability, nuclear shape and other markers to assess hepatotoxicity, and succeeded in efficiently screening a panel of 240 compounds to identify those associated with high and low toxicity.

4. In vitro models of liver disease

Similar to studies performed in mice, hepatocytes could be produced from patients with metabolic diseases and utilized to characterize these disorders[27]. While it is not possible to harvest mature hepatocytes from human patients, hiPS cell populations could be established. Once established from a patient(s) with a given metabolic liver disease, these hiPS cells could then be differentiated into hepatocytes[28], which would provide an ideal model system for studying the disorder. Furthermore, the disease of interest could potentially be cured by transplanting functioning hepatocytes into the patient, as these cells play pivotal roles in metabolism.

High plasma levels of low-density lipoprotein cholesterol (LDL-Chol) are known to cause cardiovascular disease. As such, successful reduction of LDL-Chol levels may prevent these diseases. Furthermore, hiPS cells derived from patients with familial hypercholesterolemia (FH), which is caused by mutations in the gene encoding the LDL receptor, could provide an effective model for analyzing the mechanism of this condition[29]. Wilson’s disease is a congenital metabolic disorder that results in excess copper accumulation in hepatocytes due to a mutation in the ATP7B gene [30]. Hepatocytes were recently established from hiPS cells originated from a patient with Wilson’s disease[31]. The hepatocytes from hiPS cells consistently express the mutant ATP7B gene. These findings therefore demonstrate that In vitro differentiation of hepatocytes can be utilized to characterize liver diseases and may be useful for developing novel therapies to treat these conditions.

5. In vitro model of hepatocyte infection with hepatitis viruses

Hepatitis C virus (HCV) causes liver cirrhosis and hepatocellular carcinoma (HCC). Meanwhile, unlike hiPS cells, which are not permissive to HCV infection, hepatocyte-like cells derived from hiPS cells can be infected by HCV[32,33]. Hepatocyte-like cells produced from iPS cells were transplanted into mice and were viable for greater than three months[34]. Furthermore, these hepatocyte-like cells were infected with the HCV virus, indicating that this mouse model is useful for investigating HCV infections. Likewise, hepatocyte-like cells derived from hiPS cells exert an inflammatory response to infection[35], and may therefore provide a suitable In vitro model for studying the mechanism of human HCV infection. Such a model could lead to innovative strategies for inhibiting HCV replication or survival, which would help to prevent both HCV-mediated liver cirrhosis and HCC.

Hepatitis B virus (HBV) is another significant causative agent of viral hepatitis, liver cirrhosis, and HCC. Hepatocytes differentiated from hiPS cells are also permissive to HBV infection and are therefore a useful model for studying human HBV-mediated illnesses[36].

6. In vitro model of hepatocyte differentiation

It is very hard to obtain primary hepatocytes from liver tissues because the culturing of these cells is associated with technical challenges. Knowledge of hepatocyte differentiation, however, would improve the methods for generating hepatocytes from hiPS cells In vitro. Because it is not possible to investigate human hepatocyte differentiation using an in vivo model, our knowledge of this process could be significantly enhanced by an In vitro model, such as the differentiation of hepatocytes from hiPS cells[27]. As discussed above, hepatocytes differentiated from hiPS cells are a promising and useful tool for basic research and clinical applications; however, differentiation protocols have yet to be established.

Hepatocyte differentiation

Before moving on to describing current methods for the generation of hepatocytes from human and mouse iPS cells, we will discuss the cellular mechanism that governs hepatocyte differentiation[37].

1. Growth factors and hepatocyte differentiation

Activin A, a member of the transforming growth factor-β superfamily, mimics Nodal, binds Activin receptors, and activates Smad2 by phosphorylation[38]. Furthermore, activin A enhances differentiation of human ES cells to endodermal cells[39]. The differentiation of iPS cells to endodermal cells requires the Wnt/β-catenin signaling pathway[40]. Indeed, Wnt/β-catenin modulates the patterning and organogenesis of the endoderm[41]. Wnt/β-catenin signaling also directs liver specification of cells in the foregut during liver development[42], and is essential for proliferation of hepatoblasts and differentiation of hepatoblasts to both bile duct epithelial cells and mature hepatocytes in mice[43]. Lastly, activin A and Wnt3A function synergistically to promote efficient differentiation of hiPS cells into endodermal cells In vitro[44].

Basic fibroblast growth factor (bFGF) sustains the self-renewal characteristics of human ES cells[45]. bFGF is also secreted from the developing heart during liver development[46]. bFGF causes the forgut endoderm to develop into the liver. Meanwhile, bone morphogenetic proteins (BMPs) are members of the TGF-β superfamily and are involved in spinal development[47]. BMP4 is secreted from the septum transversum during embryonic development, and it induces the expression of liver-specific genes in endodermal cells[48]. In addition, BMP4 stimulates the differentiation of hepatic stem cells to mature hepatocytes[49].

Hepatocyte growth factor (HGF) is secreted from sinusoidal endothelial cells in the liver[50]. HGF is a strong mitogen of hepatocytes and plays an important role in liver regeneration[51,52]. Indeed, HGF was found to promote differentiation of murine ES cells to hepatocytes[53]. Meanwhile, Oncostatin M (OncoM), a member of the interleukin-6 cytokine family that is required for differentiation of hepatoblasts to mature hepatocytes, is secreted from hematopoietic stem cells in fetal liver[54,55].

Dexamethasone and insulin-transferrin-selenium (ITS) are used to maintain the In vitro function of hepatocytes. These compounds promote differentiation of hepatoblasts[56], and are used to promote differentiation of hiPS cells to the hepatic lineage[57].

2. Transcription factors and hepatocyte differentiation

Transcription factors play essential roles during hepatocyte differentiation by controlling the expression levels of liver-specific genes[58]. The forkhead box A family of transcription factors consists of three members: FOXA1, FOXA2, and FOXA3[59]. These proteins, which are also known as hepatic nuclear factor 3 (HNF3) alpha (A1), beta (A2) and gamma (A3), are involved in both liver development and metabolism. Liver development is initiated with the formation of a liver bud, which is mediated by the FoxA1 and GATA4 transcriptional regulators[58]. A previous report demonstrated that FoxA1 is up-regulated in response to retinoic acid, and down-regulates Nanog[60]. This report also suggests that FoxA1 suppresses a gene strongly involved in maintenance of pluripotency while at the same time promoting differentiation toward hepatocytes. It has been predicted that induction of the expression of this transcription factor promotes differentiation of hiPS to hepatocytes. Meanwhile, a FoxA2 deficiency was lethal in mice due to severe defects in liver and pancreatic development[61]. FoxA2 and GATA4 are the first proteins that were demonstrated to bind to the albumin gene enhancer sequence in embryonic liver precursor cells[62]. These proteins relax compacted chromatin structures, and initiate hepatocyte differentiation.

FoxA3 contributes to hepatocyte differentiation and regulates the expression of liver-specific genes[63]. Together, GATA4, Fox1A, and FoxA3 induce transdifferentiation of mouse fibroblasts to functional hepatocyte-like cells[64]. In addition to the FoxA (HNF3) proteins, HNF4α is required for proper liver development[65]. Indeed, mice that are deficient in HNF4α are not viable due to the lack of normal gastrulation[66]. Furthermore, introduction of Hnf4α into knockout mice with HNF4α revealed that this compound is essential for differentiation of hepatocytes to cells with liver-specific metabolic functions. Furthermore, HNF4α regulates genes involved in drug metabolism such as P450 and uridine 5´-diphospho (UDP)-glucuronosyltransferases[65]. Interestingly, the human hepatocytes are rich with binding sites for both HNF1α and HNF4α[67]. These results support the conclusion that HNF1α and HNF4α are essential for hepatocyte differentiation.

The disruption of hematopoietically expressed homeobox (Hex) expression resulted in embryonic lethality that was attributable to insubstantial liver formation; thus, Hex is essential for liver organogenesis[68]. Moreover, Hex is essential for hepatoblast differentiation and bile duct formation[69].

The expression of sex-determining region Y (Sry) HMG box (Sox) 17 is restricted to the nascent primitive endodermal epithelium, and the absence of Sox17 leads to premature delamination and migration of the parietal endoderm[70].

CCAAT/enhancer binding protein α (C/EBPα) is a basic leucine zipper transcription factor. C/EBPα is enriched in liver cells where it governs the expression of liver specific genes, but is down-regulated in patients after partial hepatectomy[71,72]. Hepatocytes of knockout mice with C/EBPα are positive for A6 and albumin[73]. A6 is an antigen of biliary epithelial cells[74]. Albumin is a marker of hepatocytes. These results suggest that C/EBPα (-/-) hepatocytes exhibit characteristics of both hepatocytes and bile duct epithelial cells. Interestingly, suppression of C/EBPα expression in hepatoblasts promotes differentiation to bile duct epithelial cells[75]. These results suggest that C/EBPα promotes the differentiation of hepatoblasts to mature hepatocytes.

Protocols for differentiation of hiPS cells into hepatocytes

Growth factors and transcription factors play essential roles in hepatocyte differentiation. As such, the application of growth factors is critical for In vitro production of hepatocytes from hiPS cells and is therefore included in all current protocols. We will first describe the In vitro methods developed for the generation of hepatocytes using growth factors. Notably, the differentiation of hiPS cells to hepatocytes or cholangiocytes requires the application of distinct growth factors. A previous study demonstrated that cholangiocyte differentiation requires growth hormone, epidermal growth factor, interleukin-6, and sodium taurocholate[76]. Additional report suggests that the mechanism of hepatocyte differentiation from hepatoblasts is distinct from that of cholangiocytes[73,77]. Transcription factors are also essential for hepatocyte differentiation, and transfection reagents or electroporation are conventionally used to introduce genes of interest into iPS cells[78,79]; however, it remains difficult to effectively introduce transcription factors into hiPS cells using these methods[80]. Therefore, methods for hepatocyte differentiation using adenoviral vector-mediated expression of transcription factors will be discussed.

1. Generation of hepatocytes from iPS cells using growth factors

Most protocols have adopted the method of stepwise addition of growth factors to resemble the in vivo hepatocyte differentiation process during liver development[81-84] (Table 1). This process involves the progression of endodermal cells to immature hepatocytes (often referred to as hepatoblasts), and finally to mature hepatocytes. All current differentiation protocols include the use of activin A, which was shown to maintain the pluripotency of the iPS cells by inducing the expression of Nanog[85,86]; however, these reports are controversial. In these protocols, a high concentration of activin A (100 ng/mL) is applied to hiPS cells to induce hepatocyte differentiation, while 10 ng/mL is used to maintain pluripotency. Notably, the morphology of hiPS cells exposed to 10 ng/mL activin A is different from that of cells treated with 100 ng/mL[87]. It is speculated that activin A exerts different effects on iPS cells at higher and lower concentrations; however, the mechanism of this activity is unknown. Subsequently, LY294002 (a specific inhibitor of phosphatidyl-inositol 3 phosphatase), B27 supplement, or bFGF are added, depending on the purpose of the research. After 3-5 days of culturing, the iPS cells differentiate into endodermal cells. From days 5-10, a combination of BMP2 or 4 and either FGF2 or 4 is applied. FGF2 was shown to sustain the self-renewal characteristics of human ES cells[45]. Contrary to this phenomenon, however, FGF2 is added to hiPS to mediate differentiation to hepatocytes.

Chen et al[88] proposed another multistep protocol in which growth factors, but not transcription factors, are utilized. Using this method, they succeeded in differentiating hiPS cells into mature hepatocytes within only 12 days, which is significantly faster than the time required to generate hepatocytes in other studies. In their protocol, activin A (100 ng/mL) and HGF (10 ng/mL) are added from on days 1 to 3. Furthermore, hepatocyte-like cells were successfully derived when HGF was added at the first step of differentiation[88]. Sox17 and FoxA2 expression, which are induced by activin A, are important markers of endodermal differentiation. HGF and activin A may produce synergistic effects on the differentiating cells.

Similar to Chen et al, Kondo and co-workers generated hepatocyte-like cells by sequential stimulation with growth factors[89]. However, while Chen and co-workers utilized IMDM medium from day 8 to 12, the Kondo protocol requires the use of Modified Laford medium from day 12 to 21. Using their protocol, Kondo et al were successful in generating hepatocyte-like cells from two different hiPS cell lines. Furthermore, these hepatocyte-like cells are useful for drug screening as they express CYP3A4. This report was also informative in that the results indicate that the culture medium used might affect the differentiation process.

Yanagida and co-workers successfully generated hepatic progenitor-like cells upon sequential stimulation of hiPS cells with cytokines[90]. The stimulated cells were subjected to separation by flow cytometry using CD13- and CD133-specific antibodies, and the resulting cell population were found to express both hepatocyte and cholangiocyte markers. This approach is useful in that hepatic progenitor-like cells are thought to produce hepatocytes and cholangiocytes for development of the liver architecture.

Javed et al established a method by which hiPS cells harvested from patients can be utilized to generate hepatocytes[91]. In this method, Javed and co-workers sequentially stimulated the hiPS with growth factors, including activin A and bFGF. In contrast to the other protocols, however, they co-cultured the hiPS cells with non-parenchymal stellate cells of liver in embryoid bodies. Embryoid bodies have also been used for the differentiation of hepatocytes from mouse ES cells[55], and stellate cells are the source of growth factors required for liver regeneration[92,93]. This approach is reasonable in that the hiPS cells mature toward hepatocytes under more physiologically-relevant conditions.

In a separate study, Nakamura et al[94] derived mature hepatocytes from human ES and hiPS cells under feeder- and serum-free conditions. They also succeeded in producing cholangiocytes and proliferating progenitor cells. While indocyanine green was taken up by 30% of the resulting hepatocytes, 80% of the cells exhibited glycogen storage. The cells also exhibited CYP3A4 metabolic activity. These results indicate that the group successfully generated active hepatocytes.

Human serum contains multiple growth factors. Notably, in a previous study, sera from hepatectomised patients promoted hepatocyte differentiation from iPS cells[95]. As a result, this approach would be a promising method for In vitro culturing of hepatocytes. In addition, the application of proteomic approaches would elucidate the mechanism of hepatocyte differentiation upon exposure to these sera[96].

2. Generation of hepatocytes from iPS cells using transcription factors

Transcription factors play important roles in liver development and hepatocyte differentiation[97]. However, only a few protocols using transcription factors have been developed. The primary reason for this is that the introduction of genes into iPS cells is technically difficult. While transfection of human or mouse iPS cells with plasmids is generally ineffective, adenoviral vectors provide highly efficient transduction of hiPS cells[98]. Inamura et al[99] transduced hES and hiPS cells with a Hex expression vector resulting in efficient production of hepatoblasts (Table 2). Previously, this group demonstrated that transduction of hepatoblasts with an HNF4α expression vector yielded differentiation into mature hepatocytes In vitro[84]. In this protocol however, hepatoblasts were first incubated with activin A. Sox17 was then introduced to promote differentiation, followed by introduction of HNF4 to induce terminal differentiation of the hepatoblasts into hepatocytes. Mature hepatocytes appeared at approximately 20 days after the initiation of the differentiation process. These reports are interesting in that they suggest that the In vitro differentiation process of hiPS cells into hepatocytes resembles that which occurs in vivo.

In a separate study, Sekine et al[100] treated cells with the phosphoinositide 3-kinase (PI3K) inhibitor LY294002 as well as 100 ng/mL activin A. Either HGF or keratinocyte growth factor (KGF) was then applied from days 10-14. In this study, while FoxA2 and Sox17 expression were observed, the expression of alpha-fetoprotein (AFP) and albumin were not analyzed. These results indicate that PI3Ks may control the differentiation of iPS cells into endodermal cells; however, other factors would still be required to mediate this process In vitro.

Limitations associated with hepatocytes derived from hiPS cells

Cells cultured using the protocols mentioned above are referred to as hepatocyte-like cells. This is due to the fact that these cells exhibit only certain similarities to primary hepatocytes. First, hepatocyte-like cells have been shown to remain in an immature state[101]. Furthermore, the detoxification activity of these cells is lower than that of primary hepatocytes in culture[82,84]. Hepatocytes derived from hiPS cells also exhibit lower levels of FoxA1, FoxA2, FoxA3, and HNF1α expression, and Takayama et al[84] therefore speculated that In vitro growth of hepatocytes requires additional factors that have yet to be identified. Meanwhile, hepatocytes differentiated from hiPS cells express certain mRNA molecules that are normally not detected in fetal or adult liver tissues[81]. Notably, hepatocytes are differentiated more efficiently from hepatoblast-derived hiPS cells than from adult hepatocytes, suggesting that the efficiency of differentiation may depend on the origin of the iPS cells. Indeed, differentiation potential depends on the hiPS cells used[102], and there are differences between hiPS cell populations in the efficiency of differentiation to hepatocytes, as evidenced by differences in albumin expression levels[103]. The reprogramming factors themselves affect the differentiation of iPS cells to specific cell fates[104]. Although the results originate from mouse iPS cells, there are differences in the methylation sites between iPS cells reprogrammed with different factors. As a result, given that the previously mentioned liver-specific genes are important for clin ical and pharmacological applications, these protocols need to be further developed before the resulting cell populations can be used in a medical setting.

Novel approaches for the generation of hepatocytes from iPS cells

Despite the protocols described above, it is still challenging to obtain mature hepatocytes In vitro. One of the reasons for these difficulties is that the iPS cells utilized in these studies have been cultured two-dimensionally on plates or dishes[105]. To overcome this limitation, novel approaches are under investigation that can be divided into three categories: the extracellular matrix, 3D culture, and cell sheet approaches.

An extracellular matrix (ECM) provides conditions suitable for cultured cells to differentiate into hepatocytes. In one study, co-culturing of mouse ES cells with M15 cells, a mesonephric cell line, induced differentiation of the ES cells into the hepatocyte lineage[106]. Eighty percent of the mouse ES cells cultured with M15 expressed AFP, and 9% expressed albumin. Interestingly, even fixed M15 cells promoted mouse ES cell differentiation. Meanwhile, Shiraki et al reported that a synthesized basement membrane composed of human recombinant laminin 511[107] induced differentiation of mouse ES cells into hepatocyte lineages.

A 3D culture system composed of gelatin and an ECM derived from Swiss 3T3 cells[108] preserved the functions of hepatocyte-like cells differentiated from hiPS cells. The most important ECM component was determined to be type 1 collagen. In a separate study, 3D culturing promoted the maturation of hepatocytes derived from hiPS cells[109].

For this culturing technique, cells are grown in hollow fibers similar to embryoid bodies. Hollow fibers are useful because the efficiency of embryoid body formation is low compared to that of mouse ES cells, which also differentiate into hepatocytes within hollow fibers[110]. This organoid culture system allows efficient formation of mouse ES cell aggregates in their lumen, and the liver-specific functions of the mouse ES cells are comparable with those of primary hepatocytes.

Takebe et al cultured hiPS cells with human mesenchymal stem cells and human umbilical vascular endothelial cells[111]. The mixed cell populations formed three-dimensional structures that were transplantable to mice. The transplanted complexes were vascularized and resembled an organ-like structure similar to the liver. This method is promising in that the liver-like structure could be utilized for future therapeutic strategies.

Du et al assembled hepatocytes and endothelial cells in hydrogel fibers to form three-dimensional cultures[112]. Notably, the hepatocytes and endothelial cells were both differentiated from iPS cells. Their report therefore suggests that three-dimensional structures could be generated from hiPS cells harvested from patients, which could then be transplantable.

Primary rat hepatocytes have been successfully cultured for 200 days on temperature-responsive sheets[113]. Furthermore, primary rat hepatocytes maintained liver-specific functions for up to 28 days on the temperature-responsive sheets when cultured with endothelial cells[114]. These sheets attach to the bottoms of culture dishes at 37℃ and detach at 25℃, and provide a system for easy culturing and handling of cells. Likewise, this system enables easy manipulation of hiPS cells and may be used to promote differentiation of these cells into hepatocytes.

Valproic acid (VPA) is a histone deacetylase inhibitor[115], which enables efficient generation of iPS cells[116]. Indeed, Kondo et al utilized this compound to promote differentiation of hepatocytes from iPS cells[117]. Their protocol essentially involves multistep stimulation with growth factors such as activin A, HGF, OncoM, and dexamethasone. Importantly, the expression levels of liver-specific genes and of albumin were higher in the cell populations treated with VPA than in the untreated populations. Likewise, genes involved in drug-metabolism were highly expressed in the VPA-treated cells. While the mechanism is unclear, the stage at which VPA is administered and the duration of VPA treatment affected the efficiency of hepatocyte differentiation.

For practical application of hepatocytes derived from iPS cells, it is necessary to produce these cells on a large scale. Stirred-suspension bioreactors are useful for scalable In vitro generation of hepatocytes[118]. Vosough et al initiated hepatocyte differentiation by treating a stirred-suspension culture with rapamycin and activin A. The resulting cells exhibited hepatocyte characteristics such as albumin secretion and urea production. Although these cells required further maturation, this strategy provides a method with the potential to generate sizable quantities of hepatocytes from iPS cells In vitro.

The application of iPS cell-derived hepatocytes to the screening of drugs for liver toxicity requires enhanced expression of CYP3A and other genes that are important for detoxification. HNF6 up-regulates CYP3A4 in iPS cells[119], suggesting that In vitro generation of hepatocyte-like cells that perform specific functions similarly to mature hepatocytes is an option for overcoming the current limitations associated with hepatocyte-like cells that are described above. In addition, it has been suggested that transcription factors are key players in the generation of mature hepatocytes from iPS cells. Indeed, FoxA1 and HNF1α promote differentiation of hiPS cells to hepatocytes that exhibit mature functions[120].

Direct production of hepatocytes from fibroblasts

In addition to the production of hepatocytes derived from iPS cells, the generation of hepatocytes directly from fibroblasts has been reported. In a previous publication, ectopic expression of HNF1α, HNF4α, and FoxA3 in human fibroblasts using a lentiviral expression system resulted in the differentiation to hepatocytes that, upon transplantation, enhanced the survival of mice with liver damage[121]. In a separate report, HNF1A, HNF4A, HNF6, ATF5, PORX1, and C/EBPα were expressed in human fibroblasts, and the resulting hepatocytes exhibited expression of genes involved in drug metabolism, such as CYP3A4, at levels comparable to those observed in freshly-isolated human primary hepatocytes[122]. Surprisingly, hepatocytes generated from human fibroblasts were capable of repopulating mouse livers[123]. With further refinement, this approach could circumvent the need for the differentiation of hiPS cells to hepatocytes. Meanwhile, the limitation of this approach is that, because they are primary cells, the ability to passage and propagate fibroblasts is limited.

The tumorigenicity of iPS cells

A significant concern associated with the transplantation of iPS cell-derived hepatocytes is the risk of tumorigenicity[124]. This tumorigenicity was initially attributed to genomic integration of viral vectors[125]. Therefore, to reduce this risk, plasmid vectors, Sendai virus vectors, and RNA synthesized from the cDNA of the four reprogramming transcription factors have been used to induce hepatocyte differentiation[126-128]. Additionally, combinations of reprogramming factors have been investigated[129]. Despite these efforts, however, the risk of tumorigenicity has yet to be eliminated. The link between pluripotency and tumorigenicity was first reported in 1960 in a study of teratocarcinoma[130]. Both pluripotency and tumorigenicity involve mechanisms of self-renewal, proliferation, and active telomerases[131]. As a result, it is difficult to eliminate the risk of tumorigenicity due to residual hiPS cells that persist in the transplanted material. It is therefore necessary to develop methods for eradicating iPS cells within differentiated somatic cell populations.

Purification of hepatocytes from iPS cells

Flow cytometry, heat shock, suicidal gene therapy, and toxic materials have been utilized for purification of hepatocytes. Yamamoto et al introduced green fluorescent protein (GFP), driven by an albumin promoter/enhancer, into mouse ES cells[16]. Albumin is also expressed in endodermal cells[132]. Meanwhile, the genetic knock-in of a marker gene at the albumin locus is expected to enable purification of hepatocytes differentiated from hiPS cells[133]. The flow cytometry with albumin promoter/enhancer may isolate the other cells than hepatocytes, such as endodermal cells.

Immunolabeling of delta-like 1 homolog (DLK1) has been utilized for the isolation of hepatoblasts by flow cytometry [134]. The drawback of this approach, however, is that DLK1 is not expressed in the adult human liver[135]. Meanwhile, sublethal heat shock treatment induced apoptosis in human ES cells[136]; however, this approach might also damage the hepatocytes. In addition, the thymidine kinase gene, driven by the Nanog promoter, was introduced into hiPS cells[137]. The cells were subsequently ablated with ganciclovir treatment. This method is ideal for selecting differentiated hepatocytes because they do not express Nanog. One potential limitation of this approach, however, is that ganciclovir might be toxic to hepatocytes. Likewise, while, benzethonium chloride, methylbenzethonium, and N-oleoyl serinol induce apoptosis in human iPS and mouse ES cells[138,139], these compounds may also damage hepatocytes. Thus, efficient purification methods that do not result in damage to hepatocytes have yet to be developed.

Hepatocyte selection medium

Glucose is an important energy source for cell growth and survival. Glucose deprivation aids in the purification of hepatocytes because, unlike their precursor cell types, they are capable of producing this monosaccharide[140]. Pyruvate, which is the end product of glycolysis, is incorporated into the citric acid cycle, and removal of pyruvate and glucose from In vitro growth media resulted in neuronal cell death[141]. Meanwhile, galactose enters glycolysis as a substrate for galactokinase, which is expressed in the liver and kidney (Figure 1)[142,143]. Therefore, it is predicted that hepatocytes can survive in a medium containing galactose but lacking glucose and pyruvate[144,145].

Of the amino acids, arginine deprivation is least tolerated by cells cultured In vitro[146]. Arginine is produced through the urea cycle, which is exclusive to hepatocytes (Figure 2). Indeed, the removal of arginine led to the development of the first medium for purifying hepatocytes[140]. Subsequently, hepatocytes were purified from hiPS cells by culturing in a medium lacking both arginine and tyrosine, referred to as hepatocyte selection medium (HSM)[147]. HSM is advantageous in that it does not contain toxic reagents and consists only of common media components. As a result, this medium is not harmful to hepatocytes.

Conclusions

Human iPS cells are a promising source of hepatocytes, which could be utilized for drug screening, for cell transplantation, and as a model for studying human diseases. While several protocols for the differentiation of hiPS cells into hepatocytes have been published, the cell populations derived using these methods exhibit limited hepatocyte characteristics. In the future, as more sophisticated methods are developed, novel applications of these cells will be realized.

CONFLICT OF INTERESTS

There are no conflicts of interest with regard to the present study.

REFERENCES

1Michalopoulos GK. Principles of liver regeneration and growth homeostasis. Compr Physiol 2013; 3(1): 485-513 [PMID: 23720294 DOI: 10.1002/cphy.c120014]

2Habibullah CM, Syed IH, Qamar A, Taher-Uz Z. Human fetal hepatocyte transplantation in patients with fulminant hepatic failure. Transplantation 1994; 58(8): 951-952 [PMID: 7940741]

3Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, Yamanaka S. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 2007; 131(5): 861-872 [PMID: 18035408 DOI: S0092-8674(07)01471-7 [pii] 10.1016/j.cell.2007.11.019]

4Strom SC, Chowdhury JR, Fox IJ. Hepatocyte transplantation for the treatment of human disease. Semin Liver Dis 1999; 19(1): 39-48 [PMID: 10349682 DOI: 10.1055/s-2007-1007096]

5Riehle KJ, Dan YY, Campbell JS, Fausto N. New concepts in liver regeneration. J Gastroenterol Hepatol 2011; 26 Suppl 1: 203-212 [PMID: 21199532 PMCID: 3077908 DOI: 10.1111/j.1440-1746.2010.06539.x]

6Fisher RA, Bu D, Thompson M, Wolfe L, Ritter JK. Optimization of conditions for clinical human hepatocyte infusion. Cell Transplant 2004; 13(6): 677-689 [PMID: 15648738]

7Mitaka T, Sattler CA, Sattler GL, Sargent LM, Pitot HC. Multiple cell cycles occur in rat hepatocytes cultured in the presence of nicotinamide and epidermal growth factor. Hepatology 1991; 13(1): 21-30 [PMID: 1824839 DOI: 0270-9139(91)90211-D [pii]]

8Krause DS, Theise ND, Collector MI, Henegariu O, Hwang S, Gardner R, Neutzel S, Sharkis SJ. Multi-organ, multi-lineage engraftment by a single bone marrow-derived stem cell. Cell 2001; 105(3): 369-377 [PMID: 11348593 DOI: S0092-8674(01)00328-2 [pii]]

9Theise ND, Badve S, Saxena R, Henegariu O, Sell S, Crawford JM, Krause DS. Derivation of hepatocytes from bone marrow cells in mice after radiation-induced myeloablation. Hepatology 2000; 31(1): 235-240 [PMID: 10613752 DOI: S0270913900990256 [pii] 10.1002/hep.510310135]

10Theise ND, Nimmakayalu M, Gardner R, Illei PB, Morgan G, Teperman L, Henegariu O, Krause DS. Liver from bone marrow in humans. Hepatology 2000; 32(1): 11-16 [PMID: 10869283 DOI: S0270913900830116 [pii] 10.1053/jhep.2000.9124]

11Sakaida I, Terai S, Yamamoto N, Aoyama K, Ishikawa T, Nishina H, Okita K. Transplantation of bone marrow cells reduces CCl4-induced liver fibrosis in mice. Hepatology 2004; 40(6): 1304-1311 [PMID: 15565662 DOI: 10.1002/hep.20452]

12Volarevic V, Nurkovic J, Arsenijevic N, Stojkovic M. Concise review: therapeutic potential of mesenchymal stem cells for the treatment of acute liver failure and cirrhosis. Stem Cells 2014; 32(11): 2818-2823 [PMID: 25154380 DOI: 10.1002/stem.1818]

13Hang H, Yu Y, Wu N, Huang Q, Xia Q, Bian J. Induction of highly functional hepatocytes from human umbilical cord mesenchymal stem cells by HNF4alpha transduction. PLoS One 2014; 9(8): e104133 [PMID: 25137413 PMCID: 4138090 DOI: 10.1371/journal.pone.0104133 PONE-D-14-01747 [pii]]

14Li X, Yuan J, Li W, Liu S, Hua M, Lu X, Zhang H. Direct differentiation of homogeneous human adipose stem cells into functional hepatocytes by mimicking liver embryogenesis. J Cell Physiol 2014; 229(6): 801-812 [PMID: 24166453 DOI: 10.1002/jcp.24501]

15Yin Y, Lim YK, Salto-Tellez M, Ng SC, Lin CS, Lim SK. AFP(+), ESC-derived cells engraft and differentiate into hepatocytes in vivo. Stem Cells 2002; 20(4): 338-346 [PMID: 12110703]

16Yamamoto H, Quinn G, Asari A, Yamanokuchi H, Teratani T, Terada M, Ochiya T. Differentiation of embryonic stem cells into hepatocytes: biological functions and therapeutic application. Hepatology 2003; 37(5): 983-993 [PMID: 12717379]

17Basma H, Soto-Gutierrez A, Yannam GR, Liu L, Ito R, Yamamoto T, Ellis E, Carson SD, Sato S, Chen Y, Muirhead D, Navarro-Alvarez N, Wong RJ, Roy-Chowdhury J, Platt JL, Mercer DF, Miller JD, Strom SC, Kobayashi N, Fox IJ. Differentiation and transplantation of human embryonic stem cell-derived hepatocytes. Gastroenterology 2009; 136(3): 990-999 [PMID: 19026649]

18Chang HM, Liao YW, Chiang CH, Chen YJ, Lai YH, Chang YL, Chen HL, Jeng SY, Hsieh JH, Peng CH, Li HY, Chien Y, Chen SY, Chen LK, Huo TI. Improvement of Carbon Tetrachloride-Induced Acute Hepatic Failure by Transplantation of Induced Pluripotent Stem Cells without Reprogramming Factor c-Myc. Int J Mol Sci 2012; 13(3): 3598-3617 [PMID: 22489170 PMCID: 3317730 DOI: 10.3390/ijms13033598 ijms-13-03598 [pii]]

19Zhang R, Takebe T, Sekine K, Koike H, Zheng Y, Taniguchi H. Identification of proliferating human hepatic cells from human induced pluripotent stem cells. Transplant Proc 2014; 46(4): 1201-1204 [PMID: 24815160 DOI: S0041-1345(13)01417-6 [pii] 10.1016/j.transproceed.2013.12.021]

20van Wenum M, Chamuleau RA, van Gulik TM, Siliakus A, Seppen J, Hoekstra R. Bioartificial livers In vitro and in vivo: tailoring biocomponents to the expanding variety of applications. Expert Opin Biol Ther 2014; 14(12): 1745-1760 [PMID: 25366164 DOI: 10.1517/14712598.2014.950651]

21Iwamuro M, Shiraha H, Nakaji S, Furutani M, Kobayashi N, Takaki A, Yamamoto K. A preliminary study for constructing a bioartificial liver device with induced pluripotent stem cell-derived hepatocytes. Biomed Eng Online 2012; 11: 93 [PMID: 23217363 PMCID: 3549893 DOI: 1475-925X-11-93 [pii] 10.1186/1475-925X-11-93]

22Gomez-Lechon MJ, Tolosa L, Conde I, Donato MT. Competency of different cell models to predict human hepatotoxic drugs. Expert Opin Drug Metab Toxicol 2014; 10(11): 1553-1568 [PMID: 25297626 DOI: 10.1517/17425255.2014.967680]

23Takayama K, Morisaki Y, Kuno S, Nagamoto Y, Harada K, Furukawa N, Ohtaka M, Nishimura K, Imagawa K, Sakurai F, Tachibana M, Sumazaki R, Noguchi E, Nakanishi M, Hirata K, Kawabata K, Mizuguchi H. Prediction of interindividual differences in hepatic functions and drug sensitivity by using human iPS-derived hepatocytes. Proc Natl Acad Sci U S A 2014; 111(47): 16772-16777 [PMID: 25385620 DOI: 1413481111 [pii] 10.1073/pnas.1413481111]

24Kia R, Sison RL, Heslop J, Kitteringham NR, Hanley N, Mills JS, Park BK, Goldring CE. Stem cell-derived hepatocytes as a predictive model for drug-induced liver injury: are we there yet? Br J Clin Pharmacol 2013; 75(4): 885-896 [PMID: 22703588 PMCID: 3612706 DOI: 10.1111/j.1365-2125.2012.04360.x]

25Greenhough S, Medine CN, Hay DC. Pluripotent stem cell derived hepatocyte like cells and their potential in toxicity screening. Toxicology 2010; 278(3): 250-255 [PMID: 20674645 DOI: S0300-483X(10)00291-X [pii] 10.1016/j.tox.2010.07.012]

26Sirenko O, Hesley J, Rusyn I, Cromwell EF. High-content assays for hepatotoxicity using induced pluripotent stem cell-derived cells. Assay Drug Dev Technol 2014; 12(1): 43-54 [PMID: 24229356 PMCID: 3934660 DOI: 10.1089/adt.2013.520]

27Dusseaux M, Darche S, Strick-Marchand H. Animal models to test hiPS-derived hepatocytes in the context of inherited metabolic liver diseases. Methods Mol Biol 2014; 1213: 81-88 [PMID: 25173376 DOI: 10.1007/978-1-4939-1453-1_8]

28Satoh D, Maeda T, Ito T, Nakajima Y, Ohte M, Ukai A, Nakamura K, Enosawa S, Toyota M, Miyagawa Y, Okita H, Kiyokawa N, Akutsu H, Umezawa A, Matsunaga T. Establishment and directed differentiation of induced pluripotent stem cells from glycogen storage disease type Ib patient. Genes Cells 2013; 18(12): 1053-1069 [PMID: 24581426 DOI: 10.1111/gtc.12101]

29GuoCayo MA, Cai J, Delaforest A, Noto FK, Nagaoka M, Clark BS, Collery RF, Si-Tayeb K, Duncan SA. ‘JD’ iPS cell-derived hepatocytes faithfully recapitulate the pathophysiology of familial hypercholesterolemia. Hepatology 2012 [PMID: 22653811 DOI: 10.1002/hep.25871]

30GuoDusek P, Litwin T, Czlonkowska A. Wilson Disease and Other Neurodegenerations with Metal Accumulations. Neurol Clin 2015; 33(1): 175-204 [PMID: 25432729 DOI: S0733-8619(14)00074-7 [pii] 10.1016/j.ncl.2014.09.006]

31GuoYi F, Qu J, Li M, Suzuki K, Kim NY, Liu GH, Belmonte JC. Establishment of hepatic and neural differentiation platforms of Wilson’s disease specific induced pluripotent stem cells. Protein Cell 2012; 3(11): 855-863 [PMID: 22806248 DOI: 10.1007/s13238-012-2064-z]

32GuoLee SB, Seo D, Choi D, Park KY, Holczbauer A, Marquardt JU, Conner EA, Factor VM, Thorgeirsson SS. Contribution of hepatic lineage stage-specific donor memory to the differential potential of induced mouse pluripotent stem cells. Stem Cells 2012; 30(5): 997-1007 [PMID: 22378611 DOI: 10.1002/stem.1074]

33GuoWu X, Robotham JM, Lee E, Dalton S, Kneteman NM, Gilbert DM, Tang H. Productive hepatitis C virus infection of stem cell-derived hepatocytes reveals a critical transition to viral permissiveness during differentiation. PLoS Pathog 2012; 8(4): e1002617 [PMID: 22496645 PMCID: 3320597 DOI: 10.1371/journal.ppat.1002617 PPATHOGENS-D-11-02669 [pii]]

34GuoCarpentier A, Tesfaye A, Chu V, Nimgaonkar I, Zhang F, Lee SB, Thorgeirsson SS, Feinstone SM, Liang TJ. Engrafted human stem cell-derived hepatocytes establish an infectious HCV murine model. J Clin Invest 2014; 124(11): 4953-4964 [PMID: 25295540 DOI: 75456 [pii] 10.1172/JCI75456]

35GuoSchwartz RE, Trehan K, Andrus L, Sheahan TP, Ploss A, Duncan SA, Rice CM, Bhatia SN. Modeling hepatitis C virus infection using human induced pluripotent stem cells. Proc Natl Acad Sci U S A 2012; 109(7): 2544-2548 [PMID: 22308485 PMCID: 3289320 DOI: 1121400109 [pii] 10.1073/pnas.1121400109]

36GuoShlomai A, Schwartz RE, Ramanan V, Bhatta A, de Jong YP, Bhatia SN, Rice CM. Modeling host interactions with hepatitis B virus using primary and induced pluripotent stem cell-derived hepatocellular systems. Proc Natl Acad Sci U S A 2014; 111(33): 12193-12198 [PMID: 25092305 PMCID: 4143014 DOI: 1412631111 [pii] 10.1073/pnas.1412631111]

37GuoMiyajima A, Tanaka M, Itoh T. Stem/progenitor cells in liver development, homeostasis, regeneration, and reprogramming. Cell Stem Cell 2014; 14(5): 561-574 [PMID: 24792114 DOI: S1934-5909(14)00147-7 [pii] 10.1016/j.stem.2014.04.010]

38GuoSulzbacher S, Schroeder IS, Truong TT, Wobus AM. Activin A-induced differentiation of embryonic stem cells into endoderm and pancreatic progenitors-the influence of differentiation factors and culture conditions. Stem Cell Rev 2009; 5(2): 159-173 [PMID: 19263252 DOI: 10.1007/s12015-009-9061-5]

39Guo S, Mao X, He F, Liu H, Ming L. Activin A supplement in the hESCs culture enhances the endoderm differentiation efficiency. Cell Biol Int 2014; 38(7): 849-856 [PMID: 24604611 DOI: 10.1002/cbin.10274]

40Naujok O, Diekmann U, Lenzen S. The generation of definitive endoderm from human embryonic stem cells is initially independent from activin A but requires canonical Wnt-signaling. Stem Cell Rev 2014; 10(4): 480-493 [PMID: 24913278 DOI: 10.1007/s12015-014-9509-0]

41Payne C, King J, Hay D. The role of activin/nodal and Wnt signaling in endoderm formation. Vitam Horm 2011; 85: 207-216 [PMID: 21353882 DOI: B978-0-12-385961-7.00010-X [pii] 10.1016/B978-0-12-385961-7.00010-X]

42Lade AG, Monga SP. Beta-catenin signaling in hepatic development and progenitors: which way does the WNT blow? Dev Dyn 2011; 240(3): 486-500 [PMID: 21337461 DOI: 10.1002/dvdy.22522]

43Tan X, Yuan Y, Zeng G, Apte U, Thompson MD, Cieply B, Stolz DB, Michalopoulos GK, Kaestner KH, Monga SP. Beta-catenin deletion in hepatoblasts disrupts hepatic morphogenesis and survival during mouse development. Hepatology 2008; 47(5): 1667-1679 [PMID: 18393386 DOI: 10.1002/hep.22225]

44Toivonen S, Lundin K, Balboa D, Ustinov J, Tamminen K, Palgi J, Trokovic R, Tuuri T, Otonkoski T. Activin A and Wnt-dependent specification of human definitive endoderm cells. Exp Cell Res 2013; 319(17): 2535-2544 [PMID: 23954819 DOI: S0014-4827(13)00296-6 [pii] 10.1016/j.yexcr.2013.07.007]

45Xu RH, Peck RM, Li DS, Feng X, Ludwig T, Thomson JA. Basic FGF and suppression of BMP signaling sustain undifferentiated proliferation of human ES cells. Nat Methods 2005; 2(3): 185-190 [PMID: 15782187 DOI: nmeth744 [pii] 10.1038/nmeth744]

46Jung J, Zheng M, Goldfarb M, Zaret KS. Initiation of mammalian liver development from endoderm by fibroblast growth factors. Science 1999; 284(5422): 1998-2003 [PMID: 10373120 DOI: 7595 [pii]]

47Kallioniemi A. Bone morphogenetic protein 4-a fascinating regulator of cancer cell behavior. Cancer Genet 2012; 205(6): 267-277 [PMID: 22749032 DOI: S2210-7762(12)00150-0 [pii] 10.1016/j.cancergen.2012.05.009]

48Rossi JM, Dunn NR, Hogan BL, Zaret KS. Distinct mesodermal signals, including BMPs from the septum transversum mesenchyme, are required in combination for hepatogenesis from the endoderm. Genes Dev 2001; 15(15): 1998-2009 [PMID: 11485993 PMCID: 312750 DOI: 10.1101/gad.904601]

49Majumder S, Siamwala JH, Srinivasan S, Sinha S, Sridhara SR, Soundararajan G, Seerapu HR, Chatterjee S. Simulated microgravity promoted differentiation of bipotential murine oval liver stem cells by modulating BMP4/Notch1 signaling. J Cell Biochem 2011; 112(7): 1898-1908 [PMID: 21433062 DOI: 10.1002/jcb.23110]

50Maher JJ. Cell-specific expression of hepatocyte growth factor in liver. Upregulation in sinusoidal endothelial cells after carbon tetrachloride. J Clin Invest 1993; 91(5): 2244-2252 [PMID: 7683700 PMCID: 288227 DOI: 10.1172/JCI116451]

51DeLeve LD. Liver sinusoidal endothelial cells and liver regeneration. J Clin Invest 2013; 123(5): 1861-1866 [PMID: 23635783 PMCID: 3635729 DOI: 66025 [pii] 10.1172/JCI66025]

52Nakamura T, Sakai K, Matsumoto K. Hepatocyte growth factor twenty years on: Much more than a growth factor. J Gastroenterol Hepatol 2011; 26 Suppl 1: 188-202 [PMID: 21199531 DOI: 10.1111/j.1440-1746.2010.06549.x]

53Schuldiner M, Yanuka O, Itskovitz-Eldor J, Melton DA, Benvenisty N. Effects of eight growth factors on the differentiation of cells derived from human embryonic stem cells. Proc Natl Acad Sci U S A 2000; 97(21): 11307-11312 [PMID: 11027332 PMCID: 17196 DOI: 10.1073/pnas.97.21.11307. 97/21/11307 [pii]]

54Kinoshita T, Sekiguchi T, Xu MJ, Ito Y, Kamiya A, Tsuji K, Nakahata T, Miyajima A. Hepatic differentiation induced by oncostatin M attenuates fetal liver hematopoiesis. Proc Natl Acad Sci U S A 1999; 96(13): 7265-7270 [PMID: 10377403 PMCID: 22074]

55Tomizawa M, Toyama Y, Ito C, Toshimori K, Iwase K, Takiguchi M, Saisho H, Yokosuka O. Hepatoblast-like cells enriched from mouse embryonic stem cells in medium without glucose, pyruvate, arginine, and tyrosine. Cell Tissue Res 2008; 333(1): 17-27 [PMID: 18478268]

56Kamiya A, Kinoshita T, Ito Y, Matsui T, Morikawa Y, Senba E, Nakashima K, Taga T, Yoshida K, Kishimoto T, Miyajima A. Fetal liver development requires a paracrine action of oncostatin M through the gp130 signal transducer. Embo J 1999; 18(8): 2127-2136 [PMID: 10205167 PMCID: 1171297 DOI: 10.1093/emboj/18.8.2127]

57Tomizawa M, Shinozaki F, Sugiyama T, Yamamoto S, Sueishi M, Yoshida T. Single-step protocol for the differentiation of human-induced pluripotent stem cells into hepatic progenitor-like cells. Biomed Rep 2013; 1(1): 18-22 [PMID: 24648886 PMCID: 3956730 DOI: 10.3892/br.2012.2. br-01-01-0018 [pii]]

58Zaret KS, Carroll JS. Pioneer transcription factors: establishing competence for gene expression. Genes Dev 2011; 25(21): 2227-2241 [PMID: 22056668 PMCID: 3219227 DOI: 25/21/2227 [pii] 10.1101/gad.176826.111]

59Kaestner KH, Knochel W, Martinez DE. Unified nomenclature for the winged helix/forkhead transcription factors. Genes Dev 2000; 14(2): 142-146 [PMID: 10702024]

60Chen T, He S, Zhang Z, Gao W, Yu L, Tan Y. Foxa1 contributes to the repression of Nanog expression by recruiting Grg3 during the differentiation of pluripotent P19 embryonal carcinoma cells. Exp Cell Res 2014; 326(2): 326-335 [PMID: 24803390 DOI: S0014-4827(14)00178-5 [pii] 10.1016/j.yexcr.2014.04.020]

61Ang SL, Rossant J. HNF-3 beta is essential for node and notochord formation in mouse development. Cell 1994; 78(4): 561-574 [PMID: 8069909 DOI: 0092-8674(94)90522-3 [pii]]

62Cirillo LA, Lin FR, Cuesta I, Friedman D, Jarnik M, Zaret KS. Opening of compacted chromatin by early developmental transcription factors HNF3 (FoxA) and GATA-4. Mol Cell 2002; 9(2): 279-289 [PMID: 11864602 DOI: S1097276502004598 [pii]]

63Motallebipour M, Ameur A, Reddy Bysani MS, Patra K, Wallerman O, Mangion J, Barker MA, McKernan KJ, Komorowski J, Wadelius C. Differential binding and co-binding pattern of FOXA1 and FOXA3 and their relation to H3K4me3 in HepG2 cells revealed by ChIP-seq. Genome Biol 2009; 10(11): R129 [PMID: 19919681 PMCID: 3091322 DOI: gb-2009-10-11-r129 [pii] 10.1186/gb-2009-10-11-r129]

64Huang P, He Z, Ji S, Sun H, Xiang D, Liu C, Hu Y, Wang X, Hui L. Induction of functional hepatocyte-like cells from mouse fibroblasts by defined factors. Nature 2011; 475(7356): 386-389 [PMID: 21562492 DOI: nature10116 [pii] 10.1038/nature10116]

65Gonzalez FJ. Regulation of hepatocyte nuclear factor 4 alpha-mediated transcription. Drug Metab Pharmacokinet 2008; 23(1): 2-7 [PMID: 18305369 DOI: JST.JSTAGE/dmpk/23.2 [pii]]

66Li J, Ning G, Duncan SA. Mammalian hepatocyte differentiation requires the transcription factor HNF-4alpha. Genes Dev 2000; 14(4): 464-474 [PMID: 10691738 PMCID: 316377]

67Bonder MJ, Kasela S, Kals M, Tamm R, Lokk K, Barragan I, Buurman WA, Deelen P, Greve JW, Ivanov M, Rensen SS, van Vliet-Ostaptchouk JV, Wolfs MG, Fu J, Hofker MH, Wijmenga C, Zhernakova A, Ingelman-Sundberg M, Franke L, Milani L. Genetic and epigenetic regulation of gene expression in fetal and adult human livers. BMC Genomics 2014; 15: 860 [PMID: 25282492 DOI: 1471-2164-15-860 [pii] 10.1186/1471-2164-15-860]

68Keng VW, Yagi H, Ikawa M, Nagano T, Myint Z, Yamada K, Tanaka T, Sato A, Muramatsu I, Okabe M, Sato M, Noguchi T. Homeobox gene Hex is essential for onset of mouse embryonic liver development and differentiation of the monocyte lineage. Biochem Biophys Res Commun 2000; 276(3): 1155-1161 [PMID: 11027604]

69Hunter MP, Wilson CM, Jiang X, Cong R, Vasavada H, Kaestner KH, Bogue CW. The homeobox gene Hhex is essential for proper hepatoblast differentiation and bile duct morphogenesis. Dev Biol 2007; 308(2): 355-367 [PMID: 17580084]

70Artus J, Piliszek A, Hadjantonakis AK. The primitive endoderm lineage of the mouse blastocyst: sequential transcription factor activation and regulation of differentiation by Sox17. Dev Biol 2011; 350(2): 393-404 [PMID: 21146513 DOI: S0012-1606(10)01248-0 [pii] 10.1016/j.ydbio.2010.12.007]

71Xanthopoulos KG, Prezioso VR, Chen WS, Sladek FM, Cortese R, Darnell JE, Jr. The different tissue transcription patterns of genes for HNF-1, C/EBP, HNF-3, and HNF-4, protein factors that govern liver-specific transcription. Proc Natl Acad Sci U S A 1991; 88(9): 3807-3811 [PMID: 2023930 PMCID: 51542]

72Flodby P, Antonson P, Barlow C, Blanck A, Porsch-Hallstrom I, Xanthopoulos KG. Differential patterns of expression of three C/EBP isoforms, HNF-1, and HNF-4 after partial hepatectomy in rats. Exp Cell Res 1993; 208(1): 248-256 [PMID: 8359219 DOI: S0014-4827(83)71244-9 [pii] 10.1006/excr.1993.1244]

73Tomizawa M, Garfield S, Factor V, Xanthopoulos KG. Hepatocytes deficient in CCAAT/enhancer binding protein alpha (C/EBP alpha) exhibit both hepatocyte and biliary epithelial cell character. Biochem Biophys Res Commun 1998; 249(1): 1-5 [PMID: 9705820]

74Engelhardt NV, Factor VM, Yasova AK, Poltoranina VS, Baranov VN, Lasareva MN. Common antigens of mouse oval and biliary epithelial cells. Expression on newly formed hepatocytes. Differentiation 1990; 45(1): 29-37 [PMID: 2292360]

75Yamasaki H, Sada A, Iwata T, Niwa T, Tomizawa M, Xanthopoulos KG, Koike T, Shiojiri N. Suppression of C/EBPalpha expression in periportal hepatoblasts may stimulate biliary cell differentiation through increased Hnf6 and Hnf1b expression. Development 2006; 133(21): 4233-4243 [PMID: 17021047]

76Dianat N, Dubois-Pot-Schneider H, Steichen C, Desterke C, Leclerc P, Raveux A, Combettes L, Weber A, Corlu A, Dubart-Kupperschmitt A. Generation of functional cholangiocyte-like cells from human pluripotent stem cells and HepaRG cells. Hepatology 2014; 60(2): 700-714 [PMID: 24715669 DOI: 10.1002/hep.27165]

77Tomizawa M, Saisho H. Insulin-like growth factor (IGF)-II regulates CCAAT/enhancer binding protein alpha expression via phosphatidyl-inositol 3 kinase in human hepatoblastoma cell lines. J Cell Biochem 2007; 102(1): 161-170 [PMID: 17372916 DOI: 10.1002/jcb.21293]

78Takarada T, Kou M, Nakamichi N, Ogura M, Ito Y, Fukumori R, Kokubo H, Acosta GB, Hinoi E, Yoneda Y. Myosin VI reduces proliferation, but not differentiation, in pluripotent P19 cells. PLoS One 2013; 8(5): e63947 [PMID: 23691122 PMCID: 3656852 DOI: 10.1371/journal.pone.0063947. PONE-D-12-36007 [pii]]

79Liskovykh M, Chuykin I, Ranjan A, Safina D, Popova E, Tolkunova E, Mosienko V, Minina JM, Zhdanova NS, Mullins JJ, Bader M, Alenina N, Tomilin A. Derivation, characterization, and stable transfection of induced pluripotent stem cells from Fischer344 rats. PLoS 2011; 6(11): e27345 [PMID: 22076153 PMCID: 3208629 DOI: 10.1371/journal.pone.0027345. PONE-D-11-04041 [pii]]

80Vallier L, Rugg-Gunn PJ, Bouhon IA, Andersson FK, Sadler AJ, Pedersen RA. Enhancing and diminishing gene function in human embryonic stem cells. 2004; 22(1): 2-11 [PMID: 14688386 DOI: 10.1634/stemcells.22-1-2]

81DeLaForest A, Nagaoka M, Si-Tayeb K, Noto FK, Konopka G, Battle MA, Duncan SA. HNF4A is essential for specification of hepatic progenitors from human pluripotent stem cells. Development 2011; 138(19): 4143-4153 [PMID: 21852396 PMCID: 3171218 DOI: dev.062547 [pii] 10.1242/dev.062547]

82Si-Tayeb K, Noto FK, Nagaoka M, Li J, Battle MA, Duris C, North PE, Dalton S, Duncan SA. Highly efficient generation of human hepatocyte-like cells from induced pluripotent stem cells. Hepatology 2010; 51(1): 297-305 [PMID: 19998274 DOI: 10.1002/hep.23354]

83Song Z, Cai J, Liu Y, Zhao D, Yong J, Duo S, Song X, Guo Y, Zhao Y, Qin H, Yin X, Wu C, Che J, Lu S, Ding M, Deng H. Efficient generation of hepatocyte-like cells from human induced pluripotent stem cells. Cell Res 2009; 19(11): 1233-1242 [PMID: 19736565 DOI: cr2009107 [pii]. 10.1038/cr.2009.107]

84Takayama K, Inamura M, Kawabata K, Katayama K, Higuchi M, Tashiro K, Nonaka A, Sakurai F, Hayakawa T, Kusuda Furue M, Mizuguchi H. Efficient Generation of Functional Hepatocytes From Human Embryonic Stem Cells and Induced Pluripotent Stem Cells by HNF4alpha Transduction. Mol Ther 2012; 20(1): 127-137 [PMID: 22068426 PMCID: 3255576 DOI: mt2011234 [pii] 10.1038/mt.2011.234]

85Tomizawa M, Shinozaki F, Sugiyama T, Yamamoto S, Sueishi M, Yoshida T. Activin A is essential for Feeder-free culture of human induced pluripotent stem cells. J Cell Biochem 2013; 114(3): 584-588 [PMID: 22991093 DOI: 10.1002/jcb.24395]

86Shin M, Alev C, Wu Y, Nagai H, Sheng G. Activin/TGF-beta signaling regulates Nanog expression in the epiblast during gastrulation. Mech Dev 2011; 128(5-6): 268-278 [PMID: 21402155 DOI: S0925-4773(11)00038-4 [pii] 10.1016/j.mod.2011.03.001]

87Tomizawa M, Shinozaki F, Sugiyama T, Yamamoto S, Sueishi M, Yoshida T. Activin A maintains pluripotency markers and proliferative potential of human induced pluripotent stem cells. Exp Ther Med 2011; 2(3): 405-408 [PMID: 22977517 PMCID: 3440779 DOI: 10.3892/etm.2011.219. etm-02-03-0405 [pii]]

88Chen YF, Tseng CY, Wang HW, Kuo HC, Yang VW, Lee OK. Rapid generation of mature hepatocyte-like cells from human induced pluripotent stem cells by an efficient three-step protocol. Hepatology 2012; 55(4): 1193-1203 [PMID: 22095466 DOI: 10.1002/hep.24790]

89Kondo Y, Iwao T, Nakamura K, Sasaki T, Takahashi S, Kamada N, Matsubara T, Gonzalez FJ, Akutsu H, Miyagawa Y, Okita H, Kiyokawa N, Toyoda M, Umezawa A, Nagata K, Matsunaga T, Ohmori S. An efficient method for differentiation of human induced pluripotent stem cells into hepatocyte-like cells retaining drug metabolizing activity. Drug Metab Pharmacokinet 2014; 29(3): 237-243 [PMID: 24334537 DOI: DN/JST.JSTAGE/dmpk/DMPK-13-RG-104 [pii]]

90Yanagida A, Ito K, Chikada H, Nakauchi H, Kamiya A. An In vitro expansion system for generation of human iPS cell-derived hepatic progenitor-like cells exhibiting a bipotent differentiation potential. PLoS One 2013; 8(7): e67541 [PMID: 23935837 PMCID: 3723819 DOI: 10.1371/journal.pone.0067541. PONE-D-13-04177 [pii]]

91Javed MS, Yaqoob N, Iwamuro M, Kobayashi N, Fujiwara T. Generation of hepatocyte-like cells from human induced pluripotent stem (iPS) cells by co-culturing embryoid body cells with liver non-parenchymal cell line TWNT-1. J Coll Physicians Surg Pak 2014; 24(2): 91-96 [PMID: 24491001 DOI: 040579197 [pii] 02.2014/JCPSP.9196]

92Bottaro DP, Rubin JS, Faletto DL, Chan AM, Kmiecik TE, Vande Woude GF, Aaronson SA. Identification of the hepatocyte growth factor receptor as the c-met proto-oncogene product. Science 1991; 251(4995): 802-804 [PMID: 1846706]

93Marti U, Burwen SJ, Jones AL. Biological effects of epidermal growth factor, with emphasis on the gastrointestinal tract and liver: an update. Hepatology 1989; 9(1): 126-138 [PMID: 2642290 DOI: S0270913989000133 [pii]]

94Nakamura N, Saeki K, Mitsumoto M, Matsuyama S, Nishio M, Hasegawa M, Miyagawa Y, Ohkita H, Kiyokawa N, Toyoda M, Akutsu H, Umezawa A, Yuo A. Feeder-free and serum-free production of hepatocytes, cholangiocytes, and their proliferating progenitors from human pluripotent stem cells: application to liver-specific functional and cytotoxic assays. Cell Reprogram 2012; 14(2): 171-185 [PMID: 22384928 DOI: 10.1089/cell.2011.0064]

95Xing Q, Luo Y, Gao Y, Zhang S, Zhu Z, Wang Y, Yuan Q, Shu G, Lou C, Wang J, Wang P, Du Z. Hepatectomised patient sera promote hepatocyte differentiation of human-induced pluripotent stem cells. Dig Liver Dis 2014; 46(8): 731-737 [PMID: 24881854 DOI: S1590-8658(14)00336-3 [pii]. 10.1016/j.dld.2014.04.013]

96Luk JM, Liu AM. Proteomics of hepatocellular carcinoma in Chinese patients. OMICS 2011; 15(5): 261-266 [PMID: 21348761 DOI: 10.1089/omi.2010.0099]

97Zaret KS, Watts J, Xu J, Wandzioch E, Smale ST, Sekiya T. Pioneer factors, genetic competence, and inductive signaling: programming liver and pancreas progenitors from the endoderm. Cold Spring Harb Symp Quant Biol 2008; 73: 119-126 [PMID: 19028990 PMCID: 2773436 DOI: sqb.2008.73.040 [pii] 10.1101/sqb.2008.73.040]

98Xu ZL, Mizuguchi H, Sakurai F, Koizumi N, Hosono T, Kawabata K, Watanabe Y, Yamaguchi T, Hayakawa T. Approaches to improving the kinetics of adenovirus-delivered genes and gene products. Adv Drug Deliv Rev 2005; 57(5): 781-802 [PMID: 15757761 DOI: S0169-409X(05)00011-6 [pii] 10.1016/j.addr.2004.12.010]

99Inamura M, Kawabata K, Takayama K, Tashiro K, Sakurai F, Katayama K, Toyoda M, Akutsu H, Miyagawa Y, Okita H, Kiyokawa N, Umezawa A, Hayakawa T, Furue MK, Mizuguchi H. Efficient Generation of Hepatoblasts From Human ES Cells and iPS Cells by Transient Overexpression of Homeobox Gene HEX. Mol Ther 2010 [PMID: 21102561 DOI: mt2010241 [pii] 10.1038/mt.2010.241]

100Sekine K, Takebe T, Suzuki Y, Kamiya A, Nakauchi H, Taniguchi H. Highly efficient generation of definitive endoderm lineage from human induced pluripotent stem cells. Transplant Proc 2012; 44(4): 1127-1129 [PMID: 22564643 DOI: S0041-1345(12)00221-7 [pii] 10.1016/j.transproceed.2012.03.001]

101Schwartz RE, Fleming HE, Khetani SR, Bhatia SN. Pluripotent stem cell-derived hepatocyte-like cells. Biotechnol Adv 2014; 32(2): 504-513 [PMID: 24440487 PMCID: 4043206 DOI: S0734-9750(14)00005-6 [pii] 10.1016/j.biotechadv.2014.01.003]

102Koyanagi-Aoi M, Ohnuki M, Takahashi K, Okita K, Noma H, Sawamura Y, Teramoto I, Narita M, Sato Y, Ichisaka T, Amano N, Watanabe A, Morizane A, Yamada Y, Sato T, Takahashi J, Yamanaka S. Differentiation-defective phenotypes revealed by large-scale analyses of human pluripotent stem cells. Proc Natl Acad Sci U S A 2013; 110(51): 20569-20574 [PMID: 24259714 PMCID: 3870695 DOI: 1319061110 [pii] 10.1073/pnas.1319061110]

103Kajiwara M, Aoi T, Okita K, Takahashi R, Inoue H, Takayama N, Endo H, Eto K, Toguchida J, Uemoto S, Yamanaka S. Donor-dependent variations in hepatic differentiation from human-induced pluripotent stem cells. Proc Natl Acad Sci U S A 2012; 109(31): 12538-12543 [PMID: 22802639 PMCID: 3411998 DOI: 1209979109 [pii] 10.1073/pnas.1209979109]

104Sommer CA, Christodoulou C, Gianotti-Sommer A, Shen SS, Sailaja BS, Hezroni H, Spira A, Meshorer E, Kotton DN, Mostoslavsky G. Residual expression of reprogramming factors affects the transcriptional program and epigenetic signatures of induced pluripotent stem cells. PLoS One 2012; 7(12): e51711 [PMID: 23272148 PMCID: 3522693 DOI: 10.1371/journal.pone.0051711. PONE-D-12-24665 [pii]]

105Zhao Y, Yao R, Ouyang L, Ding H, Zhang T, Zhang K, Cheng S, Sun W. Three-dimensional printing of Hela cells for cervical tumor model In vitro. Biofabrication 2014; 6(3): 035001 [PMID: 24722236 DOI: 10.1088/1758-5082/6/3/035001]

106Shiraki N, Umeda K, Sakashita N, Takeya M, Kume K, Kume S. Differentiation of mouse and human embryonic stem cells into hepatic lineages. Genes Cells 2008; 13(7): 731-746 [PMID: 18513331 DOI: GTC1201 [pii] 10.1111/j.1365-2443.2008.01201.x]

107Shiraki N, Yamazoe T, Qin Z, Ohgomori K, Mochitate K, Kume K, Kume S. Efficient differentiation of embryonic stem cells into hepatic cells In vitro using a feeder-free basement membrane substratum. PLoS One 2011; 6(8): e24228 [PMID: 21887386 PMCID: 3162614 DOI: 10.1371/journal.pone.0024228. PONE-D-11-03181 [pii]]

108Nagamoto Y, Tashiro K, Takayama K, Ohashi K, Kawabata K, Sakurai F, Tachibana M, Hayakawa T, Furue MK, Mizuguchi H. The promotion of hepatic maturation of human pluripotent stem cells in 3D co-culture using type I collagen and Swiss 3T3 cell sheets. Biomaterials 2012; 33(18): 4526-4534 [PMID: 22445253 DOI: S0142-9612(12)00283-9 [pii] 10.1016/j.biomaterials.2012.03.011]

109Gieseck RL, 3rd, Hannan NR, Bort R, Hanley NA, Drake RA, Cameron GW, Wynn TA, Vallier L. Maturation of induced pluripotent stem cell derived hepatocytes by 3D-culture. PLoS One 2014; 9(1): e86372 [PMID: 24466060 PMCID: 3899231 DOI: 10.1371/journal.pone.0086372. PONE-D-13-42529 [pii]]

110Amimoto N, Mizumoto H, Nakazawa K, Ijima H, Funatsu K, Kajiwara T. Hepatic differentiation of mouse embryonic stem cells and induced pluripotent stem cells during organoid formation in hollow fibers. Tissue Eng Part A 2011; 17(15-16): 2071-2078 [PMID: 21457096 DOI: 10.1089/ten.TEA.2010.0689]

111Takebe T, Sekine K, Enomura M, Koike H, Kimura M, Ogaeri T, Zhang RR, Ueno Y, Zheng YW, Koike N, Aoyama S, Adachi Y, Taniguchi H. Vascularized and functional human liver from an iPSC-derived organ bud transplant. Nature 2013; 499(7459): 481-484 [PMID: 23823721 DOI: nature12271 [pii] 10.1038/nature12271]

112Du C, Narayanan K, Leong MF, Wan AC. Induced pluripotent stem cell-derived hepatocytes and endothelial cells in multi-component hydrogel fibers for liver tissue engineering. 2014; 35(23): 6006-6014 [PMID: 24780169 DOI: S0142-9612(14)00385-8 [pii] 10.1016/j.biomaterials.2014.04.011]

113Ohashi K, Yokoyama T, Yamato M, Kuge H, Kanehiro H, Tsutsumi M, Amanuma T, Iwata H, Yang J, Okano T, Nakajima Y. Engineering functional two- and three-dimensional liver systems in vivo using hepatic tissue sheets. Nat Med 2007; 13(7): 880-885 [PMID: 17572687 DOI: nm1576 [pii]10.1038/nm1576]

114Kim K, Ohashi K, Utoh R, Kano K, Okano T. Preserved liver-specific functions of hepatocytes in 3D co-culture with endothelial cell sheets. Biomaterials 2012; 33(5): 1406-1413 [PMID: 22118777 DOI: S0142-9612(11)01337-8 [pii] 10.1016/j.biomaterials.2011.10.084]

115Cornago M, Garcia-Alberich C, Blasco-Angulo N, Vall-Llaura N, Nager M, Herreros J, Comella JX, Sanchis D, Llovera M. Histone deacetylase inhibitors promote glioma cell death by G2 checkpoint abrogation leading to mitotic catastrophe. Cell Death Dis 2014; 5: e1435 [PMID: 25275596 DOI: cddis2014412 [pii] 10.1038/cddis.2014.412]

116Huangfu D, Maehr R, Guo W, Eijkelenboom A, Snitow M, Chen AE, Melton DA. Induction of pluripotent stem cells by defined factors is greatly improved by small-molecule compounds. Nat Biotechnol 2008; 26(7): 795-797 [PMID: 18568017 DOI: nbt1418 [pii] 10.1038/nbt1418]

117Kondo Y, Iwao T, Yoshihashi S, Mimori K, Ogihara R, Nagata K, Kurose K, Saito M, Niwa T, Suzuki T, Miyata N, Ohmori S, Nakamura K, Matsunaga T. Histone deacetylase inhibitor valproic acid promotes the differentiation of human induced pluripotent stem cells into hepatocyte-like cells. PLoS One 2014; 9(8): e104010 [PMID: 25084468 PMCID: 4119015 DOI: 10.1371/journal.pone.0104010. PONE-D-14-07021 [pii]]

118Vosough M, Omidinia E, Kadivar M, Shokrgozar MA, Pournasr B, Aghdami N, Baharvand H. Generation of functional hepatocyte-like cells from human pluripotent stem cells in a scalable suspension culture. Stem Cells Dev 2013; 22(20): 2693-2705 [PMID: 23731381 DOI: 10.1089/scd.2013.0088]

119Sasaki T, Takahashi S, Numata Y, Narita M, Tanaka Y, Kumagai T, Kondo Y, Matsunaga T, Ohmori S, Nagata K. Hepatocyte nuclear factor 6 activates the transcription of CYP3A4 in hepatocyte-like cells differentiated from human induced pluripotent stem cells. Drug Metab Pharmacokinet 2013; 28(3): 250-259 [PMID: 23291635 DOI: DN/JST.JSTAGE/dmpk/DMPK-12-RG-132 [pii]]

120 Takayama K, Inamura M, Kawabata K, Sugawara M, Kikuchi K, Higuchi M, Nagamoto Y, Watanabe H, Tashiro K, Sakurai F, Hayakawa T, Furue MK, Mizuguchi H. Generation of metabolically functioning hepatocytes from human pluripotent stem cells by FOXA2 and HNF1alpha transduction. J Hepatol 2012; 57(3): 628-636 [PMID: 22659344 DOI: S0168-8278(12)00405-9 [pii] 10.1016/j.jhep.2012.04.038]

121Huang P, Zhang L, Gao Y, He Z, Yao D, Wu Z, Cen J, Chen X, Liu C, Hu Y, Lai D, Hu Z, Chen L, Zhang Y, Cheng X, Ma X, Pan G, Wang X, Hui L. Direct reprogramming of human fibroblasts to functional and expandable hepatocytes. Cell Stem Cell 2014; 14(3): 370-384 [PMID: 24582927 DOI: S1934-5909(14)00004-6 [pii] 10.1016/j.stem.2014.01.003]

122Du Y, Wang J, Jia J, Song N, Xiang C, Xu J, Hou Z, Su X, Liu B, Jiang T, Zhao D, Sun Y, Shu J, Guo Q, Yin M, Sun D, Lu S, Shi Y, Deng H. Human hepatocytes with drug metabolic function induced from fibroblasts by lineage reprogramming. Cell Stem Cell 2014; 14(3): 394-403 [PMID: 24582926 DOI: S1934-5909(14)00009-5 [pii] 10.1016/j.stem.2014.01.008]

123Zhu S, Rezvani M, Harbell J, Mattis AN, Wolfe AR, Benet LZ, Willenbring H, Ding S. Mouse liver repopulation with hepatocytes generated from human fibroblasts. Nature 2014; 508(7494): 93-97 [PMID: 24572354 PMCID: 4161230 DOI: nature13020 [pii] 10.1038/nature13020]

124Okita K, Ichisaka T, Yamanaka S. Generation of germline-competent induced pluripotent stem cells. Nature 2007; 448(7151): 313-317 [PMID: 17554338 DOI: nature05934 [pii] 10.1038/nature05934]

125Miura K, Okada Y, Aoi T, Okada A, Takahashi K, Okita K, Nakagawa M, Koyanagi M, Tanabe K, Ohnuki M, Ogawa D, Ikeda E, Okano H, Yamanaka S. Variation in the safety of induced pluripotent stem cell lines. Nat Biotechnol 2009; 27(8): 743-745 [PMID: 19590502 DOI: nbt.1554 [pii] 10.1038/nbt.1554]

126Okita K, Nakagawa M, Hyenjong H, Ichisaka T, Yamanaka S. Generation of mouse induced pluripotent stem cells without viral vectors. Science 2008; 322(5903): 949-953 [PMID: 18845712]

127Fusaki N, Ban H, Nishiyama A, Saeki K, Hasegawa M. Efficient induction of transgene-free human pluripotent stem cells using a vector based on Sendai virus, an RNA virus that does not integrate into the host genome. Proc Jpn Acad Ser B Phys Biol Sci 2009; 85(8): 348-362 [PMID: 19838014 DOI: JST.JSTAGE/pjab/85.348 [pii]]

128Yakubov E, Rechavi G, Rozenblatt S, Givol D. Reprogramming of human fibroblasts to pluripotent stem cells using mRNA of four transcription factors. Biochem Biophys Res Commun 2010; 394(1): 189-193 [PMID: 20188704 DOI: S0006-291X(10)00384-0 [pii] 10.1016/j.bbrc.2010.02.150]

129Nakagawa M, Koyanagi M, Tanabe K, Takahashi K, Ichisaka T, Aoi T, Okita K, Mochiduki Y, Takizawa N, Yamanaka S. Generation of induced pluripotent stem cells without Myc from mouse and human fibroblasts. Nat Biotechnol 2008; 26(1): 101-106 [PMID: 18059259]

130Pierce GB, Jr., Dixon FJ, Jr., Verney EL. Teratocarcinogenic and tissue-forming potentials of the cell types comprising neoplastic embryoid bodies. Lab Invest 1960; 9: 583-602 [PMID: 13735536]

131Kooreman NG, Wu JC. Tumorigenicity of pluripotent stem cells: biological insights from molecular imaging. J R Soc Interface 2010; 7 Suppl 6: S753-763 [PMID: 20880852 PMCID: 2988279 DOI: rsif.2010.0353.focus [pii] 10.1098/rsif.2010.0353.focus]

132Abe K, Niwa H, Iwase K, Takiguchi M, Mori M, Abe SI, Abe K, Yamamura KI. Endoderm-specific gene expression in embryonic stem cells differentiated to embryoid bodies. Exp Cell Res 1996; 229(1): 27-34 [PMID: 8940246]

133Umeda K, Suzuki K, Yamazoe T, Shiraki N, Higuchi Y, Tokieda K, Kume K, Mitani K, Kume S. Albumin gene targeting in human embryonic stem cells and induced pluripotent stem cells with helper-dependent adenoviral vector to monitor hepatic differentiation. Stem Cell Res 2013; 10(2): 179-194 [PMID: 23276698 DOI: S1873-5061(12)00110-9 [pii] 10.1016/j.scr.2012.11.003]

134Tanaka M, Okabe M, Suzuki K, Kamiya Y, Tsukahara Y, Saito S, Miyajima A. Mouse hepatoblasts at distinct developmental stages are characterized by expression of EpCAM and DLK1: drastic change of EpCAM expression during liver development. Mech Dev 2009; 126(8-9): 665-676 [PMID: 19527784 DOI: S0925-4773(09)00076-8 [pii] 10.1016/j.mod.2009.06.939]

135Yanai H, Nakamura K, Hijioka S, Kamei A, Ikari T, Ishikawa Y, Shinozaki E, Mizunuma N, Hatake K, Miyajima A. Dlk-1, a cell surface antigen on foetal hepatic stem/progenitor cells, is expressed in hepatocellular, colon, pancreas and breast carcinomas at a high frequency. J Biochem 2010; 148(1): 85-92 [PMID: 20356822 DOI: mvq034 [pii] 10.1093/jb/mvq034]

136Alekseenko LL, Zemelko VI, Zenin VV, Pugovkina NA, Kozhukharova IV, Kovaleva ZV, Grinchuk TM, Fridlyanskaya, II, Nikolsky NN. Heat shock induces apoptosis in human embryonic stem cells but a premature senescence phenotype in their differentiated progeny. Cell Cycle 2012; 11(17): 3260-3269 [PMID: 22895173 PMCID: 3466525 DOI: 21595 [pii] 10.4161/cc.21595]

137Cheng F, Ke Q, Chen F, Cai B, Gao Y, Ye C, Wang D, Zhang L, Lahn BT, Li W, Xiang AP. Protecting against wayward human induced pluripotent stem cells with a suicide gene. Biomaterials 2012; 33(11): 3195-3204 [PMID: 22269649 DOI: S0142-9612(12)00042-7 [pii] 10.1016/j.biomaterials.2012.01.023]

138Conesa C, Doss MX, Antzelevitch C, Sachinidis A, Sancho J, Carrodeguas JA. Identification of specific pluripotent stem cell death--inducing small molecules by chemical screening. Stem Cell Rev 2012; 8(1): 116-127 [PMID: 21617963 PMCID: 3305812 DOI: 10.1007/s12015-011-9248-4]

139Bieberich E, Silva J, Wang G, Krishnamurthy K, Condie BG. Selective apoptosis of pluripotent mouse and human stem cells by novel ceramide analogues prevents teratoma formation and enriches for neural precursors in ES cell-derived neural transplants. J Cell Biol 2004; 167(4): 723-734 [PMID: 15545317 PMCID: 2172580 DOI: jcb.200405144 [pii] 10.1083/jcb.200405144]

140Leffert HL, Paul D. Studies on primary cultures of differentiated fetal liver cells. J Cell Biol 1972; 52(3): 559-568 [PMID: 4333210]

141Matsumoto K, Yamada K, Kohmura E, Kinoshita A, Hayakawa T. Role of pyruvate in ischaemia-like conditions on cultured neurons. Neurol Res 1994; 16(6): 460-464 [PMID: 7708138]

142Ohira RH, Dipple KM, Zhang YH, McCabe ER. Human and murine glycerol kinase: influence of exon 18 alternative splicing on function. Biochem Biophys Res Commun 2005; 331(1): 239-246 [PMID: 15845384]

143Ai Y, Jenkins NA, Copeland NG, Gilbert DH, Bergsma DJ, Stambolian D. Mouse galactokinase: isolation, characterization, and location on chromosome 11. Genome Res 1995; 5(1): 53-59 [PMID: 8717055]

144Phillips JW, Jones ME, Berry MN. Implications of the simultaneous occurrence of hepatic glycolysis from glucose and gluconeogenesis from glycerol. Eur J Biochem 2002; 269(3): 792-797 [PMID: 11846780]

145Sumida KD, Crandall SC, Chadha PL, Qureshi T. Hepatic gluconeogenic capacity from various precursors in young versus old rats. Metabolism 2002; 51(7): 876-880 [PMID: 12077734]

146Wheatley DN, Scott L, Lamb J, Smith S. Single amino acid (arginine) restriction: growth and death of cultured HeLa and human diploid fibroblasts. Cell Physiol Biochem 2000; 10(1-2): 37-55 [PMID: 10846151]

147Tomizawa M, Shinozaki F, Sugiyama T, Yamamoto S, Sueishi M, Yoshida T. Survival of primary human hepatocytes and death of induced pluripotent stem cells in media lacking glucose and arginine. PLoS One 2013; 8(8): e71897 [PMID: 23967260 PMCID: 3743790 DOI: 10.1371/journal.pone.0071897. PONE-D-13-06343 [pii]]

Peer reviewers: Nobuhiro Ohkohchi, Professor of Department of Surgery, University of Tsukuba, Vice President of Tsukuba University Hospital, Tennoudai 1-1-1, Tsukuba, 305-8575, Japan; Andrea M.P. Romani, Case Western Reserve University, Department of Physiology and Biophysics, 10900 Euclid Avenue, Cleveland, OH 44106-4970, USA.

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