**1. Introduction**

[124] Lassar AB, Paterson BM, Weintraub H. Transfection of a DNA locus that mediates

[125] Tapscott SJ, Davis RL, Thayer MJ, Cheng PF, Weintraub H, Lassar AB. MyoD1: a nu‐ clear phosphoprotein requiring a Myc homology region to convert fibroblasts to my‐

[126] Choi J, Costa ML, Mermelstein CS, Chagas C, Holtzer S, Holtzer H. MyoD converts primary dermal fibroblasts, chondroblasts, smooth muscle, and retinal pigmented epithelial cells into striated mononucleated myoblasts and multinucleated myotubes. Proceedings of the National Academy of Sciences of the United States of America.

the conversion of 10T1/2 fibroblasts to myoblasts. Cell. 1986;47:649-56.

oblasts. Science. 1988;242:405-11.

164 Pluripotent Stem Cell Biology - Advances in Mechanisms, Methods and Models

1990;87:7988-92.

Embryonic stem (ES) cells have the potential to differentiate to hepatocytes [1]. However, the use of ES cells may pose ethical problems because they are derived from human embryos. The use of human induced pluripotent stem (hiPS) cells that have been generated from adult somatic cells [2], on the other hand, does not create ethical controversies. HiPS cells are useful tools in drug discovery and regenerative medicine because they can differentiate into func‐ tional somatic cells [3]. If hiPS cells could be differentiated into hepatocytes, they would be useful for transplantation into patients suffering from hepatic failure [4]. Complications such as graft-versus-host disease as well as ethical issues could be avoided because patient-specific somatic cells could be generated from hiPS cells isolated from the patient.

The ES and hiPS cells that survive among the differentiated hepatocytes and are transplanted to patients may be tumorigenic [5]. Therefore, methods need to be developed to eliminate ES and iPS cells from the population of differentiated cells used for transplantation. To overcome these problems, a new medium, called "hepatocyte selection medium" (HSM), has been developed and will be discussed in this chapter [6].

First, pluripotency and tumorigenicity of ES and iPS cells will be discussed [7]. Next, current methods of eliminating pluripotent cells will be outlined [8, 9]. All the cells, including human iPS cells, require glucose and arginine to live [10, 11]. They will die without glucose or arginine. Hepatocytes have enzymes to produce glucose from galactose and arginine from ornithine. The unique features of hepatocytes compared with other cells will next be discussed. It was expected that hepatocytes would survive in a medium without glucose or arginine, and supplemented with galactose and ornithine [12] [13]. After this introduction, the formulation

© 2014 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

of HSM will be described [14]. Finally, the application of HSM for the selection of cells differentiated from mouse ES and human iPS cells will be presented.

were subsequently ablated by ganciclovir treatment. This method may be ideal for differenti‐ ated hepatocytes, which do not express Nanog, but the toxicity of ganciclovir may be a potential issue. Conesa et al. screened a library of 1120 small chemicals to identify molecules that caused mouse ES cells to undergo apoptosis [27], and found that benzethonium chloride and methylbenzethonium induced apoptosis in hiPS and mouse ES cells but not in human fibroblasts or mouse embryonic fibroblasts. Both reagents are quaternary ammonium salts used as antimicrobial agents; they are also used in cancer therapy and may have damaging effects on hepatocytes. *N*-oleoyl serinol (S18), which is a ceramide analogue, eliminated residual pluripotent cells in embryoid bodies [28]. Interestingly, S18 also promoted neural differentiation of embryoid body-derived cells. This strategy is promising because the reagent not only eradicates undifferentiated cells but also promotes their differentiation into the target

Hepatocyte Selection Medium http://dx.doi.org/10.5772/58394 167

Among all the amino acids, the deficiency in arginine is the least tolerated by the cells cultured *in vitro* [29]. Arginine is produced through the urea cycle, which is exclusive to hepatocytes. Indeed, an arginine-deficient medium was the first one used for the hepatocyte selection [10]. Tyrosine also is produced by hepatocytes, and H4 II E, a hepatoma cell line adapted to growth in serum-, arginine-, and tyrosine-free medium, has been established [30]. This cell line expresses ornithine transcarbamylase (OTC) involved in the urea cycle, and phenylalanine hydroxylase (PAH), which catalyzes the synthesis of tyrosine in the liver and kidney [31]. The major role of urea synthesis is the excretion of ammonium ions generated in the process of protein degradation. Urea synthesis is a cyclic process as shown in Figure 1. Ornithine plays the key role in urea synthesis, and OTC mediates the formation of L-citrulline from L-ornithine and carbamoylphosphate. Importantly, this process occurs in liver mitochondria (area bounded with green line in Figure 1). The OTC deficiency, linked to X-chromosome is the cause of hyperammonemia type 2 [32]. The elevated ammonium levels lead to infantile death or

Consequently, it could be expected that hepatocytes can be selected from ES cells in a medium

Glucose is an important source of energy for a majority of cells. Glucose deprivation aids in the hepatocyte selection process because hepatocytes are capable of synthesizing glucose [10]. Pyruvate is the final product of glycolysis, which then enters the tricarboxylic acid cycle. It was shown that pyruvate and glucose deficiency led to neural cell death [11]. Galactose enters glycolysis as a substrate for galactokinase, which is expressed in the liver and kidney [33, 34]. Therefore, it is expected that hepatocytes can survive in a medium deprived of glucose or

cell types.

**4. Arginine and urea cycle**

mental retardation later in life.

deficient in arginine and tyrosine.

**5. Glucose and gluconeogenesis**

pyruvate but supplemented with galactose [12] [13].

#### **2. Pluripotency and tumorigenicity**

The link between pluripotency and tumorigenicity was reported in 1960 based on a study of teratocarcinoma [15]. ES and iPS cells are pluripotent and are capable of self-renewal as well as differentiation into a variety of cell types. Pluripotent cells can, however, be tumorigenic because they proliferate rapidly and exhibit telomerase activity [7]. Therefore, one of the problems faced while using ES and iPS cell-derived cells for transplantation into patients is the risk of tumorigenicity [5]. For example, transplantation of mouse hepatocytes differenti‐ ated from ES cells into liver resulted in the formation of teratoma [16]. Tumorigenicity was initially attributed to genomic integration of the viral vectors used for the induction of pluripotency [17]. The Sendai virus was also used to generate iPS cells because it posed no risk of altering the host genome [18]. To reduce this risk, plasmid vectors have been used to introduce reprogramming factors such as Oct3/4, Sox2, Klf4, and c-Myc [19]. In addition, the ES cell-specific microRNA, miR-302, has been used to reduce the iPS cells tumorigenicity by suppressing cyclin E-CDK2 and cyclin D-CDK4/6 [20]. Furthermore, Yakubov et al. introduced RNA synthesized from the cDNA of the four reprogramming transcription factors [21]. Several combinations of reprogramming factors have also been investigated. Nakagawa et al. omitted c-Myc to generate iPS cells, thereby reducing the tumorigenicity because c-Myc is a wellknown oncogene [22]. Despite these efforts, the risk of tumorigenicity has not yet been eliminated. The process of pluripotency and tumorigenicity involve self-renewal, prolifera‐ tion, and active telomerase mechanisms [7]. It is, therefore, necessary to develop methods for the efficient eradication of iPS cells that survive among differentiated somatic cells.

#### **3. Methods of eliminating iPS cells**

Flow cytometry, which is commonly used to isolate target cells, was used by Yamamoto et al. to isolate hepatocytes differentiated from the mouse ES cells [8]. These workers generated ES cells expressing green fluorescent protein driven by an albumin promoter/enhancer. However, since albumin is expressed in endodermal cells as well [23], this approach led to isolation of cells other than hepatocytes, such as endodermal cells. Therefore, a different strategy was required to improve hepatocyte isolation. Flow cytometry has also been used to analyze surface antigens specific for hepatocytes. For example, delta-like 1 homolog (DLK1) has been used for isolation of hepatoblasts [9]. The issue with DLK1, however, is that this surface antigen is not expressed in the human adult liver [24]. Therefore, it may not be possible to isolate mature hepatocytes differentiated from hiPS cells using DLK-1 as a marker. In our research we focused on other methods to eliminate iPS or ES cells from heptocytes. Sub-lethal heat shock was shown to induce apoptosis in human ES cells [25], but it might also damage differentiated cells intended for transplantation. Cheng et al. reported the same strategy using suicide genes [26]. They introduced a thymidine kinase gene driven by the Nanog promoter into hiPS cells, which were subsequently ablated by ganciclovir treatment. This method may be ideal for differenti‐ ated hepatocytes, which do not express Nanog, but the toxicity of ganciclovir may be a potential issue. Conesa et al. screened a library of 1120 small chemicals to identify molecules that caused mouse ES cells to undergo apoptosis [27], and found that benzethonium chloride and methylbenzethonium induced apoptosis in hiPS and mouse ES cells but not in human fibroblasts or mouse embryonic fibroblasts. Both reagents are quaternary ammonium salts used as antimicrobial agents; they are also used in cancer therapy and may have damaging effects on hepatocytes. *N*-oleoyl serinol (S18), which is a ceramide analogue, eliminated residual pluripotent cells in embryoid bodies [28]. Interestingly, S18 also promoted neural differentiation of embryoid body-derived cells. This strategy is promising because the reagent not only eradicates undifferentiated cells but also promotes their differentiation into the target cell types.
