**4. Arginine and urea cycle**

of HSM will be described [14]. Finally, the application of HSM for the selection of cells

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.

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

differentiated from mouse ES and human iPS cells will be presented.

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

**2. Pluripotency and tumorigenicity**

**3. Methods of eliminating iPS cells**

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 mental retardation later in life.

Consequently, it could be expected that hepatocytes can be selected from ES cells in a medium deficient in arginine and tyrosine.
