**5. Glucose and gluconeogenesis**

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

**Figure 1.** Urea cycle.

Galactose is produced from lactose by hydrolysis in the gastrointestinal tract and is converted to glucose in the liver (Figure 2). Galactokinase catalyzes ATP-dependent phosphorylation of galactose to galactose 1-phosphate which then reacts with uridine diphosphate(UDP)-glucose to produce UDP-galactose converted to UDP-glucose by uridine diposphogalactose 4 epimerase. UDP-glucose is used by glycogen synthase to synthesize glycogen, which is stored in the liver and used as a source of glucose.

Pure Chemicals, Osaka, Japan); proline (30 mg/L) was added as a component necessary for DNA synthesis [35]. Aspartic acid as a nonessential amino acid was not included because it can be synthesized from ornithine and arginine. Fetal calf serum (FCS, Life Technologies) at a final concentration of 10% was used to culture mouse ES cells. For human iPS cells, 10% knockout serum replacement (KSR) (Life Technologies) was used instead of FCS to establish xeno-free conditions. Depending on the experiment, FCS and KSR were dialyzed against

Hepatocyte Selection Medium http://dx.doi.org/10.5772/58394 169

EB5, a mouse ES cell line provided by Dr. H. Niwa (Center for Developmental Biology, Riken, Kobe, Japan) was maintained in the undifferentiated state in gelatin-coated dishes without feeder cells, in Glasgow minimum essential medium (GMEM) (Sigma Aldrich Japan K.K., Tokyo, Japan) supplemented with 10% FCS (Roche Diagnostics K.K., Tokyo, Japan), 1× nonessential amino acids (NEAA), sodium pyruvate (1 mM), leukemia inhibitory factor (LIF) (1000 U/ml) (Invitrogen Japan, Tokyo, Japan), and 2-mercaptoethanol (0.1 mM) (Wako) [36]. Dissociated ES cells were cultured in hanging drops at a density of 1 × 103 cells per 30 µµl of media without LIF (ESM) to form embryoid bodies. After four days in hanging drop culture, the resulting embryoid bodies were plated onto plastic dishes (Iwaki-Asahi Techno Glass, Tokyo, Japan) precoated with gelatin (Sigma Aldrich). Seven days after their formation, the embryoid bodies transferred to HSM appeared slightly smaller than those in ESM. The cells

phosphate buffered saline (PBS) to remove amino acids and glucose.

**7. Embryoid bodies in HSM**

**Figure 2.** Galactose metabolism.

Deficiency in the enzymes such as galactokinase, galactose-1-phosphate uridyltransferase, or uridine diphosphogalactose 4-epimerase causes galactosemia. Galactose is then reduced to galactitol, which accumulates in the eye lenses causing cataracts. Deficiency in galactose-1 phosphate uridyltransferase results in accumulation of galactose-1-phosphate and depletion of inorganic phosphate in the liver causing liver failure. This is the reason why children suffering from galactosemia are kept on a galactose-free diet.

## **6. Hepatocyte selection medium**

The hepatocyte selection medium (HSM) was made from powdered amino acids following the formulation of Leibovits-15 medium (Life Technologies, Grand Island, NY). HSM did not contain arginine, tyrosine, glucose, and sodium pyruvate, but was supplemented with galactose (900 mg/L), ornithine (1 mM), glycerol (5 mM), and proline (260 mM) (all from Wako

**Figure 2.** Galactose metabolism.

Galactose is produced from lactose by hydrolysis in the gastrointestinal tract and is converted to glucose in the liver (Figure 2). Galactokinase catalyzes ATP-dependent phosphorylation of galactose to galactose 1-phosphate which then reacts with uridine diphosphate(UDP)-glucose to produce UDP-galactose converted to UDP-glucose by uridine diposphogalactose 4 epimerase. UDP-glucose is used by glycogen synthase to synthesize glycogen, which is stored

Deficiency in the enzymes such as galactokinase, galactose-1-phosphate uridyltransferase, or uridine diphosphogalactose 4-epimerase causes galactosemia. Galactose is then reduced to galactitol, which accumulates in the eye lenses causing cataracts. Deficiency in galactose-1 phosphate uridyltransferase results in accumulation of galactose-1-phosphate and depletion of inorganic phosphate in the liver causing liver failure. This is the reason why children

The hepatocyte selection medium (HSM) was made from powdered amino acids following the formulation of Leibovits-15 medium (Life Technologies, Grand Island, NY). HSM did not contain arginine, tyrosine, glucose, and sodium pyruvate, but was supplemented with galactose (900 mg/L), ornithine (1 mM), glycerol (5 mM), and proline (260 mM) (all from Wako

in the liver and used as a source of glucose.

**Figure 1.** Urea cycle.

**6. Hepatocyte selection medium**

suffering from galactosemia are kept on a galactose-free diet.

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

Pure Chemicals, Osaka, Japan); proline (30 mg/L) was added as a component necessary for DNA synthesis [35]. Aspartic acid as a nonessential amino acid was not included because it can be synthesized from ornithine and arginine. Fetal calf serum (FCS, Life Technologies) at a final concentration of 10% was used to culture mouse ES cells. For human iPS cells, 10% knockout serum replacement (KSR) (Life Technologies) was used instead of FCS to establish xeno-free conditions. Depending on the experiment, FCS and KSR were dialyzed against phosphate buffered saline (PBS) to remove amino acids and glucose.
