**9. Human iPS cells in HSM**

comprising the embryoid bodies in ESM differentiated to various cell types 28 days after the

28 days after the formation of embryoid bodies, sizes of colonies in HSM reduced, and the surviving cells appeared cuboidal (Figure 3). Some of these cells were binuclear, which is characteristic of hepatocytes; it was also previously shown that HSM was selective for hepatoblast-like cells [14]. These results suggest that HSM eliminated undifferentiated cells

Human fetal and adult hepatocytes express galactokinase and OTC, and would survive in HSM containing galactose and ornithine. If hiPS cells express similar levels of these enzymes, HSM could not be applied for selection of differentiated hepatocytes. Therefore, we compared the expression levels of galactokinase and OTC in hiPS cells with those in human fetal and adult livers. The hiPS cell line 201B7 (RIKEN Cell Bank, Tsukuba, Japan) was cultured feederfree in ReproFF medium (Reprocell, Yokohama, Japan) in dishes coated with a thin layer of Matrigel (Becton Dickinson, Franklin Lakes, NJ). Two galactokinase isoforms, GALK1 (GenBank: NM\_000154) and GALK2 (BC107153), have been identified in humans. The expression levels of GALK1, GALK2, and OTC in the 201B7 cells and fetal and adult livers were compared [6]. The expression levels of these enzymes in the 201B7 cells constituted 22.2% ± 5.0%, 14.2% ± 1.1%, and 1.2% ± 0.2% (mean ± standard deviation) of those in the adult liver, respectively, and the OTC expression was also significantly lower in the 201B7 cells than in

the fetal liver. We then cultured 201B7 cells in HSM to assess their survival rates.

formation of embryoid bodies.

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

**Figure 3.** Mouse embryonic stem cells in HSM. Scale bar, 250 μm.

and enriched the population of hepatoblast-like cells.

**8. Expression levels of GALK1, GALK2, and OTC**

The 201B7 cells were cultured in 6-well plates coated with Matrigel in the ReproFF medium, which was then changed to HSM (Figure 4). The 201B7 cells started to die and were completely eliminated in three days. Nuclear condensation and fragmentation was observed after staining with hematoxylin and eosin [6]. These nuclei also tested positive by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL). Some of the 201B7 cells that survived in HSM one day after medium change to HSM were immunostained with antibodies against Nanog, SSEA4, and TRA-1-60. The results suggested that the death of undifferentiated 201B7 cells in HSM was caused by apoptosis.

**Figure 4.** Human iPS cells cultured in HSM. Scale bar, 50 μm. Medium was changed to HSM for human iPS cells in feeder-free culture. All the human iPS cells died on day 3.
