**7. Embryoid bodies in HSM**

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

**9. Human iPS cells in HSM**

201B7 cells in HSM was caused by apoptosis.

feeder-free culture. All the human iPS cells died on day 3.

**10. Primary human hepatocytes**

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

Hepatocyte Selection Medium http://dx.doi.org/10.5772/58394 171

**Figure 4.** Human iPS cells cultured in HSM. Scale bar, 50 μm. Medium was changed to HSM for human iPS cells in

Several protocols for the differentiation of iPS cells to hepatocytes have been reported [3, 37], which describe the differentiation of iPS cells into hepatocyte-like cells which are different from primary human hepatocytes. Recently, a method to generate three-dimensional vascu‐ larized liver from iPS cells has been reported [38]. The authors induced hepatic differentiation of human iPS cells by following the protocol described by Si-Tayeb et al [37]. They mixed the iPS cells with vascular endothelial and mesenchymal stem cells, and transplanted them into a

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

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 and enriched the population of hepatoblast-like cells.
