**10. Primary human hepatocytes**

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 mouse brain. This method is sophisticated and promising, but xenograft rejection may be a problem when the generated liver is transplanted to patients with liver failure. Practical methods for the differentiation of human iPS cell to functional hepatocytes are not available. It is therefore necessary to use primary human hepatoctyes as a model of hepatocytes fully differentiated from iPS cells. Hepatocytes were isolated from a fragment of resected donor liver by using 2-step collagenase perfusion [39].

## **11. Co-culture of human iPS cells and primary human hepatocytes**

Methods have not been established regarding hepatocye differentiation from human iPS cells. It is impossible to select hepatocytes differentiated from human iPS cells from the mixture of human iPS cells. Primary human hepatocytes were used as a model of hepatocytes differen‐ tiated from human iPS cells. It was expected that human iPS cells and hepatocytes differenti‐ ated from them were mixed. Therefore, co-culure of primary human hepatocytes and human iPS cells was used as a model of the mixtures. Primary human hepatocytes were purchased from Lonza (Walkersville, MD) and cultured as per the manufacturer's instructions. Briefly, hepatocytes were thawed and spread at a density of 1.5 × 105 cells/cm2 onto CellBIND 24-well plates coated with type I collagen from the bovine dermis (Koken Co., Ltd., Tokyo, Japan) and cultured in the hepatocyte culture medium (HCM, Lonza).

The 201B7 cells and human primary hepatocytes were co-cultured as follows: human primary hepatoctyes were cultured in HCM for 24 h as described above. The 201B7 cells were added to the wells at a density of 3 × 104 cells/well. After 24 h of culture in the ReproFF medium, it was changed to HSM. Human primary hepatocytes survived in HSM, while the human 201B7 cells did not (Figure 5).

**Figure 5.** Human iPS cells co-cultured with primary human hepatocytes in HSM. Scale bar, 50 μm; arrow, hepatocytes;

Hepatocyte Selection Medium http://dx.doi.org/10.5772/58394 173

HSM can be successfully used for the selection of hepatoblast-like cells derived from mouse ES cells. HSM is an ideal medium for the elimination of hiPS cells and the isolation of differ‐ entiated hepatocytes without causing any damage. In the future, methods will be established to produce hepatocytes from human iPS cells. Residual human iPS cells are a potential hazard when the hepatocytes will be transplanted for patients with liver insufficiency because the undifferentiated cell harbor tumorigenicity. At that stage, HSM will be an indispensable

This work was supported in part by a Research Grant-in-Aid for Scientific Research (C) (Grant

medium to select hepatocytes differentiated from residual human iPS cells.

No. 23591002) from the Japan Society for the Promotion of Science (JSPS).

arrowhead, 201B7 cells.

**13. Conclusion**

**Acknowledgements**
