**13. Conclusion**

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

**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,

plates coated with type I collagen from the bovine dermis (Koken Co., Ltd., Tokyo, Japan) and

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

The HSM that we developed can be safely used for the elimination of hiPS cells because it does not contain hazardous reagents or introduce genetic material. Our results show that hiPS cells die after three days of culture in HSM. Prior to performing the experiments, we compared the hiPS cell viability in media containing crude or dialyzed KSR or combination of insulin (10 µM), dexamethasone (10 µM) and aprotitin (5000 U/ml) (IDA) Unexpectedly, the KSR dialysis and IDA had no effect on hiPS cell survival. As expected, primary human hepatocytes survived

HSM can be used in clinical practices in situations when hepatocytes differentiated from

in HSM as well as in HCM, which is the recommended medium for their culture.

human iPS cells are transplanted to patients suffering from liver failure.

cells/cm2

onto CellBIND 24-well

liver by using 2-step collagenase perfusion [39].

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

hepatocytes were thawed and spread at a density of 1.5 × 105

cultured in the hepatocyte culture medium (HCM, Lonza).

cells did not (Figure 5).

**12. Potential application of HSM**

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 medium to select hepatocytes differentiated from residual human iPS cells.
