Sullivan et al. generated functional human hepatic endoderm from human induced pluripotent stem cells (iPSCs) with four transcription factors (Oct3/4 [octamer 3/4], Sox2 [sex-determining region Y box 2], Klf4 [Kruppel-like factor 4], and C-MYC).1 Yet, we also generated human hepatocyte-like cells from human iPSCs according to the methods of Song et al.2 These human hepatocyte-like cells would be useful as research tools for liver diseases.
First, in order to better understand the possibilities of the human hepatocyte-like cells, we investigated changes of the human iPSCs during the process of differentiation induction to the human hepatocyte-like cells. However, we then used human iPSCs while maintaining higher expressions of p21 than p53 in order to avoid tumorigenicity of the human iPSCs and to increase the value of the cells as research tools.3
Second, we compared the expression of alfa-fetoprotein (AFP) and albumin between the p21 knockdown group (p21 small interfering RNA [siRNA] (+)) and the control group (p21 siRNA (−)) by reverse transcription polymerase chain reaction (Fig. 1). As a result, the expression of AFP but not albumin was found in the former in 21 days (Fig. 1). Therefore, the human iPSCs could transform to human hepatoma-like cells again during the differentiation induction process to normal hepatocyte-like cells through the knockdown of p21. However, the expression of albumin, but not AFP, was found in the latter in 21 days, indicating that the human iPSCs could also differentiate to normal human hepatocyte-like cells through the expression of albumin in 21 days without knockdown of p21 (Fig. 1).
Third, although aldo-keto reductase family 1 B10 (AKR1B10) is overexpressed in human hepatocellular carcinoma,4 a review suggests that AKR1B10 inhibits the cellular differentiation produced by retinoic acid.5 Therefore, we hypothesized that an AKR1B10 inhibitor could be used to enhance the differentiation effects of retinoic acid.
Based on our hypothesis, we tried to investigate the efficacies of acyclic retinoid (10 μM) plus tolestat as an AKR1B10 inhibitor (10 μM) therapy for the human hepatoma-like cells. As a result of this combination therapy, the expression of albumin but not AFP was found in 7 days. Furthermore, we tried to investigate the hepatotoxicities for the combination therapy by using the normal human hepatocyte-like cells. As a result, we found that the activities of glutamic oxaloacetic transaminase (GOT) and lactate dehydrogenase (LDH) in the culture medium of the normal human hepatocyte-like cells increased markedly in the case of acyclic retinoid (30 μM) plus tolestat (30 μM) compared with the case of acyclic retinoid (10 μM) plus tolestat (10 μM), although the efficacies for the combination therapy was not different.
Therefore, acyclic retinoid (10 μM) plus tolestat (10 μM) would be appropriate regimens for human hepatoma-like cells. However, by using the patient-specific hepatocyte-like cells differentiated from human iPSCs of the patients with hepatocellular carcinoma, the efficacies and toxicities of the abovementioned combination therapy for the individual patients with hepatocellular carcinoma will be evaluated more specifically in the near future.