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To the Editor:

We read with interest the article by Susseret et al.1 They successfully showed the characterization of resistance to boceprevir in patients infected with hepatitis C virus (HCV) genotype 1.1 In the near future, antiviral effects in HCV infection genotype 1 for the combination of boceprevir with various drugs (interferon, statins, and ribavirin) would be considered by replicon systems.

Si-Tayeb et al. showed a method for highly efficient generation of human hepatocyte-like cells from human induced pluripotent stem cells (iPSCs),2 and the toxicities of the aforementioned combination therapies for HCV genotype 1 should be evaluated by using the hepatocyte-like cells. By using patient-specific cells derived from patients infected with HCV genotype 1, individual toxicities for the combination therapies for HCV genotype 1 could be evaluated.

However, if the human iPSCs with tumorigenicity are used as a research tool in order to evaluate the toxicities for the combination therapies, more complex epigenetic modifications may arise in the disease-specific iPSCs. Therefore, the value of disease-specific iPSCs as a research tool may be limited. Considering this circumstance, we tried to find an indicator for the tumor transformation of human iPSCs.

As for human iPSCs, the teratomas were formed in severe combined immunodeficient (SCID) mice into which they were transplanted.2, 3 This is an important proof for pluripotency of the cells.2, 3 Furthermore, microvessel density (MVD) has been widely used as a marker of tumor angiogenesis, and has been associated with outcome of cancer therapy.4

Therefore, we investigated MVD within teratomas in SCID mice into which human iPSCs established by Oct3/4, Sox2, and Klf4, according to the methods of Nakagawa et al.,3 were transplanted.

The human iPSC lines in each group were inoculated intramuscularly into immunodeficient mice (Rag2−/−Il2rg−/−), and angiogenesis was assessed in the resulting tumors using a human-specific antibody to the endothelial marker CD31.4 Next, by using the methods of Shirako et al.,5 we performed knockdown of p21 in the human iPSC lines by p21 small interfering RNA (Fig. 1A, left side). Furthermore, we compared MVD within teratomas in mice (Rag2−/−Il2rg−/−) between the p21 knockdown group and the control group. As a result, the MVD was significantly reduced within teratomas derived from the latter human iPSCs compared to the former human iPSCs (P < 0.01, Fig. 1B). This observation shows the increased risk for cancerous transformations of human iPSCs by the p21 knockdown. Furthermore, the induction of p21 is necessary to avoid cancerous transformations of the cells. Moreover, we could find that the investigations of MVD within teratomas in SCID mice into which human iPSCs were transplanted can be useful as an indicator in order to evaluate the risk for cancerous transformations of human iPSCs when the cells are used as research tool for new drugs for HCV genotype 1 infection.

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Figure 1. (A) The expressions of p53 and p21 in human iPS cells by Western blotting. (Left side: after knockdown of p21; Right side: before knockdown of p21). (B) As for angiogenesis in teratomas from human iPS cells, microvessel density (MVD) per high-powered field (h.p.f) of teratomas was quantified by human-specific anti-CD31 immunofluorescence. n = 3-6; All values were mean ± S.E.M.

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In conclusion, human hepatocyte-like cells differentiated from human iPSCs with high qualities as determined by using this indicator should be used as a research tool.

Acknowledgements

We are grateful to members of our laboratories for technical support. Furthermore, we are also grateful to Ms. Satoko Iioka for helpful discussions.

References

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  • 1
    Susser S, Welsch C, Wang Y, Zettler M, Domingues FS, Karey U, et al. Characterization of resistance to the protease inhibitor boceprevir in hepatitis C virus-infected patients. HEPATOLOGY 2009; doi:10.1002/hep.23192.
  • 2
    Si-Tayeb K, Noto FK, Nagaoka M, Li J, Battle MA, Duris C, et al. Highly efficient generation of human hepatocyte-like cells from induced pluripotent stem cells. HEPATOLOGY 2009; doi:10.1002/hep.23354.
  • 3
    Nakagawa M, Koyanagi M, Tanabe K, Takahashi K, Ichisaka T, Aoi T, et al. Generation of induced pluripotent stem cells without Myc from mouse and human fibroblasts. Nature Biotechnol 2008; 26: 101-106.
  • 4
    Baek KH, Zaslavsky A, Lynch RC, Britt C, Okada Y, Siarey RJ, et al. Down's syndrome suppression of tumour growth and the role of the calcineurin inhibitor DSCR1. Nature 2009; 459: 1126-1130.
  • 5
    Shirako E, Hirayama N, Tsukada Y, Tanaka T, Kitamura N. Up-regulation of p21CIP1 expression mediated by ERK-dependent and -independent pathways contributes to hepatocyte growth factor-induced inhibition of HepG2 hepatoma cell proliferation. J Cell Biochem 2008; 104: 176-188.

Hisashi Moriguchi* † ‡, Raymond T. Chung†, Chifumi Sato‡, * Research Center for Advanced Science and Technology, The University of Tokyo, Tokyo, Japan, † Gastrointestinal Unit, Massachusetts General Hospital and Harvard Medical School, Boston, MA, ‡ Department of Analytical Health Science, Graduate School of Health Sciences, Tokyo Medical and Dental University, Tokyo, Japan.