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To the Editor:

We read with interest the article by Si-Tayeb et al., in which they generated human hepatocyte-like cells from human induced pluripotent stem cells (iPSCs) with four transcription factors (Oct3/4, Sox2, Nanog, and LIN28).1 They use viral transgenes for the establishment of the human iPSCs,1 and it is thought that the use of viral transgenes would contribute to the increase in tumorigenicity of iPSCs.2 Yet, as their colleagues develop vector and/or transgene-free human iPSCs,2 they could generate hepatocyte-like cells derived from the human iPSCs in the near future. However, the associations between the use of viral transgenes and the tumorigenicity of human iPSCs are as-yet not clear.

Therefore, by using our previous methods,3 we investigated microvessel density (MVD) within teratomas between the vector and/or transgene-free human iPSCs established according to the methods of Yu et al.2 and the human iPSCs established with the retroviruses encoding Oct3/4, Sox2, and Klf4, according to the methods of Nakagawa et al.4 As a result, we could not observe a significant difference in the MVD of both groups (P = 0.11). Furthermore, we investigated the expressions of p53 and p21 for the vector and/or transgene-free human iPSC lines by western blotting. Then, we investigated the MVD within teratomas between the human iPSC lines and found that the expression of p21 was controlled in comparison with p53 (Fig. 1A, column A) and the human iPSC lines that the expression of p53 was controlled in comparison with p21 (Fig. 1A, column B). As a result, the MVD was significantly reduced within teratomas derived from the latter human iPSCs compared to the former human iPSCs (P = 0.03, Fig. 1B).

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Figure 1. (A) The expressions pattern of p53 and p21 in the vector and transgene-free human iPS cells by Western blotting. In column “A”, the expression of p21 was controlled in comparison with p53. In column “B”, the expression of p53 was controlled in comparison with p21. (B) The microvessel density (MVD) in teratomas from the vector and transgene-free human iPS cell lines (the expressions of p53 and p21). As for angiogenesis in teratomas from the vector and transgene-free human iPS cell lines, microvessel density (MVD) per high-powered field (h.p.f.) of the teratomas was quantified by human-specific anti-CD31 immunofluorescence. n = 3-6; All values were mean ± standard error of the mean.

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Therefore, considering the aforementioned observations, we could find the tumorigenicity of human iPSCs derived from fibroblasts depends on the balance of gene expression levels between p21 and p53, regardless of the use of viral transgenes. The induction of p21 is necessary in order to avoid cancerous transformations of human iPSCs, and the maintaining of higher expressions of p21 than p53 is desirable in these cells. At this point, considering the induction of p21 and the control of p53 by Klf4,5 Klf4 may play an important role in order to produce human iPSCs with the least chance of tumorigenicity.

In conclusion, human hepatocyte-like cells or cardiomyocytes differentiated from the vector and/or transgene-free human iPSCs and which maintain higher expressions of p21 than p53 should be used as research tools and/or regenerative medicine for liver diseases.

Acknowledgements

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

References

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  • 1
    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.
  • 2
    Yu J, Hu K, Smuga-Otto K, Tian S, Stewart R, Slukvin II, et al. Human induced pluripotent stem cells free of vector and transgene sequences. Science 2009; 324: 797-801.
  • 3
    Moriguchi H, Chung RT, Sato C. An indicator in order to evaluate the risk for cancerous transformations of human induced pluripotent stem cells. HEPATOLOGY 2009; doi:10.1002/hep.23379.
  • 4
    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.
  • 5
    Rowland BD, Bernards R, Peeper DS. The KLF4 tumour suppressor is a transcriptional repressor of p53 that acts as a context-dependent oncogene. Nat Cell Biol 2005; 7: 1074-1082.

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.