著者

Sayaka Deguchi Kazuo Takayama Hiroyuki Mizuguchi

出版者

The Pharmaceutical Society of Japan

雑誌

Biological and Pharmaceutical Bulletin (ISSN:09186158)

巻号頁・発行日

vol.43, no.4, pp.608-615, 2020-04-01 (Released:2020-04-01)

参考文献数

50

被引用文献数

6

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Liver transplantation and hepatocyte transplantation are effective treatments for severe liver injuries, but the donor shortage is a serious problem. Therefore, hepatocyte-like cells generated from human induced pluripotent stem (iPS) cells with unlimited proliferative ability are expected to be a promising new transplantation resource. The technology for hepatic differentiation from human iPS cells has made great progress in this decade. The efficiency of hepatic differentiation now exceeds 90%, making it possible to produce nearly homogeneous hepatocyte-like cells from human iPS cells. Because there is little contamination of undifferentiated cells, there is a lower risk of teratoma formation. To date, the transplantation of human iPS cell-derived hepatocyte-like cells has been shown to have therapeutic effects using various liver injury model mice. Currently, studies are underway using model animals larger than mice. The day when human iPS cell-derived hepatocyte-like cells can be used as cellular medicine is surely approaching. In this review, we introduce the forefront of regenerative medicine applications using human iPS cell-derived hepatocyte-like cells.

著者

Mitsuhiro Machitani Fuminori Sakurai Keisaku Wakabayashi Kosuke Nakatani Kazuo Takayama Masashi Tachibana Hiroyuki Mizuguchi

出版者

公益社団法人日本薬学会

雑誌

Biological and Pharmaceutical Bulletin (ISSN:09186158)

巻号頁・発行日

vol.40, no.3, pp.272-277, 2017-03-01 (Released:2017-03-01)

参考文献数

25

被引用文献数

7

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Clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9-mediated genome engineering technology is a powerful tool for generation of cells and animals with engineered mutations in their genomes. In order to introduce the CRISPR/Cas9 system into target cells, nonviral and viral vectors are often used; however, such vectors trigger innate immune responses associated with production of type I interferons (IFNs). We have recently demonstrated that type I IFNs inhibit short-hairpin RNA-mediated gene silencing, which led us to hypothesize that type I IFNs may also inhibit CRISPR/Cas9-mediated genome mutagenesis. Here we investigated this hypothesis. A single-strand annealing assay using a reporter plasmid demonstrated that CRISPR/Cas9-mediated cleavage efficiencies of the target double-stranded DNA were significantly reduced by IFNα. A mismatch recognition nuclease-dependent genotyping assay also demonstrated that IFNα reduced insertion or deletion (indel) mutation levels by approximately half. Treatment with IFNα did not alter Cas9 protein expression levels, whereas the copy numbers of guide RNA (gRNA) were significantly reduced by IFNα stimulation. These results indicate that type I IFNs significantly reduce gRNA expression levels following introduction of the CRISPR/Cas9 system in the cells, leading to a reduction in the efficiencies of CRISPR/Cas9-mediated genome mutagenesis. Our findings provide important clues for the achievement of efficient genome engineering using the CRISPR/Cas9 system.