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Research team head, Research team member, Publisher, 私達はこれまで, 自発的再生能のない蝸牛有毛細胞の薬剤による再生と聴力改善を報告してきた(Neuron, 2013)。この"内耳再生医療"の臨床応用に向け, (1)ヒトiPS細胞由来内耳幹細胞を用いた人工蝸牛感覚上皮での薬剤スクリーニングにより臨床試験への橋渡しが可能な薬剤2剤を同定し, (2)小型霊長類コモンマーモセットモデル(特許出願)での治療効果を確認した。また, iPS研究では, ヒト内耳幹細胞においてその幹細胞性の維持と有毛細胞の成熟化の双方にWntシグナルが必須であることを見出し, マーモセットを用いて成体霊長類蝸牛における新たな再生医療の標的分子候補を複数同定した。 We previously reported auditory hair cell regeneration with a hearing improvement in mouse (Neuron, 2013). Towards translation to the clinical trials, we selected better compounds by using otic progenitor cells derived from human iPS cells. We also examined the effect of above mentioned regenerative therapy in a non-human primate common marmoset., 研究種目 : 基盤研究(B)(一般) 研究期間 : 2015～2017 課題番号 : 15H04991 研究分野 : 耳科学

type:text, Nonhuman primate embryonic stem (ES) cells have vast promise for preclinical studies. Genetic modification in nonhuman primate ES cells is an essential technique for maximizing the potential of these cells. The common marmoset (Callithrix jacchus), a nonhuman primate, is expected to be a useful transgenic model for preclinical studies. However, genetic modification in common marmoset ES (cmES) cells has not yet been adequately developed. To establish efficient and stable genetic modifications in cmES cells, we inserted the enhanced green fluorescent protein (EGFP) gene with heterotypic lox sites into the beta-actin (ACTB) locus of the cmES cells using gene targeting. The resulting knock-in ES cells expressed EGFP ubiquitously under the control of the endogenous ACTB promoter. Using inserted heterotypic lox sites, we demonstrated Cre recombinase-mediated cassette exchange (RMCE) and successfully established a monomeric red fluorescent protein (mRFP) knock-in cmES cell line. Further, a herpes simplex virus-thymidine kinase (HSV-tk) knock-in cmES cell line was established using RMCE. The growth of tumor cells originating from the cell line was significantly suppressed by the administration of ganciclovir. Therefore, the HSV-tk/ganciclovir system is promising as a safeguard for stem cell therapy. The stable and ubiquitous expression of EGFP before RMCE enables cell fate to be tracked when the cells are transplanted into an animal. Moreover, the creation of a transgene acceptor locus for site-specific transgenesis will be a powerful tool, similar to the ROSA26 locus in mice.

Nonhuman primate embryonic stem (ES) cells have vast promise for preclinical studies. Genetic modification in nonhuman primate ES cells is an essential technique for maximizing the potential of these cells. The common marmoset (Callithrix jacchus), a nonhuman primate, is expected to be a useful transgenic model for preclinical studies. However, genetic modification in common marmoset ES (cmES) cells has not yet been adequately developed. To establish efficient and stable genetic modifications in cmES cells, we inserted the enhanced green fluorescent protein (EGFP) gene with heterotypic lox sites into the beta-actin (ACTB) locus of the cmES cells using gene targeting. The resulting knock-in ES cells expressed EGFP ubiquitously under the control of the endogenous ACTB promoter. Using inserted heterotypic lox sites, we demonstrated Cre recombinase-mediated cassette exchange (RMCE) and successfully established a monomeric red fluorescent protein (mRFP) knock-in cmES cell line. Further, a herpes simplex virus-thymidine kinase (HSV-tk) knock-in cmES cell line was established using RMCE. The growth of tumor cells originating from the cell line was significantly suppressed by the administration of ganciclovir. Therefore, the HSV-tk/ganciclovir system is promising as a safeguard for stem cell therapy. The stable and ubiquitous expression of EGFP before RMCE enables cell fate to be tracked when the cells are transplanted into an animal. Moreover, the creation of a transgene acceptor locus for site-specific transgenesis will be a powerful tool, similar to the ROSA26 locus in mice.