Takahashi Saori, Lee Jiyoung, Kohda Takashi, Matsuzawa Ayumi, Kawasumi Miyuri, Kanai-Azuma Masami, Kaneko-Ishino Tomoko, Ishino Fumitoshi
Department of Epigenetics, Medical Research Institute, Tokyo Medical and Dental University (TMDU), 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan.
Department of Epigenetics, Medical Research Institute, Tokyo Medical and Dental University (TMDU), 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan Global Center of Excellence Program for International Research Center for Molecular Science in Tooth and Bone Diseases, Tokyo Medical and Dental University (TMDU), 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan.
Development. 2014 Oct;141(20):3842-7. doi: 10.1242/dev.110726. Epub 2014 Sep 24.
The recent successful establishment of mouse parthenogenetic haploid embryonic stem cells (phESCs) and androgenetic haploid ESCs (ahESCs) has stimulated genetic research not only in vitro but also in vivo because of the germline competence of these cell lines. However, it is difficult to maintain the haploid status over time without a frequent sorting of the G1 phase haploid ESCs by fluorescence-activated cell sorting (FACS) at short intervals, because haploid cells tend to readily self-diploidize. To overcome this spontaneous diploid conversion, we developed a phESC culture condition using a small molecular inhibitor of Wee1 kinase to regulate the cell cycle by accelerating the G2/M phase transition and preventing re-entry into extra G1/S phase. Here, we demonstrate that, under this condition, phESCs maintained the haploid status for at least 4 weeks without the need for FACS. This method will greatly enhance the availability of these cells for genetic screening.
最近成功建立的小鼠孤雌生殖单倍体胚胎干细胞（phESC）和雄核发育单倍体胚胎干细胞（ahESC），由于这些细胞系具有种系能力，不仅在体外而且在体内都刺激了遗传学研究。然而，如果不通过荧光激活细胞分选（FACS）在短时间间隔内频繁分选G1期单倍体胚胎干细胞，就很难长时间维持单倍体状态，因为单倍体细胞容易自发地自我二倍体化。为了克服这种自发的二倍体转化，我们开发了一种phESC培养条件，使用Wee1激酶的小分子抑制剂，通过加速G2/M期转换和防止重新进入额外的G1/S期来调节细胞周期。在此，我们证明，在这种条件下，phESC无需FACS即可维持单倍体状态至少4周。该方法将大大提高这些细胞用于遗传筛选的可用性。
