@article{df451102249c414b9c7529e29149dfe8,
title = "A culture platform to study quiescent hematopoietic stem cells following genome editing",
abstract = "Other than genetically engineered mice, few reliable platforms are available for the study of hematopoietic stem cell (HSC) quiescence. Here we present a platform to analyze HSC cell cycle quiescence by combining culture conditions that maintain quiescence with a CRISPR-Cas9 genome editing system optimized for HSCs. We demonstrate that preculture of HSCs enhances editing efficiency by facilitating nuclear transport of ribonucleoprotein complexes. For post-editing culture, mouse and human HSCs edited based on non-homologous end joining and cultured under low-cytokine, low-oxygen, and high-albumin conditions retain their phenotypes and quiescence better than those cultured under the proliferative conditions. Using this approach, HSCs regain quiescence even after editing by homology-directed repair. Our results show that low-cytokine culture conditions for gene-edited HSCs are a useful approach for investigating HSC quiescence ex vivo.",
keywords = "CRISPR-Cas9, genome editing, hematopoietic stem cell, quiescence, stem cell culture",
author = "Kohei Shiroshita and Hiroshi Kobayashi and Shintaro Watanuki and Daiki Karigane and Yuriko Sorimachi and Shinya Fujita and Shinpei Tamaki and Miho Haraguchi and Naoki Itokawa and Kazumasa Aoyoama and Shuhei Koide and Yosuke Masamoto and Kenta Kobayashi and Ayako Nakamura-Ishizu and Mineo Kurokawa and Atsushi Iwama and Shinichiro Okamoto and Keisuke Kataoka and Keiyo Takubo",
note = "Funding Information: We thank all members of the Takubo laboratory for their indispensable support, K. Brownhill for helping to prepare the manuscript, Dr. Takehiko Yamamoto of Keio University for advice regarding Nucleofector, Drs. Ravindra Majeti and Yusuke Nakauchi of Stanford University for support regarding human HSPC KI experiments, and Dr. Satoshi Yamazaki of Tsukuba University for advice regarding PVA-based medium. K.S. was supported in part by a KAKENHI grant from the Ministry of Education, Culture, Sports, Science and Technology (MEXT)/Japan Society for the Promotion of Science (JSPS) (21J11016). H.K. was supported in part by a KAKENHI grant from MEXT/JSPS (19K17847 and 21K08431) and a grant from the National Center for Global Health and Medicine. D.K. was supported in part by a KAKENHI grant from MEXT/JSPS (19K17877, 21J01690, and 22K08493), JB research grants, and the Takeda Science Foundation. K.T. was supported in part by KAKENHI grants from MEXT/JSPS (20K21621, 21H02957, and 22K19550), grants from the National Center for Global Health and Medicine, a grant from the Japan Health Research Promotion Bureau, Japan Agency for Medical Research and Development grants (JP18ck0106444, JP18ae0201014, JP20bm0704042, and JP20gm1210011), a grant from the Takeda Science Foundation, and the MEXT Joint Usage/Research Center Program at the Advanced Medical Research Center, Yokohama City University. K.S. H.K. Y.S. S.F. S.T. M.H. N.I. K.A. S.K. and K. Kobayashi performed the study and analyzed the data. S.W. D.K. Y.M. A.N.-I. M.K. A.I. S.O. and K. Kataoka provided scientific advice and materials. K.S. H.K. and K.T. wrote the manuscript. K.T. conceived the project and supervised the research. The authors declare no competing interests. Funding Information: We thank all members of the Takubo laboratory for their indispensable support, K. Brownhill for helping to prepare the manuscript, Dr. Takehiko Yamamoto of Keio University for advice regarding Nucleofector, Drs. Ravindra Majeti and Yusuke Nakauchi of Stanford University for support regarding human HSPC KI experiments, and Dr. Satoshi Yamazaki of Tsukuba University for advice regarding PVA-based medium. K.S. was supported in part by a KAKENHI grant from the Ministry of Education, Culture, Sports, Science and Technology ( MEXT )/Japan Society for the Promotion of Science ( JSPS ) ( 21J11016 ). H.K. was supported in part by a KAKENHI grant from MEXT / JSPS ( 19K17847 and 21K08431 ) and a grant from the National Center for Global Health and Medicine . D.K. was supported in part by a KAKENHI grant from MEXT / JSPS ( 19K17877 , 21J01690 , and 22K08493 ), JB research grants, and the Takeda Science Foundation . K.T. was supported in part by KAKENHI grants from MEXT / JSPS ( 20K21621 , 21H02957 , and 22K19550 ), grants from the National Center for Global Health and Medicine , a grant from the Japan Health Research Promotion Bureau , Japan Agency for Medical Research and Development grants ( JP18ck0106444 , JP18ae0201014 , JP20bm0704042 , and JP20gm1210011 ), a grant from the Takeda Science Foundation , and the MEXT Joint Usage/Research Center Program at the Advanced Medical Research Center, Yokohama City University . Publisher Copyright: {\textcopyright} 2022 The Authors",
year = "2022",
month = dec,
day = "19",
doi = "10.1016/j.crmeth.2022.100354",
language = "English",
volume = "2",
journal = "Cell Reports Methods",
issn = "2667-2375",
publisher = "Cell Press",
number = "12",
}