Journal
CELL REPORTS METHODS
Volume 2, Issue 12, Pages -Publisher
CELL PRESS
DOI: 10.1016/j.crmeth.2022.100354
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Funding
- KAKENHI grant from the Ministry of Education, Culture, Sports, Science and Technology (MEXT)/Japan Society for the Promotion of Science (JSPS) [21J11016]
- KAKENHI grant from MEXT/JSPS [19K17847, 21K08431]
- National Center for Global Health and Medicine
- Takeda Science Foundation
- KAKENHI grants from MEXT/JSPS [20K21621, 21H02957, 22K19550, 19K17877, 21J01690, 22K08493]
- Japan Health Research Promotion Bureau, Japan Agency for Medical Research and Development [JP18ck0106444, JP18ae0201014, JP20bm0704042, JP20gm1210011]
- MEXT Joint Usage/Research Center Program at the Advanced Medical Research Center, Yokohama City University
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This study presents a platform for analyzing the quiescence of hematopoietic stem cells (HSCs) by combining culture conditions and a CRISPR-Cas9 genome editing system. The study demonstrates that preculturing HSCs enhances editing efficiency and that low-cytokine culture conditions better preserve the phenotypes and quiescence of edited HSCs.
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.
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