1. A culture platform to study quiescent hematopoietic stem cells following genome editing.
- Author
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Shiroshita K, Kobayashi H, Watanuki S, Karigane D, Sorimachi Y, Fujita S, Tamaki S, Haraguchi M, Itokawa N, Aoyoama K, Koide S, Masamoto Y, Kobayashi K, Nakamura-Ishizu A, Kurokawa M, Iwama A, Okamoto S, Kataoka K, and Takubo K
- Subjects
- Animals, Mice, Humans, Cytokines metabolism, Gene Editing methods, Hematopoietic Stem Cells
- 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 ., (© 2022 The Authors.)
- Published
- 2022
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