A culture platform to study quiescent hematopoietic stem cells following genome editing

Kohei Shiroshita, Hiroshi Kobayashi, Shintaro Watanuki, Daiki Karigane, Yuriko Sorimachi, Shinya Fujita, Shinpei Tamaki, Miho Haraguchi, Naoki Itokawa, Kazumasa Aoyoama, Shuhei Koide, Yosuke Masamoto, Kenta Kobayashi, Ayako Nakamura-Ishizu, Mineo Kurokawa, Atsushi Iwama, Shinichiro Okamoto, Keisuke Kataoka, Keiyo Takubo

Research output: Contribution to journalArticlepeer-review

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.

Original languageEnglish
Article number100354
JournalCell Reports Methods
Volume2
Issue number12
DOIs
Publication statusPublished - 2022 Dec 19
Externally publishedYes

Keywords

  • CRISPR-Cas9
  • genome editing
  • hematopoietic stem cell
  • quiescence
  • stem cell culture

ASJC Scopus subject areas

  • Biotechnology
  • Biochemistry
  • Biochemistry, Genetics and Molecular Biology (miscellaneous)
  • Genetics
  • Radiology Nuclear Medicine and imaging
  • Computer Science Applications

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