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

Cell Rep Methods. 2022 Dec 5;2(12):100354. doi: 10.1016/j.crmeth.2022.100354. eCollection 2022 Dec 19.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cytokines / metabolism
  • Gene Editing* / methods
  • Hematopoietic Stem Cells*
  • Humans
  • Mice


  • Cytokines