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

Other than genetically engineered mice, few reliable platforms are available for the study of hematopoietic stem cell (HSC) quiescence. In this protocol, we present a new 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 demonstrated that preculture of HSCs enhanced the editing efficiency by facilitating nuclear transport of ribonucleoprotein complex. For post-editing culture, both mouse and human HSCs edited based on non-homologous end joining and cultured under low cytokine, low oxygen, and high albumin conditions retained their phenotypes and the quiescence better than those cultured under the proliferative condition. Using this approach, HSCs could 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.