PDF(Pubmed)
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.
摘要：
除了基因工程小鼠，很少有可靠的平台可用于造血干细胞（HSC）静止的研究。在这里，我们提出了一个平台，通过将维持静止的培养条件与针对HSC优化的CRISPR-Cas9基因组编辑系统相结合来分析HSC细胞周期静止。我们证明HSC的预培养通过促进核糖核蛋白复合物的核转运来提高编辑效率。对于后期编辑文化，基于非同源末端连接编辑并在低细胞因子下培养的小鼠和人HSC，低氧,和高白蛋白条件比在增殖条件下培养的更好地保留了它们的表型和静止性。使用这种方法，即使在通过同源定向修复进行编辑后，HSC也会恢复静止。我们的结果表明，基因编辑的HSC的低细胞因子培养条件是研究离体HSC静止的有用方法。
