From the State Key Laboratory of Cell Biology, Shanghai Key Laboratory of Molecular Andrology, Chinese Academy of Sciences Center for Excellence in Molecular Cell Science, Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai 200031, China and.
the State Key Laboratory of Membrane Biology, Tsinghua-Peking Center for Life Sciences, School of Life Sciences, Tsinghua University, Beijing 100084, China.
J Biol Chem. 2018 Apr 6;293(14):5230-5235. doi: 10.1074/jbc.RA118.002029. Epub 2018 Feb 15.
Mammalian haploid embryonic stem cells (haESCs) serve as a powerful tool for genetic analyses at both the cellular and organismal levels. However, spontaneous diploidization of haESCs limits their use in these analyses. Addition of small molecules to the culture medium to control the cell cycle can slow down diploidization, but cell-sorting methods such as FACS are still required to enrich haploid cells for long-term maintenance Here, acting on our observation that haploid and diploidized cells differ in diameter, we developed a simplified filtration method to enrich haploid cells from cultured haESCs. We found that regular cell filtration with this system reliably maintained the haploidy of mouse haESCs for over 30 passages. Importantly, CRISPR/Cas9-mediated knockout and knockin were successfully achieved in the filtered cells, leading to stable haploid cell lines carrying the desired gene modifications. Of note, by injecting haESCs into metaphase II oocytes, we efficiently obtained live mice with the expected genetic traits, indicating that regular filtration maintained the functional integrity of haESCs. Moreover, this filtration system was also feasible for derivation of mouse haESCs from parthenogenetic haploid blastocysts and for human haESC maintenance. In conclusion, we have identified a reliable, efficient, and easy-to-handle technique for countering diploidization of haploid cells, a major obstacle in haESC applications.
哺乳动物单倍体胚胎干细胞（haESCs）可作为细胞和机体水平遗传分析的有力工具。然而，haESCs 的自发二倍体化限制了它们在这些分析中的应用。在培养基中添加小分子以控制细胞周期可以减缓二倍体化，但仍需要细胞分选方法（如 FACS）来富集单倍体细胞以进行长期维持。在这里，基于我们观察到单倍体和二倍体化细胞在直径上存在差异的结果，我们开发了一种简化的过滤方法，从培养的 haESCs 中富集单倍体细胞。我们发现，使用该系统进行常规细胞过滤可可靠地维持小鼠 haESCs 的单倍体性超过 30 代。重要的是，在过滤后的细胞中成功实现了 CRISPR/Cas9 介导的基因敲除和敲入，从而产生了携带所需基因修饰的稳定单倍体细胞系。值得注意的是，通过将 haESCs 注射到中期 II 期卵母细胞中，我们有效地获得了具有预期遗传特征的活小鼠，表明常规过滤维持了 haESCs 的功能完整性。此外，该过滤系统也可用于从孤雌单倍体囊胚中衍生小鼠 haESCs 以及维持人 haESC。总之，我们已经确定了一种可靠、高效且易于处理的技术，可用于对抗 haESC 应用中的主要障碍——单倍体细胞的二倍体化。
