Abstract
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 in vitro 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.
Highlights
Mammalian haploid embryonic stem cells serve as a powerful tool for genetic analyses at both the cellular and organismal levels
Parthenogenetic haploid embryonic stem cells (haESCs) (PG-haESCs) have a similar size as AG-haESCs with same DNA content (Fig. 1A). These results suggest that 1C cells might be directly purified from a cell mixture by physical filtration based on their size
Cell suspensions with different concentrations were used for filtration, and filtrates were collected for analyzing the ratio of 1C cells
Summary
A cell flow cytometer can separate Hoechst 33342–stained haESCs into three groups according to DNA content: 1C (haploid cells in G0/G1 phase), 2C (haploid cells in G2/M phase and diploid cells in G0/G1 phase), and 4C (diploid cells in G2/M phase) [24]. Cell size analyses showed that the average diameter of 1C, 2C, and 4C in mouse haESCs is 8.3 m, 9.5 m, and 10.8 m, respectively (Fig. 1A), indicating an obvious size difference.
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