Haploid embryonic stem cells can be enriched and maintained by simple filtration

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.

Keywords: CRISPR/Cas; FACS; cell biology; cell sorting; diploidization; embryo; embryonic stem cell; filtration; haploid embryonic stem cells; haploidy.

Figure 1.

Long-term maintenance of mouse haESCs through filtration. A, diameters of mouse haESCs with 1C, 2C, or 4C (C, DNA content; mAG, mouse androgenetic haESCs; mPG, mouse parthenogenetic haESCs). B, diagram of the filtration system. C, filters with 5-μm and 8-μm pore size membranes could enrich mouse AG-haESCs efficiently. D, filters with 5-μm (left panel) and 8 μm (right panel) membranes could be used to maintain the haploidy of H19^▵DMR-IG^▵DMR-AGH for a long term.

Figure 2.

Filtered haESCs are functional. A, filtered cells had better cell vitality (scale bar = 200 μm) and colony-forming ability (scale bar = 500 μm) in the first passage after enrichment compared with FACS. B, summary of A. Total cell numbers and colony-forming efficiencies of filtered haESCs were significantly better than haESCs sorted by FACS. Ho, Hoechst 33342. **, p < 0.01. C, filtered haESCs of H19^▵DMR-IG^▵DMR-AGH with different times of filtration could efficiently support the birth of SC mice. D, newborn and adult SC mice generated using filtered haESCs from H19^▵DMR-IG^▵DMR-AGH.

Figure 3.

Generation of mutant mice using DKO-AG-haESCs enriched by filtration. A, diagram of two sgRNAs targeting two exons of Iqgap2, respectively, that may cause 35-kb deletion. Iqgap2 F and Iqgap2 R represent primers used for genotyping of Iqgap2. B, Iqgap2 with 35-kb deletion achieved efficiently in filtered H19^▵DMR-IG^▵DMR-AGH cells. C, diagram of 1-bp deletion in Crygc and the sequence of sgRNA targeting Crygc. D, filtered haploid cells carrying the 35-kb deletion in Iqgap2 supported the birth of SC mice. E, genetic trait analysis revealed that the SC mice generated in D all carried the heterozygous mutation in Iqgap2. KO cells, cells from the line of 2-1; WT cells, H19^▵DMR-IG^▵DMR-AGH.

Figure 4.

Applications of filtration in derivation of mouse PG-haESCs and maintaining human haESCs. A, two PG-haESCs were derived from mouse parthenogenetic haploid blastocysts by using filters with 5-μm or 8-μm membranes. B, diameters of human haESCs with 1C, 2C, or 4C. C, DNA content; hPG, human parthenogenetic haESCs. C, human haESCs could be enriched by using filters with 8-μm membranes.