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

Area of Science:

• Stem Cell Biology
• Developmental Biology
• Genetics

Background:

• Mammalian haploid embryonic stem cells (haESCs) are valuable for genetic analysis.
• Spontaneous diploidization of haESCs in vitro limits their application.
• Current methods to maintain haploidy, like cell sorting, are complex and require enrichment.

Purpose of the Study:

• To develop a simplified method for enriching and maintaining haploid cells.
• To overcome the challenge of spontaneous diploidization in haESCs.
• To enable long-term in vitro culture and genetic manipulation of haESCs.

Main Methods:

• Utilized the difference in diameter between haploid and diploid cells for separation.
• Developed and implemented a simplified filtration system for haESC enrichment.
• Assessed the maintenance of haploidy over multiple cell passages.

Main Results:

• The filtration method reliably maintained mouse haESC haploidy for over 30 passages.
• CRISPR/Cas9 gene editing (knockout and knockin) was successful in filtered haESCs.
• Filtered haESCs produced live mice with expected genetic traits after injection into oocytes, confirming functional integrity.

Conclusions:

• A reliable, efficient, and easy-to-handle filtration technique effectively counters haESC diploidization.
• This method supports stable genetic modification and derivation of haESCs from various sources, including human.
• The filtration system overcomes a major obstacle in the application of haESCs for genetic studies.