A fast and efficient size separation method for haploid embryonic stem cells

Abstract

Hemizygous mutations introduced in haploid genomes can directly expose a phenotype, thus facilitating gene function analysis and forward genetic screening. Recently, mammalian haploid cells could be derived from mouse, rat, monkey, and human embryos and have been applied to screens of cellular mechanisms including cell signaling, pathogen host factors, and developmental pathways. Notably, haploid cell cultures have an intrinsic tendency for diploidization and, thus, require periodic cell sorting. Here, we report a method for rapid purification of haploid mouse embryonic stem cells from mixed cell populations with high viability and yield. Our method uses membranes with micrometer pores for force-free separation and facilitates enrichment of haploid cells without flow cytometry. The separation method simplifies maintaining haploid cell cultures and has further applications in establishing haploid cell lines from embryos and isolating cell cycle phases of mammalian cells.