Mammalian haploid stem cells: establishment, engineering and applications

Abstract

Haploid embryonic stem cells (haESCs) contain only one set of genomes inherited from the sperm or egg and are termed AG- or PG-haESCs, respectively. Mammalian haESCs show genome-wide hypomethylation and dysregulated imprinting, whereas they can sustain genome integrity during derivation and long-term propagation. In addition, haESCs exhibit similar pluripotency to traditional diploid ESCs but are unique because they function as gametes and have been used to produce semi-cloned animals. More strikingly, unisexual reproduction has been achieved in mice by using haESCs. In combination with a gene editing or screening system, haESCs represent a powerful tool for studies of underlying gene functions and explorations of mechanisms of genetic and epigenetic regulation not only at the cellular level in vitro but also at the animal level in vivo. More importantly, genetically edited AG-haESC lines may further serve as an ideal candidate for the establishment of a sperm bank, which is a highly cost-effective approach, and a wide range of engineered semi-cloned mice have been produced. Here, we review the historical development, characteristics, advantages and disadvantages of haESCs. Additionally, we present an in-depth discussion of the recent advances in haESCs and their potential applications.

Keywords: CRISPR/Cas9; Genetic screen; LINE-1; Self-diploidization; Sperm bank; Unisexual mice.

Fig. 1

Schematics showing the derivation of mammalian haploid embryonic stem cells. a, b Androgenetic haploid embryos have been generated by removing the maternal pronucleus from the zygote (a) or injecting a sperm head into an enucleated oocyte (b). Then, the mouse AG-haESCs are obtained from the derived AG-ESCs using FACS. c Procedure for producing rat AG-haESCs. Allodiploid ESCs are obtained through the fusion of mouse and rat AG-haESCs. d, e Parthenogenetic haploid embryos are generated from MII oocytes activated by 10 mM SrCl₂ in calcium-free CZB medium without CB (d) or through the removal of the male pronucleus from the zygote (e). a–e Blastocysts or ES cells shown in light colors are haploid cells and darker colored cells are diploid cells

Fig. 2

Mechanism of self-diploidization in mouse haESCs. Self-diploidization mainly occurs in metaphase due to mitotic slippage, and mitosis is longer in haESCs. Specifically, the sister chromosomes do not segregate in anaphase or segregate without cytokinesis. Then, these two nuclei duplicate independently and fuse together at metaphase in the next cell cycle. Overexpression of Dnmt3b or the addition of inhibitors to the culture medium reduces the frequency of self-diploidization

Fig. 3

Schematics showing the procedures used to generate cloned or semi-cloned mammals. a Cloned animals have been produced by injecting a diploid somatic cell into an enucleated oocyte followed by activation with SrCl₂ in calcium-free CZB medium supplemented with CB. b Live semi-cloned mice have been produced by injecting a secondary spermatocyte into an intact MII oocyte. c The semi-cloned embryos have been obtained by injecting a diploid somatic cell into an intact MII oocyte followed by activation in media lacking CB. However, no viable animals have been produced using this method. d, e Semi-cloned mice (or rats) have been produced by injecting an M-phase (d) or G0/G1-phase (e) AG-haESC into an intact MII oocyte or a pre-activated MII oocyte, respectively. f The procedure used to generate semi-cloned mice from PG-haESCs in place of a maternal genome. A sperm head is first injected into an oocyte, followed by the removal of the spindle. One hour later, a G0/G1-phase PG-haESC is injected to generate a diploid embryo. Then, semi-cloned embryos and mice can be produced

Fig. 4

Schematics showing the procedures used to generate unisexual animals. a Bimaternal mice are produced through the injection of M-phase DKO PG-haESCs into intact MII oocytes. b Bipaternal embryos are generated by injecting G0/G1 phase 7KO AG-haES cells into androgenetic haploid embryos that were previously injected with a sperm head, followed by the removal of the maternal spindle. The developmental potential of these bipaternal embryos is limited to E8.5 after embryo transfer. However, bipaternal mice are able to be obtained by an alternative strategy through a tetraploid embryo complementation assay after the derivation of bipaternal diploid ESCs