Current advances in haploid stem cells

Abstract

Diploidy is the typical genomic mode in all mammals. Haploid stem cells are artificial cell lines experimentally derived in vitro in the form of different types of stem cells, which combine the characteristics of haploidy with a broad developmental potential and open the possibility to uncover biological mysteries at a genomic scale. To date, a multitude of haploid stem cell types from mouse, rat, monkey and humans have been derived, as more are in development. They have been applied in high-throughput genetic screens and mammalian assisted reproduction. Here, we review the generation, unique properties and broad applications of these remarkable cells.

Keywords: androgenetic; diploidization; functional genomics; haploidy; imprinting; parthenogenetic; stem cells.

Figure 1

Derivation of mouse haploid embryonic stem cells (haESCs). (A) Derivation strategies of parthenogenetic haESCs (phESCs) and androgenetic haESCs (ahESCs). Parthenogenetic haploid blastocysts are developed from artificially activated MII oocytes. Androgenetic embryos can be obtained by injecting sperm into the enucleated MII oocytes or removing the female pronucleus from fertilized oocytes. The resulting haploid blastocysts are subsequently cultured to develop haESC lines. (B) The haESC lines of different mammalian species have been generated

Figure 2

Diploidization of haploid cells. (A) Schematic showing that abnormal cell cycle regulation is the cause for diploidization in haploid cells. (B) Solutions for diploidization include physical approaches using fluorescence-activated cell sorting (FACS) and membranes with micrometer pores, chemical approaches via addition of kinase inhibitors and genetic manipulations in haploid cells

Figure 3

Applying haploid stem cells for functional genomics. (A) Forward genetic screens are powerful tools for the discovery and functional annotation of genetic elements. Three major steps are: mutagenesis to generate high-throughput mutant libraries, selection for the phenotype of interest, and mapping of mutations. (B) Two types of delivery systems used in gene trapping, the plasmid system and the retroviral system. (Top) Schematic diagram of splice acceptor (SA) gene trap, in which transposon elements were integrated in a plasmid vector. Splicing to upstream exons results in gene trap fusion transcript from which puromycin (puro) is transcribed by an endogenous promoter. CAG, a constitutive promoter; IRES, internal ribosome entry site; pA, poly A; ITR, inverted terminal repeat. (Bottom) Schematic diagram of ployA-trap, in which transposon elements were integrated in a retroviral vector. Insertion of a constitutive promoter-driven marker gene into introns results in a gene trap fusion transcript from which puromycin (puro) is terminated by an endogenous polyadenylation site. CMV, a constitutive promoter; SD, splice donor. LTR, long terminal repeat. (C) Schematic diagram of lentiviral expression vector for SpCas9 and sgRNA in a dual-vector form and single-vector form. psl+, Psi packaging singal; PRE, Rev response element; cPPT, central polypurine tract; EF1α, elongation factor 1α promoter; CMV, immediate-early cytomegalovirus enhancer-promoter; U6, RNA polymerase III U6 promoter; 2A, 2A self-cleavage peptide; Blast, blasticidin selection marker; Puro, puromycin selection marker; WPRE, post-transcriptional regulatory element

Figure 4

Haploid embryonic stem cells (haESCs) can be used to replace gametes for the generation of alive mice. Deletions of the specific imprinting regions in haESCs can facilitate to generate normally growing bimaternal mice and live bipaternal mice. DMRs, differentially methylated regions. ahESC, androgenetic haESC. phESC, parthenogenetic haESC