Expression data from haploid and diploid epiblast stem cells
Ontology highlight
ABSTRACT: Haploid pluripotent stem cells, such as haploid embryonic stem cells (haESCs), facilitate the genetic study of recessive traits. In vitro, fish haESCs maintain haploidy in both undifferentiated and differentiated states, but whether mammalian haESCs can preserve pluripotency in the haploid state has not been tested. Here, we report that mouse haESCs can differentiate in vitro into haploid epiblast stem cells (haEpiSCs), which maintain an intact haploid genome, unlimited self-renewal potential, and durable pluripotency to differentiate into various tissues in vitro and in vivo. Mechanistically, the maintenance of self-renewal potential depends on the Activin/bFGF pathway. We further show that haEpiSCs can differentiate in vitro into haploid progenitor-like cells.
Project description:Phenotypes of haploid embryonic stem cells (haESCs) are dominant for recessive traits in mice. However, one major obstacle to their use is self-diploidization in daily culture. Although haESCs maintain haploidy well by deleting p53, whether they can sustain haploidy in differentiated status and the mechanism behind remain unknown. To address that, we induced p53-deficient haESCs into multiple differentiated lineages keeping a haploid status in vitro. Besides, haploid cells also remained in chimeric embryos and teratomas arising from p53-null haESCs. Transcriptome analysis revealed that apoptosis genes were down-regulated in p53-null haESCs, comparing to that in wild-type haESCs. Finally, we knocked-out p73, another apoptosis gene, and observed stabilization of haploidy in haESCs, either. These results indicated that the main mechanism of diploidization was apoptosis-related genes triggered cell death in haploid cell cultures. Thus, we can derive haploid somatic cells by manipulating apoptosis gene, facilitating genetic screens of lineage-specific development.
Project description:We performed a genome-wild loss-of-function screening and revealed several gene mutations could significantly reduce the rate of self-diploidization in haESCs. We further demonstrated that CRISPR/Cas9 mediated Etl4 knockout stabilizes the haploid state in different haploid cell lines. More interestingly, Etl4 deficiency could increase the mitochondrial oxidative phosphorylation (OXPHOS) capacity and decrease glycolysis in haESCs. Collectively, our study identifies Etl4 as a novel haploidy-related factor and suggests that the changes of energy metabolism is associated with self-diploidization.
Project description:Haploid embryonic stem cells (haESCs) have been extensively applied in forward and reverse genetic screening. However, a mammalian haploid somatic cell line is difficult to achieve because of spontaneous diploidization in differentiation. As a non-human primate species, monkeys are widely used in basic and pre-clinical research in which haploid cells are restricted to ESCs. Here, we report that rhesus monkey haESCs in an optimized culture medium show naïve-state pluripotency and stable haploidy. This model facilitated the derivation of haploid neural progenitor cells (haNPCs), which maintained haploidy and differentiation potential into neurons and glia for a long period in vitro. High-throughput trapping mutations can be efficiently introduced into haNPCs via piggyBac transposons. This system proves useful when identifying gene targets of neural toxicants via a proof-of-concept experiment. Using CRISPR/Cas9 editing, we confirmed that B4GALT6, from the candidate gene list, is a resistance gene of A803467 (a tetrodotoxin-like toxicant). This model is the first non-human primate haploid somatic cell line with proliferative ability, multipotency and an intact genome, thus providing a cellular resource for recessive genetic and potential drug screening.
Project description:We generate histone modification profiles and DNA methylation profiles of mouse haploid embryonic stem cells. By comparison to mouse diploid ESCs and MEFs, we found the similar chromatin landscapes between haESCs and mESCs, which reveal the self-renew ability and pluripotency in haESCs. Besides, haESCs specific chromatin landscapes show its sperm-like functions.
Project description:We generate histone modification profiles and DNA methylation profiles of mouse haploid embryonic stem cells. By comparison to mouse diploid ESCs and MEFs, we found the similar chromatin landscapes between haESCs and mESCs, which reveal the self-renew ability and pluripotency in haESCs. Besides, haESCs specific chromatin landscapes show its sperm-like functions.
Project description:Rat embryonic stem cells (ESCs), which are widely studied, can self-renew and exhibit pluripotency in long-term culture, but the mechanism underlying how they exit pluripotency remains obscure. Rat haploid ESCs (haESCs) enable advances in the discovery of unknown functional genes owing to their homozygous and pluripotent characteristics. Herein, we performed genome-wide mutation using piggyBac transposons in rat haESCs and obtained differentiation-retarded mutants assisted by Rex1-GFP reporter selection. High-throughput sequencing analysis further revealed numerous insertions related to various pathways affecting random differentiation. Thereafter, deletion of Thop1 (one candidate gene in the screened list) arrested the differentiation of rat ESCs by inhibiting the phosphorylation of ERK1/2, whereas overexpression of Thop1 promoted rat ESCs exit from pluripotency. Our findings provide an ideal tool to study functional genomics in rats: a homozygous haploid system carrying a pluripotency reporter that facilitates robust discovery of the mechanisms involved in the self-renewal or pluripotency of rat ESCs.
Project description:Rat embryonic stem cells (ESCs), which are widely studied, can self-renew and exhibit pluripotency in long-term culture, but the mechanism underlying how they exit pluripotency remains obscure. Rat haploid ESCs (haESCs) enable advances in the discovery of unknown functional genes owing to their homozygous and pluripotent characteristics. Herein, we performed genome-wide mutation using piggyBac transposons in rat haESCs and obtained differentiation-retarded mutants assisted by Rex1-GFP reporter selection. High-throughput sequencing analysis further revealed numerous insertions related to various pathways affecting random differentiation. Thereafter, deletion of Thop1 (one candidate gene in the screened list) arrested the differentiation of rat ESCs by inhibiting the phosphorylation of ERK1/2, whereas overexpression of Thop1 promoted rat ESCs exit from pluripotency. Our findings provide an ideal tool to study functional genomics in rats: a homozygous haploid system carrying a pluripotency reporter that facilitates robust discovery of the mechanisms involved in the self-renewal or pluripotency of rat ESCs.
Project description:Mammalian haploid embryonic stem cells (haESCs) provide new possibilities for large-scale genetic screens because they bear only one copy of each chromosome. However, haESCs are prone to spontaneous diploidization through unknown mechanisms. Here, we report that a small molecule combination could restrain mouse haESCs from diploidization by impeding exit from naïve pluripotency and by shortening the S-G2/M phases. Combined with 2i and PD166285, our chemical cocktail could maintain haESCs in the haploid state for at least five weeks without fluorescence-activated cell sorting (FACS) enrichment of haploid cells. Taken together, we established an effective chemical approach for long-term maintenance of haESCs, and highlighted that proper cell cycle progression was critical for the maintenance of haploid state.
Project description:Mouse androgenetic haploid embryonic stem cells (mAG-haESCs) can be utilized to uncover gene functions, especially those of genes with recessive effects, and to produce semicloned mice when injected into mature oocytes. However, mouse haploid cells undergo rapid diploidization during long-term culture in vitro and subsequently lose the advantages of haploidy and the factors that drive diploidization are not well understood. In this study, we compared the small RNAs (sRNAs) of mAG-haESCs, normal ESCs and mouse round spermatids by high-throughput sequencing and identified distinct sRNA profiles. Several let-7 family members and miR-290-295 cluster miRNAs were found significantly differentially transcribed. Knockdown and overexpression experiments showed that let-7a and let-7g suppress diploidization while miR-290a facilitates diploidization. Our study revealed the unique sRNA profile of mAG-haESCs and demonstrated that let-7a overexpression can mitigate diploidization in mAG-haESCs. These findings will help us to better understand mAG-haESCs and utilize them as a tool in the future.
Project description:Haploid cells are amenable for genetic analysis because they contain only one set of chromosomes. Here,we report the derivation of haESCs from monkey parthenogenic blastocysts. These cells, which we designated PG-haESCs (parthenogenic haploid embryonic stem cells), express classical ESC markers, are pluripotent, and can differentiate to different cell lines from all three embryonic germ layers in vivo and in vitro. We used microarrays to compare the gene expression levels among PG-haESC, ICSI-derived ESCs and female monkey somatic fibroblasts.