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Inhibition of Apoptosis Reduces Diploidization of Haploid Mouse Embryonic Stem Cells during the Differentiation

ABSTRACT: Inhibition of Apoptosis Reduces Diploidization of Haploid Mouse Embryonic Stem Cells during the Differentiation

PROVIDER: PRJNA623293 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR11486316_1.fastq.gz	Fastqsanger.gz
	SRR11486316_2.fastq.gz	Fastqsanger.gz
	SRR11486317_1.fastq.gz	Fastqsanger.gz
	SRR11486317_2.fastq.gz	Fastqsanger.gz
	SRR11486318_1.fastq.gz	Fastqsanger.gz

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Inhibition of Apoptosis Reduces Diploidization of Haploid Mouse Embryonic Stem Cells during the Differentiation

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.

2020-04-07 | GSE148118 | GEO

Haploid embryonic stem cells

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.

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Overexpression of Let-7a Mitigates Diploidization in Mouse Androgenetic Haploid Embryonic Stem Cells

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.

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Inhibition of Apoptosis Reduces Diploidization of Haploid Mouse Embryonic Stem Cells during Differentiation.

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 it remain unknown. To address this, we induced p53-deficient haESCs into multiple differentiated lineages maintain haploid status in vitro. Haploid cells also remained in chimeric embryos and teratomas arising from p53-null haESCs. Transcriptome analysis revealed that apoptosis genes were downregulated in p53-null haESCs compared with that in wild-type haESCs. Finally, we knocked out p73, another apoptosis-related gene, and observed stabilization of haploidy in haESCs. These results indicated that the main mechanism of diploidization was apoptosis-related gene-triggered cell death in haploid cell cultures. Thus, we can derive haploid somatic cells by manipulating the apoptosis gene, facilitating genetic screens of lineage-specific development.

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Project description:Inhibition of Akt Signaling in PIK3R2 Overexpressing Colon Cancer Stem Cells Reduces Tumor Growth due Apoptosis and Decreased Mucin Production

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A genetic screen identifies Etl4-deficiency capable of stabilizing the haploidy in embryonic stem cells

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.

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Overexpression of Let-7a Mitigates Diploidization in Mouse Androgenetic Haploid Embryonic Stem Cells

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