High‐throughput screening in postimplantation haploid epiblast stem cells reveals  Hs3st3b1  as a modulator for reprogramming

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Abstract

Epiblast stem cells (EpiSCs) derived from postimplantation epiblast are pluripotent stem cells, epigenetically distinct from embryonic stem cells (ESCs), which are widely used in reprogramming studies. Recent achieved haploid cell lines in mammalian species open a new era for high‐throughput genetic screening, due to their homozygous phenotypes. Here, we report the generation of mouse haploid EpiSCs (haEpiSCs) from postimplantation chimeric embryos at embryonic day 6.5 (E6.5). These cells maintain one set of chromosomes, express EpiSC‐specific genes, and have potentials to differentiate into three germ layers. We also develop a massive mutagenesis protocol with haEpiSCs, and subsequently perform reprogramming selection using this genome‐wide mutation library. Multiple modules related to various pathways are implicated. The validation experiments prove that knockout of Hst3st3b1 (one of the candidates) can promote reprogramming of EpiSCs to the ground state efficiently. Our results open the feasibility of utilizing haEpiSCs to elucidate fundamental biological processes including cell fate alternations.

Abstract

Mouse haploid epiblast stem cells (haEpiSCs) can be derived from E6.5 postimplantation chimeric epiblast, which are pluripotent with a single genome. Genome‐scale gene trapping in haEpiSCs provides a platform to uncover essential genes regulating cellular reprograming. Knocking out one of the candidate genes, Hs3st3b1, greatly improves the efficiency of reprogramming from EpiSCs to embryonic stem cells.

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Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2

Michael Love, Wolfgang Huber, Simon Anders (2014)

In comparative high-throughput sequencing assays, a fundamental task is the analysis of count data, such as read counts per gene in RNA-seq, for evidence of systematic changes across experimental conditions. Small replicate numbers, discreteness, large dynamic range and the presence of outliers require a suitable statistical approach. We present DESeq2, a method for differential analysis of count data, using shrinkage estimation for dispersions and fold changes to improve stability and interpretability of estimates. This enables a more quantitative analysis focused on the strength rather than the mere presence of differential expression. The DESeq2 package is available at http://www.bioconductor.org/packages/release/bioc/html/DESeq2.html. Electronic supplementary material The online version of this article (doi:10.1186/s13059-014-0550-8) contains supplementary material, which is available to authorized users.

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Trimmomatic: a flexible trimmer for Illumina sequence data

Anthony M. Bolger, Marc Lohse, Bjoern Usadel (2014)

Motivation: Although many next-generation sequencing (NGS) read preprocessing tools already existed, we could not find any tool or combination of tools that met our requirements in terms of flexibility, correct handling of paired-end data and high performance. We have developed Trimmomatic as a more flexible and efficient preprocessing tool, which could correctly handle paired-end data. Results: The value of NGS read preprocessing is demonstrated for both reference-based and reference-free tasks. Trimmomatic is shown to produce output that is at least competitive with, and in many cases superior to, that produced by other tools, in all scenarios tested. Availability and implementation: Trimmomatic is licensed under GPL V3. It is cross-platform (Java 1.5+ required) and available at http://www.usadellab.org/cms/index.php?page=trimmomatic Contact: usadel@bio1.rwth-aachen.de Supplementary information: Supplementary data are available at Bioinformatics online.

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Gene Ontology: tool for the unification of biology

Michael Ashburner, Catherine A. Ball, Judith Blake … (2002)

Genomic sequencing has made it clear that a large fraction of the genes specifying the core biological functions are shared by all eukaryotes. Knowledge of the biological role of such shared proteins in one organism can often be transferred to other organisms. The goal of the Gene Ontology Consortium is to produce a dynamic, controlled vocabulary that can be applied to all eukaryotes even as knowledge of gene and protein roles in cells is accumulating and changing. To this end, three independent ontologies accessible on the World-Wide Web (http://www.geneontology.org) are being constructed: biological process, molecular function and cellular component.

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Author and article information

Contributors

Luyuan Li: liluyuan@nankai.edu.cn

Yan Liu: 30819007@nankai.edu.cn

Ling Shuai:

ORCID: https://orcid.org/0000-0003-0671-7478

lshuai@nankai.edu.cn

Journal

Journal ID (nlm-ta): Stem Cells Transl Med

Journal ID (iso-abbrev): Stem Cells Transl Med

Journal ID (doi): 10.1002/(ISSN)2157-6580

Journal ID (publisher-id): SCT3

Title: Stem Cells Translational Medicine

Publisher: John Wiley & Sons, Inc. (Hoboken, USA )

ISSN (Print): 2157-6564

ISSN (Electronic): 2157-6580

Publication date (Electronic): 29 January 2021

Publication date Collection: May 2021

Volume: 10

Issue: 5 ( doiID: 10.1002/sct3.v10.5 )

Pages: 743-755

Affiliations

[ ¹ ] State Key Laboratory of Medicinal Chemical Biology and College of Pharmacy Nankai University Tianjin People's Republic of China

[ ² ] Department of Obstetrics Tianjin First Central Hospital, Nankai University Tianjin People's Republic of China

[ ³ ] Nankai Animal Resource Center Nankai University Tianjin People's Republic of China

[ ⁴ ] Tianjin Central Hospital of Gynecology Obstetrics/Tianjin Key Laboratory of Human Development and Reproductive Regulation Tianjin People's Republic of China

Author notes

[*] [* ] Correspondence

Luyuan Li, PhD, State Key Laboratory of Medicinal Chemical Biology and College of Pharmacy, Nankai University, Tianjin 300350, People's Republic of China.

Email: liluyuan@ 123456nankai.edu.cn

Yan Liu, PhD, Department of Obstetrics, Tianjin First Central Hospital, Nankai University, Tianjin 300192, People's Republic of China.

Email: 30819007@ 123456nankai.edu.cn

Ling Shuai, PhD, State Key Laboratory of Medicinal Chemical Biology and College of Pharmacy, Nankai University, Tianjin 300350, People's Republic of China.

Email: lshuai@ 123456nankai.edu.cn

Author information

Ling Shuai https://orcid.org/0000-0003-0671-7478

Article

Publisher ID: SCT312895

DOI: 10.1002/sctm.20-0468

PMC ID: 8046116

PubMed ID: 33511777

SO-VID: b6ca3fd7-dadb-4ca2-b56e-dbe92ac869d0

License:

This is an open access article under the terms of the http://creativecommons.org/licenses/by-nc-nd/4.0/ License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non‐commercial and no modifications or adaptations are made.

History

Date revision received : 12 December 2020

Date received : 21 October 2020

Date accepted : 03 January 2021

Page count

Figures: 6, Tables: 1, Pages: 13, Words: 8859

Funding

Funded by: Tianjin Central Hospital of Gynecology Obstetrics

Funded by: Tianjin Key Laboratory of human development and reproductive regulation

Award ID: 2019XHY08

Funded by: Strategic Collaborative Research Program of the Ferring Institute of Reproductive Medicine, Ferring Pharmaceuticals and Chinese Academy of Sciences

Award ID: FIRMD181102

Funded by: National Natural Science Foundation of China , open-funder-registry 10.13039/501100001809;

Award ID: 31872841

Award ID: 31671538

Award ID: 32022025

Funded by: National Key Research and Development Program of China , open-funder-registry 10.13039/501100012166;

Award ID: 2019YFA0109901

Award ID: 2018YFC1004101

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source-schema-version-number 2.0

cover-date May 2021

details-of-publishers-convertor Converter:WILEY_ML3GV2_TO_JATSPMC version:6.0.2 mode:remove_FC converted:14.04.2021

High‐throughput screening in postimplantation haploid epiblast stem cells reveals Hs3st3b1 as a modulator for reprogramming

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Most cited references 55

Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2

Trimmomatic: a flexible trimmer for Illumina sequence data

Gene Ontology: tool for the unification of biology

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