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Related Concept Videos

Maintenance of the ES Cell State01:14

Maintenance of the ES Cell State

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The cells of the blastocyst inner cell mass only remain pluripotent for a short time. This state of pluripotency and self-renewal can be maintained in embryonic stem (ES) cell culture by adding specific chemicals or growth factors to ensure the cells can continue dividing and later differentiate into different cell types. In some cases, the cells are grown on a feeder layer of differentiated cells, which provides the growth factors and extracellular matrix components necessary for stem cell...
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A Simple Method to Identify Kinases That Regulate Embryonic Stem Cell Pluripotency by High-throughput Inhibitor Screening
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Defining Essential Enhancers for Pluripotent Stem Cells Using a Features-Oriented CRISPR-Cas9 Screen.

Hao Fei Wang1, Tushar Warrier1, Chadi A Farran2

  • 1Laboratory for Epigenetics, Stem Cells and Cell Therapy, Programme in Stem Cell, Regenerative Medicine and Aging, A(∗)STAR Institute of Molecular and Cell Biology, Singapore 138673, Singapore; Department of Biological Sciences, National University of Singapore, Singapore 117543, Singapore.

Cell Reports
|October 28, 2020
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Summary

Researchers identified essential stem cell enhancers using a CRISPR-Cas9 screen. These cis-regulatory elements (CREs) maintain pluripotency and regulate gene networks, with CRE111 modulating the LIF-JAK1-STAT3 pathway.

Keywords:
CRISPR screenJAK-STAT3LRRC31OCT4-boundessential cis-regulatory elementspluripotencysuper-enhancer

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Area of Science:

  • Genomics
  • Molecular Biology
  • Stem Cell Biology

Background:

  • Cis-regulatory elements (CREs) control gene expression and cellular functions like differentiation.
  • Significant gaps exist in understanding CRE function and their target genes in vivo.
  • Pluripotency maintenance in mouse embryonic stem cells (mESCs) relies on precise gene regulation.

Purpose of the Study:

  • To systematically identify functional CREs critical for maintaining pluripotency in mESCs.
  • To characterize the regulatory networks governed by these CREs.
  • To elucidate the role of specific CREs in key signaling pathways.

Main Methods:

  • Features-oriented CRISPR-utilized systematic (FOCUS) screen utilizing CRISPR-Cas9.
  • RNA sequencing (RNA-seq) to analyze gene expression changes.
  • Genomic 4C-seq to map CRE-target gene interactions.

Main Results:

  • A FOCUS screen identified 16 essential stem cell enhancers from 235 OCT4-bound CRE candidates.
  • RNA-seq and 4C-seq revealed complex CRE networks regulating mESC growth and self-renewal.
  • CRE111 was identified as a key enhancer regulating the LIF-JAK1-STAT3 pathway via its target gene, Lrrc31.

Conclusions:

  • The study successfully identified functional CREs crucial for mESC pluripotency.
  • A comprehensive map of CRE-target gene interactions was established, revealing regulatory networks.
  • The findings highlight CRE111 and Lrrc31 as critical regulators of the LIF-JAK1-STAT3 pathway, essential for stem cell maintenance.