Mapping germ-layer speciﬁcation preventing genes in hPSCs via genome-scale CRISPR screening

iScience, 2021 · DOI: https://doi.org/10.1016/j.isci.2020.101926 · Published: January 22, 2021

Genetics Regenerative Medicine & Stem Cells

Simple Explanation

Researchers used CRISPR screening to identify genes that prevent human pluripotent stem cells (hPSCs) from differentiating into specific germ layers. They engineered hPSCs with a reporter for a neuroectoderm marker and performed a genome-wide knockout screen. The screen identified genes involved in embryonic development, mRNA processing, metabolism, and epigenetic regulation as key to maintaining pluripotency and restricting lineage development. Loss of mesendodermal specifiers led to advanced neuroectodermal differentiation. MicroRNAs enriched in hPSCs showed germ layer-targeting specificity, and the cholesterol synthesis pathway maintained hPSCs by preventing neuroectoderm specification. This study provides insights into the genetic and biological processes controlling hPSC self-renewal and differentiation.

Study Duration

Not specified

Participants

Human pluripotent stem cells (hPSCs)

Evidence Level

Level 3: Genome-scale CRISPR screening

Key Findings

1
A genome-scale CRISPR screen in hPSCs identified lineage-specification preventing genes (LPGs) that, when knocked out, cause the cells to differentiate into specific germ layers.
2
LPGs are clustered into functional modules related to embryonic development, mRNA processing, metabolism, and epigenetic regulation, highlighting their roles in pluripotency and lineage development.
3
The cholesterol synthesis pathway was identified as a key regulator maintaining hPSCs by specifically preventing neuroectoderm specification.

Research Summary

This study uses genome-scale CRISPR screening in hPSCs to identify genes that prevent lineage specification, termed lineage-specification preventing genes (LPGs). The screening revealed that LPGs are clustered into functional modules, including embryonic development, mRNA processing, metabolism, and epigenetic regulation, indicating their importance in pluripotency and lineage development. The study also found that the cholesterol synthesis pathway maintains hPSCs by preventing neuroectoderm specification, and that hPSC-expressed miRNAs act as differentiation barriers that specifically inhibit differentiation of individual lineages.

Practical Implications

Understanding Pluripotency

Identifies key genetic and biological processes underlying hPSC self-renewal and trilineage differentiation.

Targeted Differentiation

Provides a resource of LPGs for future studies on cellular and molecular events regarding hPSC self-renewal and trilineage development, which can be used to enhance directed differentiation protocols.

Regenerative Medicine

Offers insights into cell fate determination, which is a key question in developmental biology and the basis for regenerative medicine.

Study Limitations

1
The study only used a PAX6 reporter for the neuroectodermal lineage, lacking parallel screens with mesendodermal reporters.
2
Specific validation is apparently required when comes to individual genes.
3
When taking the trilineage cell fate decision in count

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