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

Area of Science:

• Developmental Biology
• Stem Cell Biology
• Epigenetics

Background:

• Human pluripotent stem cells (hPSCs) hold potential for regenerative medicine.
• Understanding germ layer specification is crucial for directing hPSC differentiation.
• Identifying regulatory pathways controlling pluripotency and lineage commitment is essential.

Purpose of the Study:

• To define genetic pathways and biological processes that restrict germ layer specification in hPSCs.
• To identify factors governing hPSC self-renewal and trilineage development.
• To elucidate the role of specific pathways, like cholesterol synthesis, in maintaining pluripotency.

Main Methods:

• Genetically engineered hPSCs with germ layer reporters and CRISPR/Cas9.
• Performed genome-scale screening to identify regulatory genes.
• Analyzed gene clusters involved in embryonic development, metabolism, and epigenetics.

Main Results:

• Identified genes regulating pluripotency and lineage development, including embryonic development, mRNA processing, metabolism, and epigenetic regulation.
• Loss of mesendodermal specifiers accelerated neuroectodermal differentiation, indicating inter-germ layer antagonism.
• MicroRNAs exhibited germ layer-targeting specificity, and cholesterol synthesis pathway inhibited neuroectoderm formation.

Conclusions:

• A comprehensive landscape of genetic control for hPSC self-renewal and trilineage specification was identified.
• Specific pathways, including cholesterol synthesis and microRNAs, play critical roles in germ layer determination.
• Findings provide insights into the complex regulatory network governing early human development.