Derivation of naive human embryonic stem cells

Area of Science:

• Stem cell biology
• Developmental biology
• Epigenetics

Background:

• The naive pluripotent state in mice offers superior developmental potential compared to primed epiblast cells.
• Deriving human naive embryonic stem cells (hESCs) without transgenes has been a significant challenge.
• Previous methods relied on forced expression of OCT4, KLF4, and KLF2 to maintain hESCs in a naive state.

Purpose of the Study:

• To describe two novel routes for generating nontransgenic naive hESCs.
• To demonstrate that human naive cells meet established mouse criteria for the naive state.
• To confirm that hESCs can exist in a naive state without requiring transgenes.

Main Methods:

• Route 1: Reverse toggling of primed hESC lines using histone deacetylase inhibitors (butyrate and suberoylanilide hydroxamic acid) followed by culture in MEK/ERK and GSK3 inhibitors (2i) with FGF2.
• Route 2: Direct derivation of hESCs from human embryos cultured in 2i with FGF2.
• Validation of naive state using criteria including growth characteristics, antibody labeling, gene expression, X-inactivation, mitochondrial morphology, microRNA profile, and teratoma formation.

Main Results:

• Two efficient and reproducible methods for generating nontransgenic naive hESCs were established.
• The derived human naive cells successfully met established mouse criteria for pluripotency and developmental potential.
• Direct derivation, while challenging, yielded cells representing the earliest stage of human in vitro pluripotency.

Conclusions:

• Human ESCs can achieve a naive pluripotent state without the necessity of transgenes.
• The described methods provide efficient and reproducible means to generate nontransgenic naive hESCs.
• These findings open new avenues for studying early human development and regenerative medicine applications.