Induction of Human Naïve Pluripotency Using 5i/L/A Medium

Area of Science:

• Stem cell biology
• Developmental biology
• Epigenetics

Background:

• Mammalian epiblast cells exist in a naïve pluripotent state before implantation, characterized by lack of lineage priming and epigenetic restrictions.
• Conventional human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) are typically in a primed pluripotent state, resembling post-implantation epiblast.
• Reverting primed cells to a naïve state is crucial for understanding early human development and unlocking full regenerative potential.

Purpose of the Study:

• To develop a robust protocol for inducing naïve pluripotency in primed hESCs and iPSCs.
• To establish reliable methods for validating the acquisition of naïve pluripotency.
• To utilize reporter systems for real-time monitoring of naïve pluripotency induction.

Main Methods:

• A specific cocktail of five kinase inhibitors and two growth factors (5i/L/A) was employed.
• Detailed protocols for inducing and validating naïve pluripotency in primed hESCs and iPSCs were established.
• Two fluorescent reporter systems (OCT4-ΔPE-GFP and MECP2-GFP/tdTomato) were used for live-cell tracking of naïve identity.

Main Results:

• The 5i/L/A cocktail successfully induced key features of naïve pluripotency in primed hESCs and iPSCs.
• Established validation methods confirmed the naïve identity of reprogrammed cells.
• Reporter systems effectively tracked the dynamic acquisition of naïve pluripotency.

Conclusions:

• The developed 5i/L/A conditions provide a reliable method for generating naïve human pluripotent stem cells (hPSCs).
• This advancement offers insights into the unique characteristics of naïve hPSCs, including their transcriptional profile and developmental potential.
• The protocol facilitates further research into early human embryogenesis and regenerative medicine applications.