Abstract
Totipotent cells can differentiate into three lineages: the epiblast, primitive endoderm, and trophectoderm. Naturally, only early fertilized embryos possess totipotency, and they lose this ability as they develop. The expansion of stem cell differentiation potential has been a hot topic in developmental biology for years, particularly with respect to the generation totipotent-like stem cells. Here, the study describes the establishment of totipotency in embryonic stem cells (ESCs) via the deletion of a single gene, Pdzk1. Pdzk1-knockout (KO) ESCs substantially contribute to the fetus, placenta, and yolk sac in chimera assays but can also self-organize to form standard blastocyst-like structures containing the three lineages efficiently; thus, they exhibit full developmental potential as early blastomeres. Single-cell transcriptome and bulk RNA-seq comprehensive analyses revealed that Pdzk1-KO activates several lineage inducers (C1qa, C1qb, Fgf5, and Cdx2) to break down barriers between embryonic and extraembryonic tissues, making these lineages switch smoothly and resulting in a totipotent-like state. This versatile and scalable system provides a robust experimental model for differentiation potency and cell fate studies.