Search

Totipotency is the ability of a single cell to give rise to all of the differentiated cell types that build the conceptus, yet how to capture this property in vitro remains incompletely understood. Defining totipotency relies on a variety of assays of variable stringency. Here, we describe criteria to define totipotency. We explain how distinct criteria of increasing stringency can be used to judge totipotency by evaluating candidate totipotent cell types in mice, including early blastomeres and expanded or extended pluripotent stem cells. Our data challenge the notion that expanded or extended pluripotent states harbour increased totipotent potential relative to conventional embryonic stem cells under in vitro and in vivo conditions.

Totipotency refers to the ability of a cell to generate all of the cell types of an organism. Unlike pluripotency, the establishment of totipotency is poorly understood. In mouse embryonic stem cells, Dux drives a small percentage of cells into a totipotent state by expressing 2-cell-embryo-specific transcripts. To understand how this transition takes place, we performed single-cell RNA-seq, which revealed a two-step transcriptional reprogramming process characterized by downregulation of pluripotent genes in the first step and upregulation of the 2-cell-embryo-specific elements in the second step. To identify factors controlling the transition, we performed a CRISPR-Cas9-mediated screen, which revealed Myc and Dnmt1 as two factors preventing the transition. Mechanistic studies demonstrate that Myc prevents downregulation of pluripotent genes in the first step, while Dnmt1 impedes 2-cell-embryo-specific gene activation in the second step. Collectively, the findings of our study reveal insights into the establishment and regulation of the totipotent state in mouse embryonic stem cells.

SUMMARY Oct4A is a core component of the regulatory network of pluripotent cells, and by itself can reprogram neural stem cells into pluripotent cells in mouse and humans. However, its role in defining totipotency and inducing pluripotency during embryonic development is still unclear. We genetically eliminated maternal Oct4A using a Cre-lox approach in mouse and found that the establishment of totipotency was not affected, as shown by the generation of live pups. After complete inactivation of both maternal and zygotic Oct4A expression, the embryos still formed Oct4-GFP– and Nanog–expressing inner cell masses, albeit non-pluripotent, indicating that Oct4A is not a determinant for the pluripotent cell lineage separation. Interestingly, Oct4A-deficient oocytes were able to reprogram fibroblasts into pluripotent cells. Our results clearly demonstrate that, in contrast to its role in the maintenance of pluripotency, maternal Oct4A is crucial for neither the establishment of totipotency in embryos, nor the induction of pluripotency in somatic cells using oocytes.

The goal of regenerative medicine is to restore form and function to damaged tissues. One potential therapeutic approach involves the use of autologous cells derived from the bone marrow (bone marrow-derived cells, BMDCs). Advances in nuclear transplantation, experimental heterokaryon formation and the observed plasticity of gene expression and phenotype reported in multiple phyla provide evidence for nuclear plasticity. Recent observations have extended these findings to show that endogenous cells within the bone marrow have the capacity to incorporate into defective tissues and be reprogrammed. Irrespective of the mechanism, the potential for new gene expression patterns by BMDCs in recipient tissues holds promise for developing cellular therapies for both proliferative and post-mitotic tissues.