Induction of mouse totipotent stem cells by a defined chemical cocktail

In mice, only the zygotes and blastomeres from 2-cell embryos are authentic totipotent stem cells (TotiSCs), capable of producing all the differentiated cells in both embryonic and extraembryonic tissues and forming an entire organism1. However, it remains challenging whether and how TotiSCs, representing the very beginning of a life, can be established in vitro in the absence of germline cells. Here, we demonstrate induction and long-term maintenance of TotiSCs from mouse pluripotent stem cells (PSCs) by a combination of three small molecules, TTNPB, 1-Azakenpaullone, and WS6. These cells, which we designated as ciTotiSCs (chemically induced totipotent stem cells), resembled mouse totipotent 2C-embryo stage cells at transcriptome, epigenome and metabolome level. In addition, ciTotiSCs exhibited bidirectional developmental potentials and were able to produce both embryonic and extraembryonic cells in vitro and in teratoma. Furthermore, following injection into 8-cell embryo, ciTotiSCs contributed to both embryonic and extraembryonic lineages with high efficiency. Our chemical approach for TotiSCs induction and maintenance provides a defined in vitro system to manipulate and understand totipotent state towards creating life from non-germline.