Generation of chemically induced totipotent cells through multi-parametric analysis of single-cells

Pluripotent stem cells fluctuate among distinct functional cell states. Such heterogeneity undermines the potential applications of stem cells in regenerative medicine. The heterogeneity of pluripotent cell states and their molecular nature remain less understood. Here, we utilized mass cytometry to dissect pluripotent cell states by simultaneously profiling multiple proteins and their modifications in single cells. Results indicated the role of extensive signaling cross-talk among multiple pathways in regulating cell state heterogeneity. By systematically dissecting the signals and their combinations, we identify conditions through which large portions of pluripotent cells can be chemically reprogrammed into totipotent cells which can be cultured. Unlike pluripotent cells, these chemically induced totipotent cells (CiTC) give rise to both embryonic and extra-embryonic cell types. In addition, CiTCs are molecularly distinct from pluripotent cells. CiTCs provide an in-vitro model to understand the nature of totipotent cells and the plasticity of pluripotent cells. Our quantitative and integrative approach provides general method to understand similar heterogenous cell systems such as cancer. Overall design: Single-cell RNA-sequencing of a time-course of embryonic stem cells (ESC) under cell culture conditions to induce the totipotent-like cells