Derivation of totipotent-like stem cells with blastocyst-like structure forming potential

Abstract

It is challenging to derive totipotent stem cells in vitro that functionally and molecularly resemble cells from totipotent embryos. Here, we report that a chemical cocktail enables the derivation of totipotent-like stem cells, designated as totipotent potential stem (TPS) cells, from 2-cell mouse embryos and extended pluripotent stem cells, and that these TPS cells can be stably maintained long term in vitro. TPS cells shared features with 2-cell mouse embryos in terms of totipotency markers, transcriptome, chromatin accessibility and DNA methylation patterns. In vivo chimera formation assays show that these cells have embryonic and extraembryonic developmental potentials at the single-cell level. Moreover, TPS cells can be induced into blastocyst-like structures resembling preimplantation mouse blastocysts. Mechanistically, inhibition of HDAC1/2 and DOT1L activity and activation of RARγ signaling are important for inducing and maintaining totipotent features of TPS cells. Our study opens up a new path toward fully capturing totipotent stem cells in vitro.

Connecting the DOTs on Cell Identity

“…Pharmacological inhibition of DOT1L activity in 2CLC increases the appearance of the ringlike pericentric heterochromatin formation and enhances MERVL expression although H3K79me at these genes does not change ( Yang et al, 2022 ). DOT1L inhibitor was also included in a cocktail to stably increase totipotent properties in vitro ( Xu et al, 2022 ). We note that the DOT1L inhibitors used in these studies decrease chromatin association which may occur by directly opposing DOT1L function or by reducing transcription with the RNA polymerase II-associated DOT1L ( Nassa et al, 2019 ; Wu et al, 2021 ).…”

Recapitulating early human development with 8C-like cells

“…The ZGA-like transcriptional program is reactivated in 2CLCs (Iturbide and Torres-Padilla, 2020;Macfarlan et al, 2012;Rodriguez-Terrones et al, 2018). Moreover, based on the 2CLC model, we and two other groups establish the mouse totipotent-like stem cell lines that faithfully recapitulate mouse two-cell embryos and have more robust developmental potency than mESCs (Shen et al, 2021;Xu et al, 2022;Yang et al, 2022). This unique cell type with 2C-like transcriptome holds promise to identify ZGA regulators and explore the regulatory mechanisms of early mouse development.…”

Cocktails of defined chemical compounds: sufficient to induce totipotency in embryonic stem cells

“…In a recent work published in Nature , Hu et al 1 report that a cocktail of three small chemicals is sufficient to induce totipotency in murine embryonic stem (ES) cells. Earlier this year, two other groups identified different mixtures of chemicals, which also seem to do this job, 2 , 3 taken together this opens a new approach to capture totipotency in vitro, and to understand the underlying pathways and their interaction.…”

“…Recently, two other groups showed the induction of totipotency by different cocktails of small chemicals. As Hu et al, they used MERVL reporter constructs for screening, 2 , 3 Xu et al also used a Zscan4-Emerald reporter. 2 Xu et al achieved totipotent potential stem (TPS) cells from mouse extended pluripotent stem cells and 2-cell mouse embryos by screening a chemical library and defining a cocktail of compounds, which includes CD1530, VPA, EPZ004777, and CHIR 99021.…”

“…As Hu et al, they used MERVL reporter constructs for screening, 2 , 3 Xu et al also used a Zscan4-Emerald reporter. 2 Xu et al achieved totipotent potential stem (TPS) cells from mouse extended pluripotent stem cells and 2-cell mouse embryos by screening a chemical library and defining a cocktail of compounds, which includes CD1530, VPA, EPZ004777, and CHIR 99021. 2 The molecules CD1530, VPA, EPZ004777 regulate totipotency synergistically.…”