In the primary screen, we focused on small molecules that can increase the percentage of Zscan4-Emerald GFP + cells, and identified > 50 primary hits. ![]() N2B27 medium was used for screening and Wnt signaling agonist CHIR 99021 was also added to promote cell proliferation. ![]() As a positive control, Dux overexpression was induced in these reporter cells (Supplementary information, Fig. To identify small molecules that can induce totipotent features in mouse EPS cells, we used cell lines carrying a MERVL-tdTomato or a Zscan4-Emerald GFP reporter. Identification of a chemical cocktail that can establish totipotent-like stem cells in vitro Moreover, they can generate both embryonic and extraembryonic lineages in vivo at the single-cell level and form blastocyst-like structures in vitro. The induced totipotent-like stem cells can be stably maintained long term in vitro, with molecular features resembling 2–4-cell blastomeres. In this study, through chemical screening and combination, we identified a chemical cocktail that can directly establish totipotent-like stem cells from 2-cell embryos in vitro, as well as convert from EPS cells. In principle, such stem cells should be derived directly from totipotent embryos, which represents the gold criterion to capture authentic totipotency. 19 Therefore, it remains a major challenge to capture and maintain bona fide totipotent stem cells in vitro. 19 In addition, their extraembryonic differentiation potentials are still limited compared to totipotent embryos. 18 Despite their expanded developmental potentials, these cells are still transcriptomically distinct from 2-cell embryos. 16, 17 Importantly, extended pluripotent stem (EPS) cells can be induced into blastocyst-like structures. 13 In recent years, our group and others have shown that small molecule combinations can expand the developmental potentials of pluripotent stem cells toward extraembryonic lineages. 14, 15 The major limitation of 2C-like cells is that they are only a minor population from mouse ES cell cultures and reside in a transient intermediate state, and thus these cells cannot be stabilized in vitro. Previous studies have shown that a rare 2-cell embryo (2C)-like subpopulation exists in mouse embryonic stem (ES) cell cultures that express 2-cell blastomere molecular markers and have embryonic and extraembryonic developmental potentials. 10, 11, 12 Compared to these blastocyst-derived stem cell types, totipotent embryonic cells harbor the greatest developmental potency, and there has been great scientific interest in capturing such stem cells from totipotent embryos in vitro, 13 whose unrestricted potency would have broad applications for stem cell biology and regenerative medicine. Importantly, the developmental potentials of these stem cell types are lineage restricted in that they have difficulties in crossing the embryonic and extraembryonic lineage boundaries especially in vivo. 3, 4, 5 Different self-renewing stem cells, including pluripotent stem cells, trophoblast stem cells and extraembryonic endoderm cells, can be derived from the blastocyst, 6, 7, 8, 9 which preserve the developmental potentials of epiblast, trophectoderm and primitive endoderm, respectively. 1, 2 The totipotent potential of early-stage blastomeres is gradually restricted at the blastocyst stage, when differentiation into epiblast, trophectoderm and primitive endoderm occurs. Our study opens up a new path toward fully capturing totipotent stem cells in vitro.ĭuring development, early-stage blastomeres are totipotent cells that have the potential to generate an entire individual, including both embryonic and extraembryonic components, at the single-cell level. Mechanistically, inhibition of HDAC1/2 and DOT1L activity and activation of RARγ signaling are important for inducing and maintaining totipotent features of TPS cells. Moreover, TPS cells can be induced into blastocyst-like structures resembling preimplantation mouse blastocysts. In vivo chimera formation assays show that these cells have embryonic and extraembryonic developmental potentials at the single-cell level. TPS cells shared features with 2-cell mouse embryos in terms of totipotency markers, transcriptome, chromatin accessibility and DNA methylation patterns. 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. It is challenging to derive totipotent stem cells in vitro that functionally and molecularly resemble cells from totipotent embryos.
0 Comments
Leave a Reply. |
Details
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |