![]() Another road to totipotency seems to be the spliceosome inhibition via pladienolide B. ![]() CHIR 99021 is valuable for promoting the proliferation of totipotent stem cells and essential for maintaining the in vivo developmental potency of these cells. 1-Azakenpaullone is a selective inhibitor of GSK-3β, which regulates multiple signal transduction pathways, and is also a key component of the network responsible for maintaining stem cell pluripotency, and WS6 is an inhibitor of Erb3 binding protein 1 and the NFκB kinase pathway. TTNPB is an analog of retinoic acid (RA) that selectively activates RAR and improves the reprogramming efficiency of the cells. One road of totipotency induction occurs due to inhibition of DOT1L and HDAC, and activation of RARγ signaling. AS8351 supports the transition to totipotency via inhibition of histone demethylase. Both TPSs and TLSCs were able to self-organize to form blastoids, which had the potential to implant and initiate decidualization, however without the formation of a fetus.Īmong these molecules, CD1530 is an agonist of retinoic acid receptor γ (RARγ), VPA is an inhibitor of histone deacetylase (HDAC), and EPZ004777 and SCG0946 are DOT1L inhibitors, whereas CHIR 99021 play an important role in activating wingless and Int-1 (Wnt) signaling by inhibiting glycogen synthase kinase 3β (GSK-3β) signaling pathways. These molecules increased the expression of MERVL and totipotency-associated genes in mouse pluripotent stem cells and promotes the transition from the pluripotent to TLSCs. For the remodeling of chromatin, a combination of small molecules such as SGC0946 (a potent, selective inhibitor of DOT1L (disruptor of telomeric silencing 1-like, a histone H3K79 methyltransferase)) and AS8351 (an inhibitor of lysine demethylase 5B) was used. 3 reported that the remodeling of pericentromeric region of heterochromatin and H3K4me3 domains reprograms mouse ES cells to totipotent-like stem cells (TLSCs). 2 The molecules CD1530, VPA, EPZ004777 regulate totipotency synergistically. 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. Recently, two other groups showed the induction of totipotency by different cocktails of small chemicals. ![]() 1 Strikingly, ciTotiSCs are capable to produce germline chimeras after aggregation with eight-cell embryos. 1 These results were verified with single-cell RNA-sequencing, which also revealed that ciTotiSCs contributed to the formation of extraembryonic trophoblast and yolk sac cell types, and confirmed the exclusive expression of lineage-specific marker such as visceral yolk sac cells (Apoa4, Fxyd2, Entpd2), spongiotrophoblast cells (Tpbpa, Rhox9) and syncytiotrophoblast cells (Itm2a). The reporter-labeled cells integrated into epiblast, extraembryonic ectoderm, and ectoplacental cone, and in the later stage of embryonic development (E13.5) these cells contributed to the formation of parts of the placenta and entire yolk sac. For a stringent evaluation of totipotency, reporter-labeled (with ubiquitous expression) ciTotiSCs were aggregated with mouse eight-cell stage embryos and transferred to pseudo-pregnant female mice, and chimerism was evaluated in E7.5 conceptuses. ![]() The ciTotiSCs were able to differentiate into embryoid bodies in vitro and formed teratomas, and formed both embryonic and extraembryonic lineages after aggregation with host embryos. In addition to transcriptome and epigenome analyses, metabolome evaluation revealed that ciTotiSCs exhibited metabolic features similar to totipotent cells. 1 Single-cell transcriptome analysis of the ciTotiSCs suggests that they are closer to the two-cell stage than the previously described totipotent blastomere-like cells (TBLCs). DNA methylation analysis was performed, and the ciTotiSCs showed a hypomethylated genomic architect similar to zygotes or two-cell mouse embryos. The transcriptome analyses and genome-wide chromatin accessibility of ciTotiSCs evaluated by transposase-accessible chromatin sequencing support the close resemblance of ciTotiSCs with mouse two-cell blastomere. ![]() Upregulation of totipotent genes and downregulation of pluripotent genes were confirmed with bulk and single-cell RNA-sequencing. The resulting chemically induced totipotent stem cells (ciTotiSCs) were able to maintain long-term totipotency in in vitro culture with a stable karyotype, expressed totipotency marker genes such as Zscan4, Zfp352, Tcstv1, Tcstv3, and Sp110, and exhibited downregulation of pluripotency marker genes such as Oct4, Nanog, Sox2, Tdgf1, and Zfp42. ![]()
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