The cleavage of zygotes generates totipotent blastomeres. In human 8-cell blastomeres, zygotic genome activation (ZGA) occurs to initiate the ontogenesis program. However, capturing and maintaining totipotency in human cells pose significant challenges. Here, we realize culturing human totipotent blastomere-like cells (hTBLCs). We find that splicing inhibition can transiently reprogram human pluripotent stem cells into ZGA-like cells (ZLCs), which subsequently transition into stable hTBLCs after long-term passaging. Distinct from reported 8-cell-like cells (8CLCs), both ZLCs and hTBLCs widely silence pluripotent genes. Interestingly, ZLCs activate a particular group of ZGA-specific genes, and hTBLCs are enriched with pre-ZGA-specific genes. During spontaneous differentiation, hTBLCs re-enter the intermediate ZLC stage and further generate epiblast (EPI)-, primitive endoderm (PrE)-, and trophectoderm (TE)-like lineages, effectively recapitulating human pre-implantation development. Possessing both embryonic and extraembryonic developmental potency, hTBLCs can autonomously generate blastocyst-like structures in vitro without external cell signaling. In summary, our study provides key criteria and insights into human cell totipotency.

Fully grown oocytes have the natural ability to transform 2 terminally differentiated gametes into a totipotent zygote representing the acquisition of totipotency. This process wholly depends on maternal-effect factors (MFs). MFs stored in the eggs are therefore likely to be able to induce cellular reprogramming to a totipotency state. Here we report the generation of totipotent-like stem cells from mESCs using 4MFs Hsf1, Zar1, Padi6, and Npm2, designated as MFiTLSCs. MFiTLSCs exhibited a unique and inherent capability to differentiate into embryonic and extraembryonic derivatives. Transcriptomic analysis revealed that MFiTLSCs are enriched with 2-cell-specific genes that appear to synergistically induce a transcriptional repressive state, in that parental genomes are remodeled to a poised transcriptional repression state while totipotency is established following fertilization. This method to derive MFiTLSCs could help advance the understanding of fate determinations of totipotent stem cells in a physiological context and establish a foundation for the development of oocyte biology-based reprogramming technology.

Though totipotency and pluripotency are transient during early embryogenesis, they establish the foundation for the development of all mammals. Studying these in vivo has been challenging due to limited access and ethical constraints, particularly in humans. Recent progress has led to diverse culture adaptations of epiblast cells in vitro in the form of totipotent and pluripotent stem cells, which not only deepen our understanding of embryonic development but also serve as invaluable resources for animal reproduction and regenerative medicine. This review delves into the hallmarks of totipotent and pluripotent stem cells, shedding light on their key molecular and functional features.

Mammalian development commences with the zygote, which can differentiate into both embryonic and extraembryonic tissues, a capability known as totipotency. Only the zygote and embryos around zygotic genome activation (ZGA) (two-cell embryo stage in mice and eight-cell embryo in humans) are totipotent cells. Epigenetic modifications undergo extremely extensive changes during the acquisition of totipotency and subsequent development of differentiation. However, the underlying molecular mechanisms remain elusive. Recently, the discovery of mouse two-cell embryo-like cells, human eight-cell embryo-like cells, extended pluripotent stem cells and totipotent-like stem cells with extra-embryonic developmental potential has greatly expanded our understanding of totipotency. Experiments with these in vitro models have led to insights into epigenetic changes in the reprogramming of pluri-to-totipotency, which have informed the exploration of preimplantation development. In this review, we highlight the recent findings in understanding the mechanisms of epigenetic remodeling during totipotency capture, including RNA splicing, DNA methylation, chromatin configuration, histone modifications, and nuclear organization.

Regeneration refers to the process by which organisms repair and replace lost tissues and organs. Regeneration is widespread in plants and animals; however, the regeneration capabilities of different species vary greatly. Stem cells form the basis for animal and plant regeneration. The essential developmental processes of animals and plants involve totipotent stem cells (fertilized eggs), which develop into pluripotent stem cells and unipotent stem cells. Stem cells and their metabolites are widely used in agriculture, animal husbandry, environmental protection, and regenerative medicine. In this review, we discuss the similarities and differences in animal and plant tissue regeneration, as well as the signaling pathways and key genes involved in the regulation of regeneration, to provide ideas for practical applications in agriculture and human organ regeneration and to expand the application of regeneration technology in the future.

Protoplasts are single cells isolated from tissues or organs and are considered a suitable system for cell studies in plants. Embryogenic cells are totipotent stem cells, but their regeneration ability decreases or becomes lost altogether with extension of the culture period. In this study, we isolated and cultured EC-derived protoplasts (EC-pts) from carrots and compared them with non-EC-derived protoplasts (NEC-pts) with respect to their totipotency. The protoplast isolation conditions were optimized, and the EC-pts and NEC-pts were characterized by their cell size and types. Both types of protoplasts were then embedded using the alginate layer (TAL) method, and the resulting EC-pt-TALs and NEC-pt-TALs were cultured for further regeneration. The expression of the EC-specific genes SERK1, WUS, BBM, LEC1, and DRN was analyzed to confirm whether EC identity was maintained after protoplast isolation. The protoplast isolation efficiency for EC-pts was 2.4-fold higher than for NEC-pts (3.5 × 106 protoplasts·g−1 FW). In the EC-pt group, protoplasts < 20 µm accounted for 58% of the total protoplasts, whereas in the NEC-pt group, small protoplasts accounted for only 26%. In protoplast culture, the number of protoplasts that divided was 2.6-fold higher for EC-pts than for NEC-pts (7.7 × 104 protoplasts·g−1 FW), with a high number of plants regenerated for EC-pt-TALs, whereas no plants were induced by NEC-pt-TAL. Five times more plants were regenerated from EC-pts than from ECs. Regarding the expression of EC-specific genes, WUS and SERK1 expression increased 12-fold, and LEC1 and BBM expression increased 3.6−6.4-fold in isolated protoplasts compared with ECs prior to protoplast isolation (control). These results reveal that the protoplast isolation process did not affect the embryogenic cell identity; rather, it increased the plant regeneration rate, confirming that EC-derived protoplast culture may be an efficient system for increasing the regeneration ability of old EC cultures through the elimination of old and inactivate cells. EC-derived protoplasts may also represent an efficient single-cell system for application in new breeding technologies such as genome editing.

Totipotent stem cells are cells with the capacity to form an entire embryo. Many attempts have been made to convert other types of cells to totipotent stem cells which we called induced totipotent stem cells. Various aspects of these cells such as transcriptional and epigenetics networks are unique. By taking advantage of these aspects, efficient methods have been provided to induce totipotent stem cells. Although this advancement is significant, many aspects of induction such as the underlying mechanism remain to be elucidated. On the other hand, embryonic stem cells usually are the source of induction which raise important questions regarding if these methods are induction or promotion of 2C intrinsic totipotent cells in ESC culture. Here, we review the latest mouse progress in underling mechanism of induction of totipotent stem cells. In addition, we follow up on the progress of Blastoids derived from totipotent stem cells.

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 unknown whether and how totipotent stem cells can be established in vitro in the absence of germline cells. Here we demonstrate the induction and long-term maintenance of TotiSCs from mouse pluripotent stem cells using a combination of three small molecules: the retinoic acid analogue TTNPB, 1-azakenpaullone and the kinase blocker WS6. The resulting chemically induced totipotent stem cells (ciTotiSCs), resembled mouse totipotent 2-cell embryo cells at the transcriptome, epigenome and metabolome levels. 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 embryos, ciTotiSCs contributed to both embryonic and extraembryonic lineages with high efficiency. Our chemical approach to totipotent stem cell induction and maintenance provides a defined in vitro system for manipulating and developing understanding of the totipotent state and the development of multicellular organisms from non-germline cells.

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.

Totipotent stem cells (TSCs), can develop into complete organisms, are used in biological fields such as regenerative medicine, mammalian breeding, and conservation. However, it is difficult to maintain the developmental totipotency and self-renewal capacity of cells cultured from early-stage embryos, which becomes a key factor limiting the research of TSCs. Fortunately, a breakthrough in the study of induced pluripotent stem cells returning to their totipotent state has been made, resulting in the establishment of multiple TSCs and igniting a new wave of stem cell research. Furthermore, the blastocyst-like structures can be generated by the established TSCs, which lays a foundation for synthetic embryos in vitro. In this review, we summarize the totipotent stage of early embryos, the establishment and cultivation of TSCs, and the developmental ability exploration of TSCs to promote further research of TSCs.