On May 4, 2022, denghongkui’s research group, Li Cheng’s research group and Xujun’s research group of School of life sciences and life joint center of Peking University published a research paper entitled”derivation of totipotent like stem cells with blastocyst like structure forming potential” in cell research magazine. In this study, a new culture condition for totipotent stem cells was established through the combination of chemical small molecule screening, which can support the establishment of totipotent stem cell lines from mouse two cell embryos and extended pluripotent stem cells (EPS cells). The new totipotent stem cells can be stably cultured in vitro for a long time. They are highly similar to mouse two cell embryos in molecular characteristics and developmental potential, and can be induced to form a blastocyst like structure similar to in vivo blastocysts at the transcriptome level.
From left to right are Licheng, denghongkui and Xujun (source:Official Website of Peking University)
How to prepare totipotent stem cells in vitro has long been an important scientific issue in the field of stem cells. In mice, only fertilized eggs and two cell embryos have totipotency:a single cell can form a complete individual. The subsequent blastocyst cells can be used to establish pluripotent stem cells, trophoblast stem cells and primitive endoderm stem cells. However, the developmental potential of these stem cells is limited, and they cannot develop into both intraembryonic and extraembryonic tissues at the same time. Recent studies have found that there are a very small number of cells expressing mervl, a molecular marker of mouse two cell embryos, in the mouse pluripotent stem cell population, known as 2-cell like cells, which have some molecular characteristics of two cell embryos (1). However, such cells cannot be stably cultured in vitro. In addition, recent studies have found that there are still great differences between two cell like cells and two cell embryos in vivo. As a model for studying totipotency in vitro, it still has great limitations (2).
Denghongkui team of Peking University has long been committed to establishing new methods to regulate the developmental potential of stem cells by means of chemical small molecule regulation (3-6). In 2017, denghongkui’s team reported a new small molecule combination (LCDM), which can establish expanded pluripotent stem cells (EPS cells) in human and mice (4). EPS cells have the potential to develop in and out of the embryo, and can be induced to form a blastoid structure (7). However, compared with mouse two cell embryos, the molecular characteristics of these cells are still quite different from those of two cell embryos, and the extraembryonic differentiation potential of the cells is also limited. There are a high proportion of intermediate and mesoderm like cells in the induced blastocyst like structure (8). Recently, Du Peng of Peking University and Wang Jichang of Sun Yat sen University reported the induction conditions of totipotent stem cells (9-10). At present, how to establish totipotent stem cells directly from mouse totipotent embryos is still the”gold standard” of totipotent stem cell research.
In this study, the team identified chemical small molecules that can induce and increase the proportion of mervl and zscan4 positive cells in EPS cells through high-throughput screening of chemical small molecules. Through further combination optimization, we found a small molecular combination cd1530, VPA, epz004777, chir 99021 (CPEC combination) that can induce EPS cells into totipotent stem cells. The induced totipotent stem cells can be cultured in vitro stably for a long time. More importantly, CPEC combination can support the establishment of totipotent stem cell lines directly from mouse two cell embryos in vitro. The researchers named the totipotent potential stem cells (TPS cells) established with the support of CPEC.
The researchers further analyzed the molecular characteristics and developmental potential of TPS cells from the aspects of transcriptome, apparent characteristics and chimeric ability. They found that TPS cells expressed a large number of totipotent genes at the single cell level and down regulated the molecular markers of pluripotency. Further single cell transcriptome analysis showed that there was a cell subgroup (about 10%) in TPS cell population that was highly similar to that of metaphase two cell embryos at the transcriptome level. They quantitatively analyzed the TPS cells, the totipotent stem cell subsets in tBLC, the transcriptome similarity between two cell like cells and two cell embryos reported by dupeng team, and found that the totipotent stem cell subsets in TPS cells were the most similar to two cell embryos. ATAC SEQ and genome-wide methylation analysis also showed that TPS cells had the characteristics of apparent modification of two cell embryos. In terms of developmental potential analysis, they proved that a single TPS cell has the ability to develop in and out of embryo at the same time through single cell chimerism experiments at different developmental stages. In order to strictly prove the extraembryonic development potential of TPS cells in vivo, they analyzed the single cell transcriptome of E17.5 chimeric placenta. The results showed that TPS derived cells could differentiate into a variety of extraembryonic trophoblast cell types. In addition, they found that in the chimeric placenta formed by tdtomato labeled TPS cells and GFP labeled receptor embryos, there were a large number of tdtomato single positive chimeric cells, which highly expressed the molecular markers of trophoblast cells, ruling out the possibility of false positives caused by cell fusion. These results indicate that TPS cells have similar molecular characteristics and developmental potential as two cell embryos.
The ability of self-assembly to form blastocyst like structures is one of the most critical functional criteria for evaluating cell totipotency. The researchers have proved that TPS cells can be induced to form blastocyst like structure efficiently by regulating the signal pathway of early embryonic development. Single cell transcriptome analysis showed that the structure of TPS induced blastocysts was similar to that of mouse E4.5 blastocysts in the presence of cells in the ectoderm, trophoblast and primitive endoderm, which were highly similar at the transcriptome level. Through quantitative analysis of transcriptome data, the researchers further compared the trophoblast cells in TPS blastocyst like structure and the trophoblast cells in blastocysts induced by the combination of mouse trophoblast stem cells/pluripotent stem cells. It was found that the trophoblast cells in TPS blastocyst like structure were more similar to the mouse trophoblast ectodermal cells in preimplantation blastocysts. Moreover, unlike the blastocyst like structure induced by EPS cells, TPS blastocyst like structure does not contain a large number of intermediate cells and mesoderm like cells. The implantation of TPS derived blastocyst like structure in vivo can induce decidualization reaction, but it still can not develop into individuals like normal blastocysts, suggesting that the scheme of inducing blastocyst like structure still needs to be optimized.
Finally, the researchers analyzed the molecular mechanism of CPEC combination inducing and regulating totipotency in TPS cells. They found inhibition of hdac1/2 and dot1l activity and specific activation of rar γ Pathway plays an important role in the induction and maintenance of TPS cells. Interestingly, when small molecules combined with CPEC were used to treat mouse two cell embryos, they found that these small molecules could help maintain the expression of totipotent molecular markers in mouse embryos to some extent. These results indicate that hdac1/2, dot1l, rar γ The synergistic regulation of pathways plays an important role in the regulation of totipotency in mice.
In conclusion, this study established a new type of totipotent stem cells from mouse two cell embryos by chemical regulation. The cells have similar molecular characteristics and two-way development potential to two cell embryos, and can form a blastocyst like structure more similar to preimplantation blastocysts in vivo. This work not only provides a more suitable and reliable model for the study of totipotency in vitro, but also takes an important step towards the goal of using totipotent embryos to capture and maintain totipotent stem cells in different mammalian species.
Professor denghongkui, researcher Licheng and researcher Xujun are the co correspondents of this research achievement. Xuyaxing, zhaojingshu, renyixuan, wangxuyang and lvyulin of Peking University are the first authors of the research results. This work has been supported by the life science joint center, the national key R & D program, the National Natural Science Foundation, etc.