In vitro cultured stem cells correspond to different stages of embryonic development. Mammalian embryonic development begins with a fertilized egg, the zygote, which undergoes cleavage through the two-cell, four-cell, and eight-cell stages, followed by polarization and compaction to form a morula. The morula further develops into a blastocyst with three cell lineages: inner cell mass (ICM), primitive endoderm, and trophectoderm 1, 2. An important milestone during embryonic development is zygotic genomic activation (ZGA), which marks the point when the fertilized embryo begins independent transcriptional activation. ZGA is divided into minor and major phases. Major ZGA mainly occurs in two-cell and eight-cell blastomeres, respectively, in mice and humans. Embryos at the pre-ZGA stage are mainly featured by parental characters (mainly maternal). During ZGA, almost all the parental features, such as epigenetics, are erased to prepare for the rewriting of a new life cycle. ZGA-specific genes, such as Zscan4, Dux, and Zfp352, are normally used to define and mark totipotency 3, 4.

Pluripotent stem cells (PSCs) have the ability to differentiate into all three germ layers of the embryo. Pluripotent embryonic stem cells (ESCs) were first derived from the ICM 40 years ago 5, 6, and PSCs have been widely applied in early embryonic research and regenerative medicine. The definition of totipotent cells can be categorized into two types: one is the strict definition, where a single cell can develop into a complete organism; the other is a broader definition, where the cell has the ability to differentiate into all lineages, including both embryonic and extraembryonic lineages. In recent years, in vitro cultured totipotent stem cells capable of simulating the totipotent stage have been achieved 7, 8•, which offer greater developmental potential and broader prospects for clinical applications. It is important to note that these in vitro cultured totipotent stem cells meet the broader definition of totipotency. They closely resemble the transcriptome of in vivo totipotent cells at the molecular level and have been shown to possess bidirectional developmental potential in in vitro and in vivo functional assays, differentiating into both embryonic and extraembryonic lineages. Furthermore, it should be emphasized that in vitro cultured totipotent stem cells, which simulate in vivo conditions, are not entirely equivalent to in vivo totipotent embryonic cells. Zygotes and early blastomeres possess a unique developmental microenvironment, including maternal factors from the oocyte 9, 10, undergoing rapid cleavage to increase cell numbers without self-renew. In contrast, in vitro cultured totipotent stem cells maintain the totipotent stage through methods, such as spliceosome inhibition and epigenetic manipulation or by reprogramming PSCs into totipotent stem cells 7, 8•. During subsequent culture, without the influence of maternal substances from the oocyte, these cells can self-renew. Therefore, in vivo and in vitro totipotent cells differ in morphology, size, proliferation, transcriptome, and epigenetics.

Various criteria of differing stringency can be employed to assess totipotency [11]. On the one hand, molecular comparisons at various levels are crucial, particularly between in vitro totipotent cells and in vivo totipotent blastomeres. On the other hand, and more importantly, the functional capacity of totipotent cells must be considered. The most stringent criterion would be the ability of a single totipotent stem cell to give rise to an entire embryo or even a new organism, both in vitro and in vivo. However, due to ethical restrictions surrounding human embryos, providing evidence of the potential to differentiate into both embryonic and extraembryonic lineages through in vitro differentiation assays and in vivo chimerism is currently the most commonly used method of evaluation. Therefore, a comprehensive approach that integrates molecular, functional, and ethical considerations is essential when evaluating the totipotent capacity of stem cells.

This review aims to comprehensively summarize the existing methods and molecular characteristics of human and mouse totipotent stem cells, discuss the evaluation criteria for totipotent stem cells, and provide an outlook on the future applications of totipotent stem cells.