In early mammalian embryonic development, from zygote to two-cell-stage blastomeres (in mice) or to 8-cell-stage blastomeres (in humans) are considered totipotent, which is the ability to give rise to an embryo and all its supportive extra-embryonic tissues ^[1][2][3][1,2,3]. Along blastomeres being fated to inner cell mass (ICM) or trophectoderm (TE), ICM is further committed to epiblast and hypoblast (also named primitive endoderm). The epiblast, the source of pluripotent stem cells (PSCs), keeps developmental potential to give rise to three germ layers tissues. However, they are incapable of extra-embryonic linage differentiation. The cultured PSCs can be divided into two classes according to their potency. First-class PSCs can differentiate into both embryonic and extra-embryonic cell lineages. The second-class PSCs, derived from the epiblast, can only differentiate into embryonic lineages. Interestingly, a low-population cluster in mouse PSCs can be found to share features with the two-cell-stage mouse embryos in transcriptome, epigenome, and developmental potential. Thus, they are named two-cell-like cells (2CLCs) [1]. Recently, human 8C-like cells (8CLCs), which could produce embryonic and extra-embryonic lineages, were identified [2]. Furthermore, extended pluripotent stem cells (EPSCs) with both embryonic and extra-embryonic potency were established from human and mouse pluripotent stem cells [4]. In addition, the so-called expanded potential stem cells (EPSCs) from mouse eight-cell-stage blastomeres were also established. These EPSCs are enriched with molecular signatures of blastomeres and possess developmental potency for all embryonic and extra-embryonic cell lineages [5]. Notably, porcine EPSCs and human EPSCs were also successfully established [6].

Second-class pluripotent stem cells derived from epiblasts show three states: naive, formative, and primed; although these cells are capable of differentiation into three germ layers, they exhibit many differences in cell morphology, signaling requirement, epigenetic modifications, metabolome, and transcription profiling ^[7][8][7,8].

Researchers have dedicated significant effort to capturing PSCs at different stages in culture dishes and characterizing them. In this endeavor, high-throughput sequencing, single-cell analysis, and other advanced technologies have been especially useful in assessing the fidelity of in-vitro PSCs to their in-vivo counterparts ^[9][10][9,10].

The three states of pluripotent stem cells, the naive, formative, and primed states, can be obtained from both mouse and human embryos or through epigenetic resetting and reprogramming ^{[8][9][11][12][13][14][15][16]}[8,9,11,12,13,14,15,16]. Mouse embryonic stem cells (mESCs) can self-renew indefinitely in vitro while preserving the developmental potential to reconstitute all embryonic cell types. mESCs are considered to be in the naive state since they are derived from pre-implantation embryos. They are characterized by high pluripotency and can contribute to chimeras and X activation in females [7]. Chimeras refer to an organism comprising at least two populations of genetically distinct cells. They are an invaluable tool for studying mammalian development and PSCs’ potential ^[17][18][17,18]. Naive mESCs can be reintroduced into host embryos to contribute to the mouse development. Moreover, the naive pluripotent stem cells must undergo a period of conversion (called capacitation) and enter the early post-implantation epiblast-like state before they are enabled to respond to cues for trilineage germ-layer differentiation ^[19][20][19,20]. Epiblast stem cells (EpiSCs) are derived from the columnar epithelial epiblast of the post-implantation embryo. They are defined as a primed state, with random X inactivation in females and cannot form chimera ^[12][21][12,21]. Between preimplantation and the epithelialized stage of the egg cylinder of late post-implantation, other intermediate stem cell states are mostly heterogeneous and have not been fully characterized ^[9][22][9,22]. These intermediate stem cells are referred to as formative pluripotency, corresponding to the cells of the early post-implantation epiblast [8]. Formative stem cells can rapidly respond to the induction of lineage specification (especially the germline lineage), cues for cell polarity, and do not show lineage priming [8]. There have been many efforts to capture stem cells in their formative state in vitro. Recently, a population of intermediate stem cells from E5.5-E6 mouse embryos was captured as formative stem (FS) cells. Interestingly, FS cells can be directly induced into primordial germ cells (PGCs) and form chimera at lower efficiency [13]. Similarly, two other groups reported their success in the derivation of formative cell lines (namely fPSCs and XPSCs). These cells share common features with FS cells, including PGCs induction and transcription profiling ^[15][16][15,16]. Additionally, rosette-like stem cells (RSCs) derived from epiblast RSCs express naive markers KLF4, NANOG, and ESRRB, while upregulating Otx2, Tcf7l1, Podxl, and Cgn and repressing primed marker OCT6 [14].

It is well known that integrated cooperation among signals, transcription factors (TF), and epigenetic modifications is required for the maintenance of pluripotent stem cells ^{[7][12][13][15][16][21]}[7,12,13,15,16,21]. Hence, uncovering the molecular mechanisms underlying pluripotency states is a pre-requisite to stem cell therapy and regenerative medicine. Further, stem cells in different pluripotent states can be an excellent model to study embryo development as naive, formative, and primed cells perfectly mimic embryos at pre-implantation, early post-implantation, and late post-implantation stages.

Pluripotent stem cells are derived from the epiblast at the preimplantation stage to the mid-gastrulation stage during early embryo development ^[23][82]. Capturing pluripotent stem cells can not only provide excellent platforms to study early embryonic development and cell fate conversions for regenerative medicine applications. Naive ESCs derived from the ICM of a blastocyst are not biased to differentiate and can produce chimeras. These ESCs require minimal extrinsic signaling and can be stably cultured under the 2i/LIF condition or any combination of these three factors (2i + LIF, 2i, LIF + CH, LIF + PD) ^[7][24][25][7,58,61]. Furthermore, ESCs can efficiently transit from one culture condition to another without affecting pluripotency or cell survival ^[24][58]. Though the chromatin state of naive ESCs is less restricted and more open, naive ESCs cannot differentiate directly into PGCs ^[26][31]. It is noteworthy that genomic instability and retrotransposon activity is elevated while telomere maintenance declines during the conversion of naive ESCs to primed EpiSCs ^[27][83]. This suggests that the compromised potency in EpiSCs is determined by the changes in chromatin states.

Formative stem cells possess an intermediate state of pluripotency between the naive and the primed PSCs ^[13][15][13,15]. PGCs and lineage differentiation can be more quickly induced from formative stem cells than from naive ESCs. Formative stem cells require FGF and activin A activation but not the LIF/2i condition. They can generate chimeras at lower efficiencies and can differentiate into PGCs, showing that formative stem cells are beginning to exit from full pluripotency. EpiSCs can neither form chimeras nor produce PGCs, showing that EpiSCs derived from post-implantation egg cylinder represent fate-biased pluripotency [7].

Notably, the conversion among diverse pluripotent states can be achieved in vitro. Consistent with embryo development direction, naive ESCs can be easily induced into formative stem cells or EpiSCs and formative stem cells also easily transit to EpiSCs [13]. However, reversion-primed EpiSCs back into naive ESCs requires a combination of Klf4 over-expression with the 2i/LIF condition ^[28][63]. Whether over-expression of certain pluripotent-state-specific genes can be universally utilized to reprogram more primed stem cells back into a naiver state remains unknown and is worth trying.

It is noteworthy that researchers also put efforts into capturing PSCs in intermediate states between naive pluripotency and totipotency. Further, 2C-like cells that express totipotency genes Zscan4s and Mervl are the first reported cells capable of producing intra- and extra-embryonic tissues, although they are not stable in the medium and only exist in a very low percentage ^[1][3][1,3]. In 2017, two research groups reported the establishment of extended/expanded pluripotent stem cells (EPSCs) using different combinations of small molecules ^[4][5][4,5]. The extended pluripotent stem cells capable of generating both embryonic and extraembryonic lineages from both humans and mice can be captured by using an LCSD chemical cocktail (LIF, CHIR99021, (S)-(+)-dimethindene maleate (DiM) and minocycline hydrochloride (MiH). These mouse EPSCs show robust and superior chimeric ability at the single-cell level, while the human EPSCs show interspecies chimeric competency in mouse conceptuses [4]. The EPLSCs can also be established by the cocktail (LIF, CHIR99021, PD0325901, JNK Inhibitor VIII, SB203580, A-419259, and XAV939), which can modulate key developmental pathways. Importantly, the EPSCs hold bidirectional differentiation potential in both intra- and extra-embryonic differentiation [5]. Furthermore, totipotent blastomere-like cells (TBLCs), which are close to 2-cell and 4-cell stage embryos, can be established in vitro by inhibiting the spliceosome ^[29][84]. The less compromised pluripotency also provides a good model for research and applications.

Interestingly, hESCs that are also derived from the pre-implantation epiblast, are considered as being in a primed state since hESCs share similar features with primed mEpiSCs but not mESCs ^[30][31][85,86]. This might be due to species differences between mouse and man. Mouse embryos offer a longer window for isolating naive ESCs [7]. Many efforts have been put into gaining naive hESCs from human embryos or reprograming primed hESCs ^[19][32][33][19,87,88]. Exogenous KLF2/NANOG transgenes can trigger the conversion from primed to naive pluripotency [11]. Subsequently, several methods have been established to maintain naive hPSCs in vitro using combinations of inhibitors and agonists, such as 2i (MERi; GSKi), HDACi, TGF-β, and FGF agonists ^{[32][34][35][36][37]}[81,87,89,90,91]. In addition, Guo et al. successfully captured human naive pluripotent stem cells directly from isolated cells of the human ICM with a series of kinase inhibitors (PD0325901, CHIR99021, Gö6983, Y-27632 and human LIF) ^[38][92]. The cell lines exhibit naive features, such as global DNA hypomethylation, expression of naive pluripotency markers KLF4, TFCP2L1, and DPPA3, active mitochondria, and reduced glucose dependence, and can be propagated by enzymatic dissociation to single cells ^[38][92].

Early embryo development is a fascinating process where the one zygotic cell develops into an entire embryo. Grasping the knowledge of the embryo development is crucial for the reusearchers to fully understand human health. However, due to the ethics and the scarcity of research materials, scientists cannot directly study human embryo development. Pluripotent stem cells provide an excellent in-vitro model to study early embryo development. The divergent pluripotent stem cells the researchers we discussed above perfectly represent different pluripotent stem cells in developmental embryos at different stages. Thus, understanding the differences and transition among these pluripotent stem cells is important in biomedical research.

Since PSCs have the ability that differentiates into specific types of cells under specific stimulation, all pluripotent cells are valuable resources for stem-cell-based therapy and tissue replacement. The global resources of transplantable organs are in short supply. The directed differentiation of PSCs brings hope for future cell therapy and organ transplantation. It is always a challenge to obtain functional and high-purity cells and organs for clinical applications. The divergent pluripotent stem cells provide more options of stem cells resources for cellular differentiation, including extraembryonic cell types, including placenta and umbilical cord cells. In addition, PSCs have always been used for drug screening and disease modeling. Notably, the advanced genome editing technologies can greatly facilitate the utilization of PSCs in genetic modification and gene therapy.

In short, the subtle genetic/epigenetic difference among stem cells in different pluripotent states not only lets the reusearchers link in-vitro observation with in-vivo developmental processes but also allows the researchersus to comprehend the underlying mechanism of maintenance, exit, and acquisition of pluripotency. Such knowledge is invaluable as the researcherswe seek to manipulate stem cells for regenerative medicine.