Pluripotent stem cells (PSCs) are a unique type of stem cell that have the ability to develop into almost any type of cell in the body. It has the ability to self-renewal and differentiation potential as key characteristics of PSCs. Self-renewal of PSCs can produce more of themselves, maintaining an undifferentiated state for an extended period of time. This characteristic is important for generating large populations of these cells in the culture dish. The ability of PSCs to differentiate into cells of all three germ layers (endoderm, mesoderm and ectoderm), making them capable of forming any cell in the body. However, they cannot produce an entire organism unlike totipotent cells.

PSCs can be distinguished by their sources. Embryonic stem cells (ESCs) derived from the inner cell mass (ICM) of a blastocyst, a pre-implantation embryo, are representative PSCs that are widely studied due to their excellent regenerative and therapeutic potential. Epiblast stem cells (EpiSCs) are typically derived from the epiblast layer of post-implantation embryo. These cells are similar to ESCs, but their biological characteristics are different. Induced pluripotent stem cells (iPSCs), which use specific factors to reprogram adult somatic cells into pluripotent states, have presented new possibilities for stem cell research. They are freed from ethical concerns associated with the use of embryos in ESCs (Tian et al., 2023).

PSCs can be widely used in regenerative medicine, disease modeling, drug testing and stem cell therapy due to its infinite self-renewal and differentiation potential into specialized cells or tissues (Yamanaka, 2020; Wu et al., 2022b). However, PSCs are currently facing multiple challenges, not just these advantages. The use of PSCs raises ethical issues regarding the destruction of embryos, tumors (teratomas) can form when they are transplanted into a recipient therapeutically, and they are derived from sources that may be genetically different from the recipient, there is a risk of immune rejection when these cells are used therapeutically.

Therefore, we describe the classification of pluripotent stem cells (PSCs) based on differentiation potential, the types of PSCs (ESCs, EpiSCs and iPSCs), their pluripotent status (naïve vs. primed) and alkaline phosphatase (AP) activity in PSCs and finally PSCs in domestic animals.

Cell potential is referred to as the varying ability of stem cells to differentiate into specialized cell types (Hima and Srilatha, 2011). Cells with the greatest potential are able to produce more cells types than those with lower potential. Therefore, stem cells can be classified into totipotency, pluripotency, multipotency and unipotency based on their cell differentiation potential (Jaenisch and Young, 2008).

Firstly, totipotent stem cells can give rise to any of the 220 cell types found in an embryo as well as extra-embryonic cells (placenta). In the early stages of embryogenesis, individual blastomeres isolated from zygote, 2, 4, 8-celled embryos have the potential to develop into separate healthy offspring. Totipotency is the ability of a single cell to give rise to a complete, fully formed individual. However, about 30 years ago, when the nuclei of adult sheep’s differentiated somatic cells were transplanted into enucleated oocyte’s cytoplasm, these oocytes developed into normal lambs (Wilmut et al., 1997). Therefore, all cells may have the potential for totipotency if exposed to the appropriate environmental conditions. Totipotency has been not demonstrated when whole blastomeres beyond the 16-cell stage are used.

Secondly, pluripotent stem cells, which are more differentiated than the totipotency, can give rise to all cell types of the body but not the placenta. Althouigh the ability to give rise to an individual has been lost, it has the potential to differentiate into any cells, tissues or organs that make up the body (Shamblott et al., 1998). Therefore, pluripotent stem cells are referred to as the ability of self-renewal and the ability to differentiate into all somatic lineages (Evans and Kaufman, 1981). Pluripotent stem cells originate from cells derived from the inner cell mass (ICM) of pre-implantation fertilized embryos at the blastocyst stage or cells derived from the epiblast of embryos after post-implantation. The former is called embryonic stem cells (ESCs), and the latter is called epiblast stem cells (EpiSCs). Besides these pluripotent stem cells, there are two different types of pluripotent stem cells in the mouse. One is embryonic germ cells (EGCs) and the other is embryonic carcinoma cells (ECCs). EGCs originate from the primitive germ cells of the embryo during their migration from the egg yolk to the genital ridge after gastrulation. Therefore, EGCs can be harvested from a colony of primitive germ cells in the genital ridge and cultured in a state of maintaining pluripotency in vitro. However, successful production of EGCs has not been reported, especially in domestic animals, except in mice. In ECCs, when the ICM of mouse blastocysts is transplanted into another immuno-deficient mouse, it develops into specific tumors, called teratocacinomas. Transplanted ICMs in immuno-deficient mice are differentiated or remain undifferentiated, albeit in small amounts. When undifferentiated cells are isolated and then cultured in vitro, they are maintained as cells with pluripotent characteristics. Production of ECCs has not yet been reported in domestic animals except mice, such as EGCs.

About twenty years ago, new forms of pluripotent stem cells, such as embryonic stem cells, were reported, called induced pluripotent stem cells (iPSCs) (Takahashi and Yamanaka, 2006). This is a technology that converts a combination of pluripotent-related genes into pluripotent stem cells by transferring them to differentiated somatic cells using viral transduction system. In mice, it was produced by transduction of a combination of four genes (Oct-3/4, Sox2, c-Myc and klf4) and humans were also produced by viral transduction of a combination of different genes (Oct-3/4, Sox2, Nanog and Lin28) than those used by mice. However, when the viral transduction system is used, even when the somatic genome is inserted, it remains undifferentiated, which maintains the pluripotent state even when differentiation is attempted or there is a risk that pluripotent-related genes can be differentiated into tumor cells, including oncogenes.

Multipotent stem cells are able to develop into a limited number of cell types in a particular lineage. They are more restricted than pluripotent cells, but they still have significant differentiation potential. Hematopoietic stem cells found in bone marrow are multipotent and can give rise to all types of blood cells such as red blood cells, white blood cells and platelets. Additionally, neural stem cells can give rise to astrocytes, neurons and oligodendrocytes within the nervous system.

Unipotent stem cells are the most restricted type of stem cells. They can only differentiate into one type of cell. Despite their limited differentiation potential, they are still considered stem cells because they have the ability to self-renew (Blanpain et al., 2007). Muscle stem cells are unipotent and can only differentiate into muscle cells. Also, skin stem cells found in the epidermis can only produce the skin cells.

Both embryonic stem cells (ESCs) and epiblast stem cells (EpiSCs) are pluripotent stem cells (PSCs), implying they have the ability to differentiate into various cell types in the body (endoderm mesoderm and ectoderm). However, they respond to different efficiencies and signals because they differ in their origins, properties and the degrees to which they can differentiate. Therefore, we will explain their different characteristics of pluripotency.

Embryonic stem cells (ESCs) were initially described in mice but later PSCs with similar characteristics were described in humans and rat (Evans and Kaufman, 1981; Martin, 1981; Thomson et al., 1998; Li et al., 2008). In the mouse, the initial ESCs were derived from in vivo fertilized embryos on the day 3.5 (Evans and Kaufman, 1981; Martin, 1981) and then derived consecutively from totipotent blastomeres of earlier cleavage embryos (Eistetter, 1988). ESCs derived from the inner cell mass (ICM) of blastocyst-stage embryos have the ability to differentiate into almost any cell types in the body as well as the ability of self-renewal. Unlike domestic animals, the mouse embryo at the blastocyst stage enters into diapause, a quiescent period of intrauterine embryo development. Generally, diapause of mouse embryos depends upon the presence of cytokines in signaling molecules during the presence of receptors. Additionally, they have a normal karyotype, maintain high telomerase activity and exhibit remarkable long-term proliferative potential, providing the possibility for unlimited expansion in culture (Thomson et al., 1998; Odorico et al., 2001). ESCs are considered to be in a naïve pluripotent state, meaning they are closer to the undifferentiated state of the blastocyst-stage embryos. They typically require specific culture conditions, such as the presence of a feeder layer or defined culture media, to maintain their pluripotency. In general, ESCs possess a broader capacity for differentiation. Under the appropriate conditions, they can contribute to all tissues in the body (endoderm mesoderm and ectoderm), including germ cells. ESCs need the expression of specific transcription factors (Oct-3/4, Nanog and Sox2) to help maintain the naïve state and self-renewal capability. Authentic mouse ESCs require leukemia inhibitory factor (LIF) and bone morphogenetic protein 4 (BMP-4) for maintaining a pluripotency state and self-renewal activity (Ying et al., 2003). Above all, the LIF/STAT3 signaling pathway plays a crucial role in maintaining the ground/naïve state in mouse ESCs (Nichols and Smith, 2009). ESCs are widely used in basic research, drug screening and have potential applications in regenerative medicine, especially because they are easier to reprogram to a naïve state.

Epiblast stem cells (EpiSCs) were derived from the epiblast layer of post-implantation embryos around day 5.5 to day 7 in mice (Brons et al., 2007). Mouse EpiSCs exhibited distinctly different cellular and molecular characteristics from ESCs (Brons et al., 2007; Tesar et al., 2007; Han et al., 2010; Hanna et al., 2010b). Unlike ESCs in mice, EpiSCs are in a primed state, which is a more differentiated, meaning they are somewhat less versatile in terms of the cell types they can differentiate into under certain conditions.

When mouse EpiSCs were passaged by the single-cell dissociation method, they showed a flat-shaped colony rather than a dome shape and did not proliferation well. Also, they require different culture conditions, often involving more complex media with factors that support their primed state, such as Activin A, FGF (fibroblast growth factor) and Nodal, to maintain their distinct pluripotent properties (Nichols and Smith, 2009). Generally, EpiSCs have more restricted differentiation ability than ESCs, which appears to be a preference for differentiating into mesodermal and ectodermal lineages rather than endodermal tissues. This is linked to their primed pluripotent state, where they are already closer to specific lineage commitments than ESCs. While still expressing OCT-3/4 and NANOG, EpiSCs also exhibit additional markers that indicate a primed pluripotent state. These include FGF4, GATA6, and OTX2, and they respond to different bFGF/Activin/Nodal signaling pathway. Being closer to a differentiated state, EpiSCs are often more relevant for studying later stages of development and for applications where primed pluripotency is required, such as in creating more specific cell types for therapy (especially for tissues derived from mesoderm and ectoderm).

To sum up the above, ESCs are more flexible and are used in earlier stages of development or in situations requiring broad differentiation, while EpiSCs are used when studying later, more differentiated states of pluripotency and for generating certain specialized cell types.

In the mouse, naïve PSCs were found in the ICM of the blastocyst, and these cells were also the precursors of the epiblast, which gives rise to all embryonic tissues. On the contrary, the primed state was thought to be a later stage of PSCs, often considered a more mature state than the naïve stage. In this state, some commitment towards specific developmental lineages had already begun, although they still retained the ability to become any type of body cell (Nichols and Smith, 2009). These PSCs have different cytokine-dependency to maintain the undifferentiated state. Table 1 represents characteristics differences between embryonic stem cells (ESCs) and epiblast stem cells (EpiSCs). The naïve state refers to a very early stage of PSCs, characterized by their highest potential for differentiation and self-renewal. These cells were considered uncommitted and had the ability to give rise to all the cell types of the body without having been influenced by any differentiation signals. Naïve PSCs expressed specific transcription factors (OCT-3/4, NANOG and SOX2) and had a unique epigenetic landscape compared to more differentiated stem cells. To date, it has been reported that naïve ESCs exist only in mice. PSCs in the naïve state depend on the cytokines leukemia inhibitory factor (LIF) and bone morphogenetic protein 4 (BMP-4) in culture (Smith et al., 1988; Ying et al., 2003; Yu et al., 2021). On the contrary, PSCs in the primed state depend on basic fibroblast growth factor (bFGF) and Activin A in culture (Dahéron et al., 2004; Vallier and Pedersen, 2005). Among the characteristics of naïve PSCs, high clonogenicity is achieved in single cells after trypsinization (Bayerl et al., 2021). In addition, the doubling times of the cells in vitro is relatively short at 10-14 hours, showing the morphology of a dome-like structure (Romito and Cobellis, 2016). Most importantly, when naïve PSCs are injected into well-defined locations in immune-deficient mice, they develop into teratoma, which are tumors composed of various differentiated tissues, and all three representative embryonic germ layers (Yamanaka, 2020). To prove the authenticity of PSCs, ESCs in which green fluorescent protein (GFP) are constitutively expressed are injected into the blastocyst cavity of other mice, followed by transfer of the mosaic embryos into the uterus of surrogate foster mothers. The injected ESCs collectively hybridized with the ICM of surrogate embryos and later contributed to multiple organs and tissues of the resulting offspring (Liu et al., 2014). GFP expression was confirmed in ultraviolet (UV) light to detect injected ESCs in cells derived from the surrogate ICM. The production of spermatozoa and oocytes in the adult chimeric animal demonstrates complete pluripotency when these injected ESCs give to rise to a portion of the primordial germ cells in chimeric mosaic embryos (Leitch et al., 2014; Wu et al., 2022a). The production of chimeric offspring reveals the ultimate verification of the pluripotent state of ESCs. While faithful chromatin changes during differentiation are required in ESCs, the fate of X chromosome state in differentiating ESCs is not clear. Naïve ESCs also form female cell lines with two X active chromosomes (XaXa) (Heard, 2004; Patel et al., 2017). The primed state implies a later stage of PSCs, often considered more mature than the naïve stage. In this state, the stem cells have already beginning to show some commitment towards specific developmental lineages, but they still maintain the ability to become any type of somatic cells. Primed PSCs showed more change in the expression of lineage-specific factors and a change in chromatin environment, which was shown to more suitable for differentiation. Unlike the mouse ESCs in the naïve state, PSCs derived from human ESCs or mouse EpiSCs, which are developed one step further than the blastocyst-stage embryo, exist in a primed state. In humans, the primed state was closer to the developmental stage of the epiblast in mice, corresponding to the post-implantation epiblast. Clonogenicity of single cells isolated from primed PSCs after treatment of collagenase IV was very low (Han et al., 2010; Najm et al., 2011; ten Berge et al., 2011). The primed PSCs exhibited morphology of flat-like structure and could differentiate into all three germ layers in teratoma formation, but the efficient contribution of germline transmission tissue was not achieved in chimera formation (Brons et al., 2007; Rossant, 2008). In contrast to naïve state, primed PSCs undergo X chromosome inactivation (XaXi) in early egg cylinder epiblast cells (Heard, 2004).

Table 1. Summary of characteristics differences between embryonic stem cells (ESCs) and epiblast stem cells (EpiSCs).

Feature	Embryonic stem cells (ESCs)	Epiblast stem cells (EpiSCs)
Source	Inner cell mass of pre-implantation embryo	Epiblast of post-implantation embryo
State of pluripotency	Naïve	Primed
Differentiation potential	More versatile, can differentiate into all three germ layers	More restricted, mainly mesoderm and ectoderm
Culture conditions	Feeder cells or defined media	Activin A, FGF and Nodal
Molecular markers	OCT-3/4, NANOG, SOX2	OCT-3/4, NANOG, GATA6, OTX2
Use in research	Regenerative medicine, drug screening, gene therapy	Developmental biology, tissue-specific differentiation

Naïve and primed states reflect different stages of pluripotency, with the naïve state representing a more “undifferentiated” and flexible condition, and the primed state representing a slightly more committed state of pluripotent stem cells. Both states share the ability to differentiate into any cell type of the body but have different characteristics that impact their potential uses in research and therapy. Naïve pluripotent cells are more difficult to maintain in culture and are primarily used for basic research and regenerative medicine. Primed cells, being more stable, are more commonly used in clinical and research settings.

In 2006, induced pluripotent stem cells (iPSCs) in mice were generated from mouse embryonic fibroblasts by ectopic expression of four transcription factors (Oct-3/4, Sox2, klf4 and c-Myc) (Takahashi and Yamanaka, 2006). Soon after, even in humans, iPSCs were generated using a set of slightly different transcription factors (Oct-3/4, Sox2, Nanog and Lin28) (Takahashi et al., 2007). iPSCs in mice showed morphology, proliferation and teratoma formation similar to the characteristics of mouse ESCS and SSEA-1, a specific surface factor of mouse ESCs, was also expressed. However, human iPSCs were similar to human ESCs in many aspects. They showed a colony of flat morphology and expressed SSEA-3, SSEA-4, Tra-1-60 and Tra-1-81 as specific surface factors for humans.

Human iPSCs are able to avoid immune rejection, a limitation of conventional PSCs, so they can be applied especially to the development of patient-specific therapy (Yamanaka, 2007; Karagiannis et al., 2019; Aboul-Soud et al., 2021; Wu et al., 2022b). However, the technology of iPSCs is currently limited in use due to various safety issues (Takahashi and Yamanaka, 2006; Yamanaka, 2007). Transduction of exogenous genes using retrovirus vectors and permanent expression of these genes integrated into the genome of the host cells were exposed to risks such as mutations and tumors in iPSCs (Li et al., 2009; Choi et al., 2014). Numerous efforts are being made to avoid the risk of tumorigenicity or side-effects caused by viral integration, such as plasmids, Sendai viruses, adenoviruses, synthetic RNAs and proteins not integrated into the host cell’s genome (Fusaki et al., 2009; Kim et al., 2009; Okita and Yamanaka, 2010; Stadtfeld and Hochedlinger, 2010; Warren et al., 2010).

Several researchers have attempted to establish iPSCs in pigs based on variations of the technology introduced firstly in mice (Esteban et al., 2009; Alberio et al., 2010). Like mouse and human iPSCs, since porcine iPSCs were continuously expressed exogenous transgenes, these cells should establish its own characteristics that differ from conventional mouse or human iPSC. However, porcine iPSCs are still used by adopting human ESCs culture conditions (Esteban et al., 2009; Ezashi et al., 2009; Wu et al., 2009). Porcine iPSCs in a primed state showed intrinsic biases of differentiation and limited developmental capacity (Brons et al., 2007). It had been reported that LIF culture condition with two kinase inhibitors maintain pluripotency and self-renewal in porcine iPSCs and can induce naïve state similar to those of mouse ESCs (Buecker et al., 2010; Hanna et al., 2010a; Thomson et al., 2012).

Alkaline phosphatase (AP) is a catalytic enzyme that removes phosphate groups by cleaving phosphate bonds in various molecules (nucleotides, proteins and alkaloids) under alkaline conditions. It plays an important role in many biological processes including bone mineralization and liver function (Vimalraj, 2020; Levitt et al., 2022). The activity of AP is a commonly used marker to identify PSCs and assessing their undifferentiated state. Therefore, PSCs such as embryonic stem cells (ESCs), embryonic germ cells (EGCs), embryonic carcinoma cells (ECCs) or induced pluripotent stem cells (iPSCs) exhibit high levels of AP activity when they are in early undifferentiated stage (Surrati et al., 2016; Baek et al., 2023). In addition, this enzyme is widely used to assess the real-time pluripotent status of PSC cultures in laboratory settings. This enzyme’s activity provides valuable insights into the status of PSCs cultures, playing a critical role in research and potential clinical applications. The high expression of AP in PSCs indicated that the cells are in naïve and undifferentiated states (Trusler et al., 2018; Rostovskaya et al., 2019). Monitoring AP activity of PSCs before application of passage or differentiation can be very important in determining the status of PSCs. It had been shown that maintenance of the activity in AP-positive (+) colony formation considerably correlates with the clonogenic and self-renewal potential of undifferentiated human ESCs in cultures (O’Connor et al., 2008). However, low activity of AP had been detected in pluripotent epiblast stem cells (EpiSCs) (Brons et al., 2007; Tesar et al., 2007). In our study, 9 porcine EpiSCs lines were established: 7 lines were AP positive (+) and 2 lines were AP negative (Baek et al., 2021). Interestingly, it was proved that clonogenic, pluripotency-related marker expression and in vitro differentiation into vascular endothelial cells were better in AP negative (-) porcine EpiSCs than AP positives (+) (Baek et al., 2021; Jeon et al., 2021; Shin et al., 2022). Therefore, the evaluation of AP activity of PSCs should be carried out to identify the differences according to the animal species and source timing. When these cells begin to differentiate into specialized cell types, there is usually a decrease in AP activity. The activity of AP was down-regulated reciprocally with differentiation processes involving PSCs (Štefková et al., 2015).

Establishing PSCs from domestic animals, including pigs and cattle, are of great importance to develop biomedical models (Niemann and Kues, 2007; Kues and Niemann, 2011; Nowak-Imialek et al., 2011; Gandolfi et al., 2012). Numerous attempts have been made to establish ESCs lines in domestic animals but no authenticated success has been reported so far. Since several rigorous characterizations are required for the authenticity of ESCs, cells derived from domestic animals have been reported with only a few limited characteristics. Therefore, despite decades of efforts, the establishment of PSCs from domestic animals had remained an elusive goal (Telugu et al., 2010). Pigs were considered an excellent model for developing therapeutic tools because they are anatomically and physiologically similar to human (Kobayashi et al., 2017). It was very difficult to establish PSCs in pigs because specific markers of porcine PSCs are quite different from those identified in conventional mice or human PSCs, and the culture conditions were also different. Therefore, understanding species-specific characteristics of PSCs between species and knowing the proper derivation timing would help establish an authentic PSCs in domestic animals.

Putative porcine ESCs were initially derived from a blastocyst of in vivo fertilized embryo on the day 7-9, but their characteristics was poor defined (Evans et al., 1990). At the same time, it was also reported that porcine PSCs are isolated and cultured through the immunosurgical protocol from a blastocyst of in vivo fertilized embryos, but these cells did not exceed 10 passages (Piedrahita et al., 1990). Using immunosurgical protocols, it has been continuously reported that they establish putative porcine ESCs from 4 to 8-celled embryos in the early stages of development, morula as well as expanded blastocysts close to hatching (Strojek et al., 1990; Hochereau-de Reviers and Perreau, 1993; Chen et al., 1999; Keefer et al., 2007). Furthermore, these ESCs were established from the embryonic disc of pig conceptuse on the day 10.5-12 of pregnancy, dependent on bFGF/Activin/Nodal signaling pathway (Strojek et al., 1990). Putative porcine ESCs resemble characteristics to human ESC rather than naïve mouse ESCs. Numerous efforts have also been made to establish PSCs from bovine fertilized embryos by the method used in pigs (Pant and Keefer, 2009; Gong et al., 2010; Pashaiasl et al., 2010; Nowak-Imialek et al., 2011). However, like pigs, putative bovine ESCs failed to form teratomas, and the standardization of bovine stem cell-specific markers and culture conditions are still uncertain (Ozawa et al., 2012).

ESCs have also been successfully reported in primates such as monkeys and humans (Thomson et al., 1995; Thomson et al., 1998; Reubinoff et al., 2000; Lee et al., 2005). Like ESCs in mice, ESC in primates were also derived from the ICM of expanded blastocyst-stage embryos (Thomson et al., 1998). However, human ESCs show pluripotent characteristics similar to those of mouse epiblast stem cells (EpiSCs), not those of mouse ESCs. Therefore, human ESCs are not in a naïve state, but in a primed state.

Like other animal PSCs, human ESCs exhibited flat morphology rather than dome morphology, and relied on the bFGF/Activin/Nodal signaling pathway to maintain pluripotent state and self-renewal activity (Ginis et al., 2004; Tesar et al., 2007). Human ESCs in the primed state did not have the ability to inactivate one of X-chromosomes and form chimeric in females (Nichols and Smith, 2009).

Pluripotent stem cells (PSCs) offer tremendous potential in regenerative medicine, disease research, and new drug development in the field of stem cell biology. Applying PSCs to clinical practice has the potential to open a new era in customized and regenerative medicine by changing the way various diseases and diseases are treated. In addition, it can be seen that this field of PSCs is not far from being used as a substitute for new disease model animal production using livestock or insufficient human organs in the field of animal resources.

None.

Conceptualization, I-W.L., S-K.B., and J-H.L.; data curation, I-W.L., and S-K.B.; formal analysis, I-W.L., and S-K.B.; investigation, I-W.L., and S-K.B.; methodology, Y-J.L., T-S.K., and B-G.S.; project administration, J-H.L.; resources, Y-J.L., T-S.K., and B-G.S.; supervision, C.H., and J-H.L.; writing - original draft, I-W.L., and S-K.B.; writing - review & editing, C.H., and J-H.L.

This work was supported by the National Research Foundation of Korea funded by the Korean Government (2020R1l1A3072689) Republic of Korea. In-Won Lee, Yeon-Ji Lee and Bo-Gyeong Seo were supported by the scholarship from the BK21Plus Program, Ministry of Education, Republic of Korea.

Not applicable.

Not applicable.

Not applicable.

Not applicable.

No potential conflict of interest relevant to this article was reported.