Although the pursuit of bovine embryonic stem cells started more than 26 years ago for the purpose of gene-targeting, true pluripotent stem cells in this economically important species are still elusive. With the rapid advances in genome-editing and cloning using homologously recombined somatic cells, the need for pluripotent stem cells for precise genetic modification in any species became questionable. With the pig being the better model for human regenerative biology, the identification of the commonalities and uniqueness of the pluripotency circuitry across mammalian species may be the main objective for studying pluripotent stem cells in the bovine.

Pluripotent stem cells (PSCs) are characterized by their capacity for high self-renewal and multiple differentiation potential and include embryonic stem cells, embryonic germ cells and induced PSCs. PSCs provide a very suitable model for the studies of human diseases, drugs screening, regenerative medicine and developmental biology research. Pigs are considered as an ideal model for preclinical development of human xenotransplantation, therapeutic approaches and regenerative medicine because of their size and physiological similarity to humans. However, lack of knowledge about the derivation, characterization and pluripotency mechanisms of porcine PSCs hinders progress in these biotechnologies. In this review, we discuss the latest progress on porcine PSCs generation, evaluation criteria for pluripotency, the scientific and technical questions arising from these studies. We also introduce our perspectives on porcine PSC research, in the hope of providing new ideas for generating naive porcine PSCs and animal breeding.

Primordial germ cells (PGCs) are regarded as unipotent cells that can produce only either spermatogonia or oocytes. However, PGCs can be converted into the pluripotent state by first dedifferentiation to embryonic germ cells and then by reprogramming to induce them to become pluripotent stem cells (iPSCs). These two stages can be completely implemented with mouse cells. However, authentic porcine iPSCs have not been established. Here, we discuss recent advances in the stem cell field for obtaining iPSCs from PGCs. This knowledge will provide some clues which will contribute to the regulation of reprogramming to pluripotency in farm species.

Female infertility represents a major challenge for improving the production efficiency in the dairy industry. Historically, fertility has declined whereas milk yield has increased tremendously due to intensive genetic selection. In vivo evidence reveals about 60% pregnancy loss takes place during the first month following fertilization. Meanwhile, early embryo development is significant for somatic cell nuclear transfer in cattle as a large proportion of cloned embryos fail to develop beyond peri-implantation stage. Oocyte quality is of utmost importance for the early embryo to develop to term for both fertilized and cloned embryos. Epigenetic reprogramming is a key process occurring after fertilization and critical roles of epigenetic modifiers during preimplantation development are now clear. Incomplete epigenetic reprogramming is believed to be a major limitation to cloning efficiency. Treatment of cloned embryos with epigenetic modifying drugs (e.g., Trichostatin A) could greatly improve cloning efficiency in both mice and cattle. Recently, the rapid progress in high-throughput sequencing technologies has enabled detailed deciphering of the molecular mechanisms underlying these events. The robust efficiency of genomic editing tools also presents an alternative approach to the functional annotation of genes critical to early development.

Cryopreservation has undergone tremendous advances and is widely used in animal production based on decades of study of cellular permeability, freezability and empirical generalization. Several improvement are particularly important: the cryopreservation protocol has been continuously refined over the years to achieve greater reproductive performance; cryoprotective agents are more effective and less toxic than previously; there has been significant innovation in advanced cryopreservation systems and carriers. Despite this, there are still problems that urgently require practical solutions, such as remedies for cryodamage and encouraging the use of frozen–thawed porcine sperm in pig production.

Somatic cell nuclear transfer (SCNT)-derived piglets have significantly higher stillbirth rate and postnatal mortality rate than artificial insemination (AI)-generated piglets. The question whether the low survival rate of SCNT piglets was related to birth weight, umbilical cord or placenta development was investigated. In this study, stillbirth rate, neonatal death rate, birth weight, umbilical cord status, placental parameters and placental gene expression patterns were compared between SCNT and AI piglets. Results showed that mortality rates at birth and during the neonatal stage of SCNT piglets were significantly higher than those of AI piglets. The incidence of abnormal umbilical cord in SCNT and SCNT-liveborn (SCNT-LB) piglets was significantly higher than in AI and AI-liveborn (AI-LB) piglets. Birth weight, placental weight, placental surface area and placental efficiency in SCNT and SCNT-LB piglets were significantly lower than those of AI and AI-LB piglets. Placental expression profiles of imprinting, angiopoiesis and nutrient transport-related genes were defective in SCNT-LB piglets compared with those in AI-LB piglets. Thus, the low survival rate of SCNT piglets may be associated with abnormal umbilical cord and placenta development. These characteristics may have resulted from aberrant expression of angiogenesis, nutrient transport, and imprinting-related genes in the placentas.

Enucleation is a crucial procedure for mammalian somatic cell nuclear transfer (SCNT), especially for domestic animal cloning. Oocytes of domestic animals such as pigs and cattle contain dark lipid droplets that hinder localization and removal of the nucleus. Using an oocyte enucleation technique that can obtain a high enucleation rate but has minimal negative effects on the reprogramming potential of oocyte for cloning is beneficial for enhancing the outcome of SCNT. In this study, we compared the pig cloning efficiency resulting from blind aspiration-based (BA-B) enucleation and spindle imaging system-assisted (SIS-A) enucleation, and compared the pig SCNT success rate associated with BA-B enucleation and blind aspiration plus post-enucleation staining-based (BAPPS-B) enucleation. SIS-A enucleation achieved a significantly higher oocyte enucleation success rate and tended to obtain a higher in vivo full term development rate of SCNT embryos than BA-B enucleation. BAPPS-B enucleation also obtained significantly higher in vitro as well as in vivo full term development efficiency of cloned porcine embryos than BA-B enucleation. These data indicate that SIS-A and BAPPS-B enucleation are better approaches for pig SCNT than BA-B enucleation.

Porcine viral diarrhea is an acute and highly contagious enteric disease of pigs that causes huge economic losses worldwide. Porcine epidemic diarrhea virus (PEDV) and transmissible gastroenteritis virus (TGEV) are the main pathogens responsible for piglet viral diarrhea. However, currently there is no specific drug available for the effective treatment of viral diarrhea. Therefore, it is necessary to seek an effective method to diminish PEDV and TGEV infection rates. RNA interference has been applied successfully to inhibit the virus replication. It provides a potential strategy for breeding resistant pigs. In this study, four promoters and four short hairpin RNA (shRNA) vectors with LoxP sites at each end of the selectable marker genes were constructed to target PEDV and TGEV. These vectors were then transfected into porcine fetal fibroblasts, G418 resistant transfectants were confirmed by PCR and transgenic SCNT porcine blastocysts were obtained. These results have paved the way for future production of marker-free transgenic resistant to PEDV and TEGV pigs by SCNT.

Using a data set from our laboratory, we assessed the effects of several factors on pig cloning efficiency. The results demonstrated that cells at high confluence (>90%) used as donor cell resulted in higher pregnancy rate, delivery rate and overall cloning efficiency (number of live offspring born per reconstructed embryo transferred to recipients) compared with the cells at 60% to 79% confluence and 80% to 89% confluence. Cells with four, five and six passages compromised the pregnancy and delivery rates compared with first passage cells. The number of blastocysts transferred by somatic cell nuclear transfer (SCNT) did not significantly affect the cloning efficiency, but transfer of blastocyst derived from in vitro culture 5 d after SCNT achieved a significantly higher pregnancy rate compared with one to two cell SCNT embryos from overnight culture. The highest pregnancy rate, delivery rate and the largest litter size were obtained when Bama Miniature pig fibroblasts were used as donor cells and Landrace/Yorkshire hybrid gilts were used as recipients. Recipients treated with chemicals for estrus synchronization had higher pregnancy rates compared with untreated recipients. Our data might be helpful for improving SCNT efficiency in pigs.

Sall4 as one of the spalt family members contains several alternative splicing variants, which are differentially expressed and has a key role in maintaining pluripotent stem cells. However, the molecular features and function of SALL4 have not been well elucidated in porcine induced pluripotent stem cells (piPSCs). In this study, we identified SALL4 splice variants and found two SALL4 splicing variants through analysis of the porcine transcriptome data derived from piPSCs. SALL4A was only detected in piPSCs but SALL4B was globally expressed in porcine tissues and piPSCs. The level of SALL4B was significantly reduced when piPSCs differen-tiation occurred, however, the expression of SALL4A was not affected, indicating that SALL4B may be essential for the maintenance of piPSCs self-renewal. Overexpression of SALL4A and SALL4B in PEF cells could significantly stimulated expression of endogenous pluripotent genes, when SALL4B significantly promoted OCT4 expression. Conversely, SALL4A significantly promoted KLF4 expression. Additionally, both SALL4A and SALL4B could repress OTX2 promoter activity in a dose-dependent manner. Conversely, OTX2 also negatively regulated SALL4 expression. These observations indicate that a negative feedback regulatory mechanism may exist between SALL4 and OTX2, which is useful for the maintenance of the self-renewal of piPSCs.