Measuring pluripotency is a fundamental component of every stem cell-based study. Assays testing pluripotency in vitro include relative quantification of the expression of pluripotency genes at the mRNA level and immunocytochemistry to detect specific pluripotency markers at the protein level. Moreover, embryoid body (EB) formation assays to test the ability of the cells to form three embryonic germ-layers can be conducted. Teratoma formation in immunodeficient mice is widely used as an index of pluripotency, as it assesses the capability of the cells to differentiate into the three embryonic germ layers in vivo, and it provides a reliable and comprehensive validation of the functional pluripotency of the cells (15).

Descriptions of the assays used to characterize each ruminant cell line discussed are detailed in Table 1.

Several groups have reprogrammed bovine cells from various developmental stages and tissues, including non-conventional cell sources such as amnion-derived cells, Wharton's jelly cells, and multipotent stem cells such as neural stem cells and mesenchymal stromal cells (MSCs). These groups primarily relied on bovine, human, or murine reprogramming factors consisting of POU class 5 homeobox1 (OCT4), SRY-box transcription factor 2 (SOX)2, KLF transcription factor 4 (KLF4) and MYC proto-oncogene (c-MYC) (OSKM) or OSKM plus Lin-28 homolog A (LIN28) and Nanog homeobox (NANOG) (OSKMNL) (Table 1). Variations such as overexpression of Lysine demethylase 4A (KDM4A) or forced expression of SV40 large T antigen, together with reprogramming factors (16, 17) or antigen reprogramming with micro RNAs (18), have also been employed. Generally, reprogramming elements were delivered via viral vectors, but the use of transposon systems encoding reprogramming factors (19) and electroporation of plasmid DNA containing a single reprogramming gene have been explored as well (20). The majority of bovine iPSCs were maintained in a dual-factor culture medium consisting of both FGF2 and leukemia inhibitory factor (LIF) for proliferation in an undifferentiated state.

Characterization of these bovine iPSCs demonstrated the expression of endogenous pluripotency factors such as ZFP42 Zinc Finger Protein (REX1), Estrogen related receptor beta (ESRRB) and Developmental pluripotency associated 3 (STELA) at the transcript level, and ALP, Fucosyltransferase 4 (SSEA-1) and LIN28 at the protein level. Two studies demonstrated bovine iPSC longevity by maintaining cultures for more than 40 passages (21, 22). Both groups supplemented the culture medium with FGF2 and LIF, and one added kinase inhibitors (22). Another group reported the maintenance of bovine iPSCs for over 50 passages using FGF2 and LIF supplemented culture medium (23). In addition, bovine iPSCs cultured for over 70 passages was achieved with a culture medium containing doxycycline, histone methyltransferase, and WNT inhibitors (17, 19). Additionally, few groups demonstrated epigenetic validation of iPSCs by showing demethylation of NANOG and OCT4 promoter regions in the host cells' genomes (17, 18, 21). Finally, many cultures were not subjected to experimentation to demonstrate differentiation into EBs in vitro and/or formation of teratomas in vivo, measures of functional pluripotency (Table 1). While most studies did not focus on the pluripotency state, two research groups reported the generation of bovine naïve-like iPSCs (16, 19), and both naïve state-like and primed state-like status of these cells was achieved using culture conditions for mouse and human iPSCs (17, 19).

Two independent groups published the first reports of ovine iPSCs in 2011 (24, 25). They used viral vectors to introduce OSKM or OSKM plus additional pluripotency genes into fibroblasts collected at different stages of development and showed that the generated iPSCs expressed multiple pluripotency markers and formed EB and teratomas. Since these two initial reports, additional groups have introduced pluripotency genes into ovine cells using a PiggyBac transposon system (26) and microRNAs (27). In addition to ovine fibroblasts, kidney cells have also been used for reprogramming (28, 29). A variety of culture media has been used across ovine iPSC studies, and different pluripotency markers have been assessed (Table 1). Most ovine iPSC cultures could form EB, some formed teratomas, and other cultures were found to contribute to early blastocysts (26, 30) and live-born chimeric lambs (31).

All caprine iPSCs produced thus far were derived from fetal, embryonic, or adult fibroblasts. In most of the cases, lentiviral or retroviral vectors containing OSKM or OSKM plus additional pluripotency factors were utilized (Table 1). Caprine iPSCs have also been produced via mRNA transduction (32) and chemical induction using small molecules (33). Many different culture conditions have been used, which either included LIF or FGF2, or both. Most of the caprine iPSC cultures displayed a colony morphology like mouse iPSCs, stained positively for alkaline phosphatase (ALP), and exhibited goat-specific pluripotency markers at the transcriptional and/or protein level. Although most caprine iPSC cultures could develop into EBs, very few were able to induce teratomas in vivo. One study showed that directed differentiation of caprine iPSCs resulted in the in vitro production of trophoblast-like cells, yolk-sac endoderm-like cells and neuronal cells (34).

Almost all buffalo iPSC cultures have been generated using fetal fibroblasts as the reprogramming cell source, except for one study which used adipose-derived MSCs (Table 1). In addition to viral or non-viral delivery of buffalo, mouse, or human reprogramming factors (OSKM or OSKMNL), the epigenetic modifier valproic acid has been used to enhance the reprogramming efficiency (35). Chicken egg extract added to the culture medium was also shown to be adequate to generate putative buffalo iPSCs colonies (36). Although most of these buffalo iPSC cultures were able to differentiate into EBs, only one group reported generation of in vivo teratomas and epigenetically validated buffalo iPSCs (35, 37).

Finding alternatives to conventional farming practices is crucial, given the rising reliance on animal products for human nutrition. Compared to plant-based food sources, conventionally produced animal-based material has a larger environmental footprint, requires more soil and water, and leads to the emission of more greenhouse gases (45). Moreover, antibiotic overuse in livestock farming results in the emergence of antimicrobial-resistant bacterial strains, a significant human health concern (46). Cellular agriculture, defined as the production of animal-sourced food from cultured cells, has the potential to replace traditional farming with more environmentally friendly practices. The production of meat in vitro using iPSCs is proposed as a clean and prominent alternative to reduce the global burden of the livestock industry (47). In 2012, meat derived from bovine stem cells was used to create the first lab-made hamburger (48). Moreover, a commercial meat producer in the UK reported the first lab-made strips of bacon and pork belly in 2020 (49). In addition, production of cell-based seafood from fish cells and tissue cultures is also becoming popular to address the challenges associated with industrial aquaculture systems and marine capture (50). More recently, laboratory-grown meat, slaughter-free chicken, received clearance from U.S. Food and Drug Administration for human consumption (51). Additionally, generation of other iPSC-derived animal products, such as skin and fur, could reduce our dependence on industrial farming and minimize the associated environmentally harmful effects.

The recent advancements in iPSC generation along with targeted genome editing technologies, especially the CRISPR-Cas9 system (52), have facilitated the introduction of desired genetic modifications and, combined with somatic cell nuclear transfer (SCNT) or blastocyst complementation, represent a powerful platform for transgenic animal production (53, 54).

Genetically modifying ruminants can enhance growth rates and production, improve nutrients in animal products, increase disease resistance, and enhance reproductive efficiency and fecundity. Moreover, transgenic ruminant livestock, especially those animals used for milk production such as cattle, buffalo, and goats, that are generated by genetically modified iPSCs could be used as bioreactors to produce therapeutic proteins of pharmaceutical interest. To this end, cloned transgenic cattle, which produce recombinant proteins in milk such as human coagulation factor IX has been reported (55). Additionally, transgenic ruminant livestock has the potential to significantly reduce the environmental footprint of livestock husbandry by increasing productivity and efficiency through transgenesis, which results in reduced use of land and water resources while safeguarding the soil and groundwater.

Significant paradigm changes in reproduction have been made possible by prominent developments in stem cell biology. Germ cells have been derived successfully from mouse stem cells (56) and although protocols for differentiating buffalo embryonic stem cells into germ cells (57) and animal embryo-stem cell livestock laboratory breeding systems have been proposed (58), the differentiation of ruminant iPSCs to functional gametes in vitro has not been achieved yet.

Derivation of ruminant iPSCs may open the possibility of in vitro breeding. For example, selected cell lines could be differentiated to create functional gametes, which would then be used to create a new generation of embryos through in vitro fertilization. Such breeding schemes could substantially reduce generation intervals, enhance selection intensity, achieve more genetic gain, and preserve rare ruminant breeds and highly valuable genotypes. In addition, these cells could be expanded for the banking of genetic material and be used as donor cells for SCNT.

Ruminant diseases are widespread and have detrimental consequences on the herd and consumer health (59). The lack of appropriate ruminant disease models hampers the study of disease pathogenesis and the development of strategies to control these diseases. iPSCs are valuable tools for tissue and disease modeling, as well as preclinical therapeutic development in both human and mouse models (60). However, the use of iPSCs for ruminant disease modeling is currently limited, in part because differentiating ruminant iPSCs into clinically relevant lineages has not been well explored. One study demonstrated the potential of using iPSC technology for generating bovine mammary tissue in vitro (61). In this study, bovine iPSCs were successfully generated from the bovine mammary epithelium, and mammary epithelium-derived-iPSCs were differentiated back to a mammary phenotype characterized by epithelial cells expressing cytokeratin 14, cytokeratin 18, and smooth muscle actin, after treatment with progesterone (61). These studies could be complemented by generating iPSC-derived mammary organoids that can be used to explore the pathogenesis and prevention of important bovine udder diseases. Additionally, rare genetic disorders such as bovine citrullinemia and bovine leukocyte adhesion deficiency found in Holstein-Friesian cattle (62), and Chediak-Higashi syndrome found in Hereford, Brangus, and Japanese black cattle (63), may be studied using bovine iPSCs models, based on the unique advantages that iPSC cultures have shown in the modeling of rare human genetic disorders (64).

Endocrine-disrupting chemicals (EDCs) may significantly impact the reproductive functioning of ruminant livestock, which greatly impacts agricultural production (65). Bovine iPSCs have been used to study the effects of phthalate esters, synthetic organic chemicals used in the plastic industry (20). These esters were found to significantly downregulate androgen receptors on iPSCs, which supported apoptosis (20). Ruminant iPSCs may also be used in toxicological studies investigating how pharmaceuticals, potential toxins, teratogens, and EDCs affect livestock species and humans.

A composite organism of at least two genetically distinct cell populations is called a chimera. With the use of iPSC technology, the production of chimeric ruminants would allow for the genetic engineering of farm animals to improve traits of agricultural importance and the generation of biomedical models. Reports on interspecies ruminant chimeras such as sheep-goat (66) and cattle-buffalo (67) are already available. Moreover, human-ruminant chimeras could be created for use as models to study human organ development and disease pathogenesis, as well as to meet the increasing demand for and reduce the shortage of human organs. For example, human iPSCs have been engrafted in cattle pre-implantation blastocysts (68). However, enormous technical challenges and complex ethical issues must be considered and overcome before producing human organs in ruminants or any other mammals becomes feasible. The risks of human consciousness, human traits, and the creation of human gametes by such chimeras are the primary ethical concerns (69), and any attempt to create human-ruminant chimeras must be thoroughly risk-assessed, technically evaluated, and closely supervised.