Canine induced pluripotent stem cells (ciPSCs) hold significant promise for veterinary regenerative medicine and as translational models for human diseases. Deriving these cells from somatic sources also circumvents ethical concerns associated with embryonic material. However, progress in the field is impeded by challenges such as low reprogramming efficiency, lack of standardization, unique epigenetic barriers, and ambiguous pluripotency states. This narrative review provides a comprehensive and up-to-date overview of current approaches to ciPSC generation and characterization, emphasizing recent advancements in donor cell selection, reprogramming methodologies, culture conditions, and analytical techniques. Furthermore, the review explores emerging applications in regenerative medicine and disease modeling, discussing both future potential and current limitations. Addressing these challenges through improved protocols and effective differentiation strategies is crucial to fully unlocking ciPSC technology's research and therapeutic value.

Background: Direct reprogramming of somatic cells into induced neural stem cells (iNSCs) holds strong potential for regenerative medicine, especially in large animal models like pigs, which are crucial for translational and preclinical research. However, the molecular mechanisms underlying porcine fibroblast-to-iNSC reprogramming and subsequent differentiation remain poorly understood at the proteomic level. Methods: To map the proteomic landscapes associated with reprogramming and differentiation, we performed unbiased label-free discovery proteomics (nano-LC-MS/MS) and targeted SWATH-MS quantification. Proteomes of porcine tail fibroblasts (PTFs; passage 3), two porcine iNSC lines (piNSCs; VSMUi002-B and VSMUi002-E, passage 20), and their differentiated progeny (piNSCs-NGs; VSMUi002-B-NGs and VSMUi002-E-NGs, representing piNSCs at passage 20 cultured for an additional 14 days under differentiation conditions) were compared. Two previously established piNSC lines (VSMUi002-B and VSMUi002-E), generated via Sendai virus-mediated reprogramming, were used as the cellular models. Results: The piNSC lines displayed hallmark neural stem cell (NSC) morphology and expressed canonical markers (PAX6+, SOX2+, NES+, VIM+, OCT4−). Upon differentiation, they generated neuronal and glial cells expressing TUJ1, MAP2, SYP, TH, and GFAP, confirming their multipotency. A total of 4,094 proteins were identified across the three cell states. Multivariate analysis revealed distinct proteomic signatures separating fibroblasts, iNSCs, and their neuronal/glial progeny. The proteomic shift from the fibroblast to the piNSC state was marked by increased expression of stathmin 1 (STMN1), neurofilament light polypeptide (NEFL), aconitate hydratase (ACO2), electron transfer flavoprotein subunit beta (ETFB), fructose-bisphosphate aldolase B (ALDOB), and transketolase (TKT), alongside suppression of actin-related protein 2/3 complex subunit 5 (ARPC5) and LIM domain and actin-binding protein 1 (LIMA1). These shifts indicate a dismantling of the fibroblast cytoskeleton and a broad upregulation of cellular energy and biosynthetic metabolism, reflecting a loss of fibroblast identity and the acquisition of an NSC state. Upon differentiation into piNSCs-NGs, 19 proteins were consistently upregulated. These included neuronal structural proteins (INA, STMN1), cytoskeletal regulators (PFN1), signaling modulators (MBIP), and proteins involved in lysosomal function (NCOA7), cell adhesion (CDHR2), and calcium signaling (ANXA4). Pathway and network analyses highlighted post-transcriptional regulation—particularly involving RNA processing and the RNA exosome complex (e.g., EXOSC3)—as a key feature of differentiation. Conclusion: This study provides the first comprehensive proteomic map of piNSC reprogramming and differentiation in a large animal model. Our findings uncover critical regulatory proteins and pathways governing cytoskeletal organization, metabolism, and RNA processing, offering valuable insights into neural fate states. This resource advances the understanding of neural reprogramming in translational models and supports future regenerative and comparative neuroscience efforts.

Porcine epidemic diarrhea virus (PEDV) continues to cause devastating economic losses in the swine industry. However, the lack of species-specific in vitro models and reliance on costly animal models hinders development of effective interventions. We established a high-fidelity platform using porcine induced pluripotent stem cells (piPSCs; line VSMUi001-C) via a 28-day stepwise differentiation protocol. Successful lineage commitment was validated by OCT4 downregulation and robust FOXA2/SOX17 expression at day 5. By day 28, the piPSC-derived intestinal epithelial cells (piPSC-IECs) exhibited organized LGR5+ niches, MUC2+ goblet cells, and Villin+ enterocytes, mirroring native tissue architecture. Barrier functionality was confirmed by the continuous distribution of the tight junction protein zonula occludens-1 (ZO-1). Our results demonstrate that piPSC-IECs are permissive to PEDV and support productive infection, encompassing the complete viral life cycle from entry to release of infectious progeny. Productive replication was confirmed by direct immunodetection of viral antigen within inoculated cells. Viral challenge induced characteristic cytopathic effects and a specific pro-inflammatory elevation of tumor necrosis factor-alpha (TNF-α). Notably, infection fragmented ZO-1, providing a mechanistic explanation for the leaky gut phenotype observed in vivo. This platform serves as a robust system for investigating viral kinetics and host–pathogen interactions with high species-specific relevance.

Post-weaning diarrhea caused by pathogenic Escherichia coli is a significant economic challenge in global swine production. Rising antimicrobial resistance linked to traditional antibiotics necessitates safe, sustainable alternatives. This study isolated and characterized host-associated Bacillus species from the gastrointestinal tracts of healthy pigs to evaluate their probiotic potential. Fifty-nine Bacillus isolates were screened for safety, gastrointestinal tract survivability, mucosal adhesion, and antimicrobial efficacy. Several isolates demonstrated robust tolerance to simulated gastrointestinal stress (acidic pH and bile salts), non-hemolytic profiles, and strong mucosal adhesion. Multiple isolates displayed inhibitory activity against pathogenic Escherichia coli in vitro. The most promising candidate, Bacillus subtilis 1BP1Co1, underwent Nanopore whole-genome sequencing. Genomic profiling confirmed its safety, revealing a complete absence of known virulence factors and high-risk antimicrobial resistance genes. Furthermore, genome annotation identified multiple biosynthetic gene clusters responsible for the production of antimicrobial secondary metabolites, including surfactin, fengycin, bacillaene, and subtilosin A, which mechanistically supports its pathogen-inhibitory properties. Collectively, these findings highlight indigenous Bacillus strains, particularly Bacillus subtilis 1BP1Co1, as promising probiotic candidates with the potential to be developed to mitigate Escherichia coli-associated enteric disorders in swine production systems.