Proteomic Landscape of Porcine Induced Neural Stem Cell Reprogramming and Differentiation

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 driving porcine fibroblast-to-iNSC conversion and subsequent differentiation remain poorly understood at the proteomic level. Methods: To map the proteomic changes during 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), porcine iNSCs (piNSCs; passage 20), and their neuronal and glial differentiated progeny (piNSCs-NGs; passage 21, after 14 days of neural induction) 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 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 transition from fibroblast to piNSC was marked by increased expression of STMN1, LIMA1, TKT, and NEFL alongside suppression of ARPC5, ACO2, ETFB, and ALDOB. These shifts indicate profound cytoskeletal remodeling and metabolic reprogramming, reflecting a loss of fibroblast identity and the acquisition of a NSC state. During piNSC-to-piNSCs-NGs differentiation, 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 transitions. This resource advances the understanding of neural reprogramming in translational models and supports future regenerative and comparative neuroscience efforts.