Proteomic landscape of porcine induced neural stem cell reprogramming and differentiation

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.

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.

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.

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.