Integrated transcriptomic and proteomic analysis reveals isolation and culture associated molecular changes in neonatal porcine pancreatic cell clusters

BACKGROUND: While porcine Neonatal Pancreatic Cell Clusters (NPCCs) were potential alternative source of islets, their immaturity and heterogeneity make them inefficiently ameliorate hyperglycemia in rodents. Our previous work showed that NPCCs regress to a transitional-like state immediately after isolation and undergo dynamic transcriptional changes during short-term in vitro culture. The present study aimed to further delineate the molecular cues underlying these changes during porcine pancreatic tissue isolation and cultivation. METHOD: Transcriptomic and proteomic analysis were performed to compare Neonatal Porcine Pancreata (NPP), freshly isolated NPCCs (NPCCs-0D) and 3-day in vitro cultured NPCCs (NPCCs-3D). Using an in-house analysis pipeline, molecular differences between groups were identified. RESULTS: NPCCs‑0D showed lower molecular abundance compared with NPP and NPCCs‑3D groups under the tested conditions. Gene Sets Enrichment Analysis (GSEA) revealed that pathways related to receptor signaling and cell-microenvironment interactions were enriched in NPP and NPCCs‑3D, highlighting the importance of external regulation in maintaining pancreatic tissue architecture. In contrast. organelle- and catalysis-associated gene sets were enriched in NPCCs‑0D, suggesting a transcriptionally active and adaptive state. CONCLUSION: Collectively, these findings indicate that molecular differences among NPP, NPCCs-0D, and NPCCs-3D reflect changes in tissue organization, cellular composition, and adaptation to isolation and culture conditions, rather than a linear differentiation process.