Optimized Lipid Nanoparticles Delivery Enables Efficient Circular RNA‐Mediated Forward Programming and Lineage Specification of Induced Pluripotent Stem Cells

Abstract Directed differentiation of induced pluripotent stem cells (iPSCs) into functionally mature hepatocytes holds great potential for liver regeneration. However, current multi‐step protocols are inefficient and often fail to generate fully functional cells. In this study, a lipid nanoparticle (LNP)‐mediated circular RNA (circRNA) delivery platform is developed to efficiently program iPSCs into endothelial cells (iECs) and forward‐programmed hepatocyte‐like cells (cFop‐Heps). Systematic formulation screening identifies LNP4 as significantly enhancing circRNA expression compared to commercial alternatives. Mechanistic studies demonstrate that delivery efficiency closely correlates with protein corona composition and dioleoylphosphatidylethanolamine (DOPE)‐facilitated membrane fusion, which promotes cellular uptake and endosomal escape. Using this platform, lineage‐specific circRNAs encoding transcription factor ETV2 (circ‐ETV2) and Liver Enriched Transcription Factors (circ‐LETFs) are constructed to drive cell fate conversion. circ‐ ETV2 induces functional iECs (CD31⁺/CD144⁺) with tube‐forming capability, while optimized circ‐LETFs generate hepatocyte‐like cells that exhibit albumin secretion, lipoprotein uptake, and glycogen storage. Transcriptomic and functional analyses reveal a transient AMOT ‐Hippo‐ SNAI2 axis that promotes hepatic specification, whereas inhibition of Hippo signaling pathway suppresses hepatic marker expression. This circRNA‐based strategy enables efficient, scalable, and controllable stem cell engineering, offering broad potential applications in liver disease modeling, drug screening, and regenerative medicine.