Immunomagneto-activated hepatocyte-specific tissue vesicle profiling reveals message diversification from regenerating liver

Background & Aims: The liver possesses a remarkable regenerative capacity which involves intricate intercellular communication. However, the mechanisms underlying the complex intercellular interactions remain incompletely understood. Extracellular vesicles (EVs) are emerging as important messengers in diverse pathophysiological conditions. Nevertheless, tissue-derived, cell-specific functional EV populations in regeneration have not been robustly analyzed. Methods: Single-cell RNA sequencing (scRNA-seq) analysis of the regenerating liver after partial hepatectomy (PHx), ultrastructural examinations of in situ liver tissue extracellular vesicles (LT-EVs), and nanoscale and proteomic profiling of hepatocyte-specific EVs were integrated. Especially, a feasible approach for investigating antigen-specific endogenous EVs was developed after hepatic enzymatic digestion followed by differential centrifugation and immunomagnetic sorting of LT-EVs. Results: scRNA-seq analysis revealed hepatocytes as the critical source of intercellular communication in the regenerating liver. In particular, hepatocytes are programmed to be enriched for biogenesis and release of EVs in liver regeneration. By analyzing in situ EVs using transmission electron microscope (TEM), LT-EVs were discovered to be emergingly present in the extracellular interstitial space adjacent to hepatocytes in the regenerating liver. Moreover, asialoglycoprotein receptor (ASGPR)-marked, magnetic microbead-sorted LT-EVs (named ASGPR+mLT-EVs) were collected. These hepatocyte-specific LT-EVs were further demonstrated with strengthened release and distinct protein signatures during liver regeneration. Importantly, the EVs demonstrated parental hepatocyte-derived information diversification, which not only inherited regulated proliferative and metabolic signals at the cellular level but also showed vesicular enrichment of organelle components. Conclusions: These findings provide the first knowledge that hepatocyte-specific tissue vesicles are indeed phenotypically and functionally involved in liver regeneration. Our study paves an avenue for in-depth biological and mechanistic research of regenerative LT-EVs and sheds light on extensive dissection of antigen-marked, physiological, and endogenous tissue EV populations. ### Competing Interest Statement The authors have declared no competing interest.