Contribution of extracellular vesicles from Adult-derived human liver stem cells to the correction of Urea Cycle Disorders

Introduction Adult-derived human liver stem cells (ADHLSCs) are currently in clinical development for the treatment of Urea Cycle Disorders (UCD). Clinical and preclinical data seem to indicate a higher clinical effect than what could be expected from the number of cells that have engrafted, suggesting that other mechanisms may be at play. We have previously demonstrated that ADHLSCs produce Extracellular Vesicles, EVs (MP, microparticles; and EXO, exosomes), which have been shown to mediate intracellular communication in other systems by delivering proteins, lipids and/or genetic information (coding and non-coding RNAs) to recipient cells. Therefore, the aim of this study was to determine the precise role of EVs in ADHLSC-mediated correction of UCD. Methods ADHLSCs were cultured for 2 days in DMEM supplemented with 10% EXO-free FBS and 1% P/S. The conditioned medium was collected, and MP and EXO fractions were harvested by serial ultracentrifugation. Transmission electron microscopy (TEM), western blotting and nanoparticle tracking analysis were used to evaluate the presence, purity and abundance of MP and EXO. RNA from EVs was stained with SytoRNA, which only fluoresces upon integration into RNA, to investigate RNA transfer from EVs to rat hepatocytes. Droplet digital PCR (ddPCR) was performed on RNA extracted from the MP and EXO as well as rat hepatocytes previously incubated with EVs to investigate the presence of human mRNAs of interest. Results We confirmed that ADHLSCs produce both MP and EXO. Characterization of the mRNA by ddPCR showed expression of ASL, ASS, and CPS1 in EVs, mainly in MPs. SytoRNA staining of the EV RNA allowed us to show transfer of EV RNA to over 60% of rat hepatocytes in vitro. Finally, we demonstrated transfer of human mRNAs of interest from EVs to rat hepatocytes using ddPCR. Summary/Conclusion In summary, our study shows that ADHLSC-derived EVS contain mRNA encoding for some of the deficient enzymes in UCD and are capable of transfering their mRNA content to recipient cells. mRNA transfer via EVs may therefore be one of the modes of action of ADHLSCs in UCD.