Abstract
IDENTIFICATION OF ANTI-FIBROTIC microRNAs ENRICHED IN EXTRACELLULAR VESICLES DERIVED FROM BONE MARROW MESENCHYMAL STEM CELLS IN PATIENTS WITH DECOMPENSATED LIVER CIRRHOSIS.
Background: We have been developing a liver regeneration therapy for decompensated liver cirrhosis (DLC) using cultured autologous bone marrow mesenchymal stem cells (BMSCs). Currently, we are conducting a clinical trial called "Self-contained liver cirrhosis regeneration therapy (jRCT2063200014)". In our clinical trial, hepatic arterial infusion of cultured autologous BMSCs has ameliorated liver function and fibrosis in patients with DLC. However, the mechanism of anti-fibrotic action of BMSCs remain to be fully determined. It was recently reported that extracellular vesicles derived from MSCs (MSC-EVs) exhibited antifibrotic properties. We therefore examined the effect of MSC-EVs on extracellular matrix (ECM) production by hepatic stellate cells, and identified anti-fibrotic microRNAs (miRNAs) enriched in MSC-EVs using BMSCs derived from patients with DLC in our clinical trial with aim of developing adequate quality standards for MSCs in liver regeneration therapy.
Methods: Human hepatic stellate cells (HHSteCs) were treated with TGFβ for activation and subsequently treated with EVs isolated from the culture media of human BMSCs and ECM gene expression determined. We used a microarray to perform a comprehensive analysis of miRNA expression profiles and compared miRNA expression profiles in MSC-EVs derived from healthy individuals or patients with DLC in our clinical trial and HHSteCs. To identify the antifibrotic miRNAs enriched in MSC-EVs, each mimic of miRNAs that highly expressed in MSC-EVs and lowly expressed in HHSteCs was transfected into HHSteCs and ECM gene expression determined.
Results: MSC-EVs inhibited expression of ECM genes (COL1A1, COL1A2, COL3A1 and ELN) by activated HHSteC (p<0.05). Thirty-six miRNAs were extracted from the microarray data that showed normalized intensity of >1,000 in both MSC-EVs of healthy individuals and that of patients with DLC in our clinical trial. Among these miRNAs, ten miRNAs highly expressed in MSC-EVs and lowly expressed in HHSteCs. Each miRNA mimic of the ten miRNAs was transfected into activated HHSteCs and we identified five miRNAs which suppress expression of any of ECM genes (p<0.05). Furthermore, transfection of a combination of the five miRNAs into activated HHSteC resulted in a significant decrease in expression of COL1A1, COL1A2, COL3A1, and ELN (p<0.05).
Conclusion: This study identified five anti-fibrotic miRNAs enriched in MSC-EVs and provided insight into mechanisms of action of MSC-EVs in fibrosis regression. Hence, miRNAs in MSC-EVs may be potential biomarkers for functional assessment of MSCs in liver regeneration therapy.