Human induced pluripotent stem cell–derived extracellular vesicles reduce hepatic stellate cell activation and liver fibrosis
Davide Povero, Eva M. Pinatel, Aleksandra Leszczynska, Nidhi P. Goyal, Takahiro Nishio, Jihoon Kim, David Kneiber, Lucas de Araujo Horcel, Akiko Eguchi, Paulina M. Ordonez, Tatiana Kisseleva, Ariel E. Feldstein
Davide Povero, Eva M. Pinatel, Aleksandra Leszczynska, Nidhi P. Goyal, Takahiro Nishio, Jihoon Kim, David Kneiber, Lucas de Araujo Horcel, Akiko Eguchi, Paulina M. Ordonez, Tatiana Kisseleva, Ariel E. Feldstein
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Research Article Cell biology Hepatology

Human induced pluripotent stem cell–derived extracellular vesicles reduce hepatic stellate cell activation and liver fibrosis

Abstract

Progression of fibrosis and the development of cirrhosis are responsible for the liver-related morbidity and mortality associated with chronic liver diseases. There is currently a great unmet need for effective antifibrotic strategies. Stem cells play a central role in wound-healing responses to restore liver homeostasis following injury. Here we tested the hypothesis that extracellular vesicles (EVs) isolated from induced pluripotent stem cells (iPSCs) modulate hepatic stellate cell (HSC) activation and may have antifibrotic effects. Human iPSCs were generated by reprogramming primary skin fibroblasts. EVs were isolated by differential centrifugation, quantified by flow cytometry (FACS), and characterized by dynamic light scattering and transmission electron microscopy (TEM). Primary human HSCs were activated with TGF-β (10 ng/mL) and exposed to iPSC-EVs. Efficacy of iPSC-EVs was tested on HSCs in vitro and in 2 murine models of liver injury (CCl4 and bile duct ligation). Characterization of iPSC-derived EVs by flow cytometry identified a large population of EVs released by iPSCs, primarily with a diameter of 300 nm, that could be visualized by TEM as round, cup-shaped objects. Fluorescent tracing assays detected iPSC-EVs in HSC cytosol after a short incubation, and EV uptake by HSCs resulted in both decrease of profibrogenic markers α–smooth muscle actin, CollagenIα1, fibronectin, and tissue inhibitor of metalloproteinases–1 and HSC profibrogenic responses, such as chemotaxis and proliferation. Genomics analyses of iPSC-EV miRNA cargo revealed 22 highly expressed miRNAs, among which miR-92a-3p was the most abundant. Transcriptome analysis identified 60 genes downmodulated and 235 upregulated in TGF-β–primed HSCs in the presence or absence of iPSC-EVs. Intravenous injection of iPSC-EVs in CCl4-induced and bile duct ligation–induced liver fibrosis resulted in antifibrotic effects at protein and gene levels. Results of this study identify iPSC-EVs as a potentially novel antifibrotic approach that may reduce or reverse liver fibrosis in patients with chronic liver disease.

Authors

Davide Povero, Eva M. Pinatel, Aleksandra Leszczynska, Nidhi P. Goyal, Takahiro Nishio, Jihoon Kim, David Kneiber, Lucas de Araujo Horcel, Akiko Eguchi, Paulina M. Ordonez, Tatiana Kisseleva, Ariel E. Feldstein

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Figure 5

Intravenous administration of m–iPSC-EVs reduces development of fibrosis and HSC activation in an experimental model of cholestatic liver fibrosis.

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Intravenous administration of m–iPSC-EVs reduces development of fibrosis...
(A) Representative microphotographs and (B) corresponding quantification graph of Picrosirius red staining of liver sections harvested from mice that received common bile duct ligation (BDL) for 2 weeks (n = 5–6 mice/group) and were administered, by tail vein, mouse iPSC-EVs (BDL iPSC-EVs) or vehicle control (BDL vehicle) for the last 6 days of the study. Mice that underwent abdomen laparotomy but did not receive BDL were used as no-disease controls (sham). Original magnification, ×10. (C) Immunoblots and (D) corresponding quantification graph of αSMA protein levels over 2 weeks in BDL mice treated with mouse iPSC-EVs or with vehicle for the last 6 days of the study. Tubulin was used as a loading control for Western blots. Values represent mean ± SD. *P < 0.05, and **P < 0.005; Kruskal-Wallis test with post hoc Mann-Whitney test and Bonferroni’s correction were used for statistical analysis.