Human induced pluripotent stem cell–derived extracellular vesicles reduce hepatic stellate cell activation and liver fibrosis
Davide Povero, … , Tatiana Kisseleva, Ariel E. Feldstein
Davide Povero, … , Tatiana Kisseleva, Ariel E. Feldstein
Published June 11, 2019
Citation Information: JCI Insight. 2019;4(14):e125652. https://doi.org/10.1172/jci.insight.125652.
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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 2

iPSC-derived EVs are internalized by HSCs and reduce HSC profibrogenic phenotype.

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iPSC-derived EVs are internalized by HSCs and reduce HSC profibrogenic p...
Representative confocal microphotographs of human HSCs exposed to (A) PKH26-labeled EVs (red) or (B) EV-free supernatant for 6 hours. Original magnification, ×40 (A), ×10 (B) (by confocal microscopy). (C) Quantitative PCR expression graphs of profibrogenic genes α–smooth muscle actin (αSMA), CollagenIα1, and tissue inhibitor of metalloproteinases–1 (TIMP-1) in HSCs primed with the profibrogenic agent transforming growth factor–β (TGF-β) and exposed to iPSC-EVs or EV-free supernatant for 24 hours. β2m, β2-microglobulin. (D) Gene expression levels of proliferation marker cyclin D determined by quantitative PCR. β2m was used as a housekeeping gene for quantitative PCR. (E) Representative Western blot gels and corresponding densitometry analyses of fibrosis markers αSMA and fibronectin in HSCs primed with TGF-β and exposed to iPSC-EVs or EV-free supernatant for 24 hours. Actin was used as a loading control. (F) Quantification graph of proliferation assay assessed in human HSCs primed with TGF-β and exposed to iPSC-EVs or EV-free supernatant for 48 hours. (G) Representative microphotographs and (H) quantitation histogram of chemotaxis assay performed on human HSCs primed with TGF-β and exposed to iPSC-EVs or EV-free supernatant for 6 hours. Original magnification, ×10. Values represent mean ± SD from at least 3 independent experiments. *P < 0.05; Kruskal-Wallis test with post hoc Mann-Whitney test and Bonferroni’s correction were used for statistical analysis.