HUMAN INDUCED PLURIPOTENT STEM CELL DERIVED EXTRACELLULAR VESICLES REVERSE HEPATIC STELLATE CELL ACTIVATION
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1 HUMAN INDUCED PLURIPOTENT STEM CELL DERIVED EXTRACELLULAR VESICLES REVERSE HEPATIC STELLATE CELL ACTIVATION Dr. Davide POVERO LABORATORY of Prof. ARIEL E. FELDSTEIN, M.D. UNIVERSITY OF CALIFORNIA SAN DIEGO DEPARTMENT OF PEDIATRICS DIVISION OF GASTROENTEROLOGY, HEPATOLOGY AND NUTRITION Key role of hepatic stellate cells in liver fibrosis Viral (HBV, HCV) Alcoholic Metabolic Autoimmune Genetic Myofibroblast-like Hepatic Stellate Cell Quiescent Hepatic Stellate Cell Cell Responses -Chemotaxis - Proliferation - ECM remodeling Mesenchymal stem cells as anti-fibrotic strategy Experimental liver fibrosis models Mesenchymal stem cells (MSCs) 1
2 Biogenesis of induced pluripotent stem cell-derived extracellular vesicles (ipsc-evs) Pluripotency induction Skin fibroblasts EV Reprogramming factors (Sox2, Oct4, KLF4) Inducible pluripotent stem cells (ipsc) Isolation of EVs by differential centrifugation Hypothesis Activated Hepatic Stellate Cells Extracellular Vesicles Human Inducible Pluripotent Stem Cells Inactivated Hepatic Stellate Cells QUESTION: Extracellular vesicles (EVs) are released by ipscs and are internalized in hepatic stellate cells (HSCs)? APPROACH: Human Inducible Pluripotent Stem Cells (ipscs) EV Characterization: - Flow Cytometry (FACS) - Dynamic Light Scattering (DLS) - Electron Microscopy (TEM) - Internalization into HSCs (Povero et al., Science Signaling, 213, Povero et al., JCMGH, 215) Cell media EVs isolation by differential centrifugation 2
3 ip-stem cells release Extracellular vesicles (EVs) 2 FACS TEM Calcein + EVs / ml media ipsc-ev EV-free sup Intensity (%) DLS ,. size (d.nm) Stem cell-derived EVs are internalized into Hepatocyte-EVs 5h hhscs ipsc-evs 5h Povero et al., JCMGH, 215 QUESTION: Do ipsc-derived extracellular vesicles deactivate HSC pro-fibrogenic responses. APPROACH: ipsc- Extracellular Vesicles Chemotaxis (Boyden s Chamber Assay) TGF-β Proliferation (BrDU Assay) 3
4 ipsc-derived EVs reduce HSC chemotaxis TGF-β 5 ipsc-evs EV-free Migrated hhsc (number / 1x field) TGF- EV+TGF- EV-free+TGF- ipsc-derived EVs reduce HSC proliferation Fluorescence intensity (fold change) TGF- ipsc-ev EV-free + TGF- + TGF- QUESTION: Do ipsc-derived extracellular vesicles reverse hepatic stellate cell (HSC) activation? APPROACH: ipsc-extracellular Vesicles Quiescent HSC Activated HSC Inactivated HSC Selected Gene Profile Adapted from Liu et al. Curr Pathobiol Rep,
5 Normalized -SMA mrna/b2m (fold change) Stem cell-derived EVs are internalized into α- 4 hhscs 3 Coll1α1 SMA TGF- ipsc-ev EV-free Normalized TIMP-2 mrna/b2m (fold change) TIMP-2 Normalized Collagen1 1 mrna/b2m (fold change) TGF- ipsc-ev EV-free 2 1 TGF- ipsc EV EV-free Exposure to ipsc-evs induces inactivation of HSCs Normalized PPAR- mrna/b2m (fold change) Normalized IL7R mrna/b2m (fold change) PPAR-γ IL7R TGF- ipsc EV EV-free TGF- ipsc-ev EV-free Normalized Csf2rb mrna/b2m (fold change) Normalized Ly86 mrna/b2m (fold change) Csf2rb Ly86 TGF- ipsc-ev EV-free TGF- ipsc-ev EV-free Summary Human pluripotent stem cells (ipscs) release extracellular vesicles (EVs) with a anti-fibrotic potential. EVs released and isolated by ipsc induce a reversal from activated to inactivated hepatic stellate cells (HSCs). ipsc-derived EVs prevents HSCs migration and proliferation, two of the main cell responses of activated HSCs in liver fibrosis. 5
6 Future Directions Investigate the mirna cargo of ipsc-evs. Investigate the gene profile of hhsc exposed to ipsc-derived EVs. Inject mouse ipsc-derived EVs in fibrotic murine models to stop or reverse liver fibrosis. UNIVERSITY OF CALIFORNIA SAN DIEGO Department of Pediatrics Division of Gastroenterology, Hepatology and Nutrition Funding: 6
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