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Scalable Generation of Nanovesicles from Human-Induced Pluripotent Stem Cells for Cardiac Repair

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posted on 2023-01-24, 04:59 authored by Jonathan LOZANO SALGADOJonathan LOZANO SALGADO, Alin RaiAlin Rai, JG Lees, H Fang, Bethany ClaridgeBethany Claridge, SY Lim, David GreeningDavid Greening
Extracellular vesicles (EVs) from stem cells have shown significant therapeutic potential to repair injured cardiac tissues and regulate pathological fibrosis. However, scalable generation of stem cells and derived EVs for clinical utility remains a huge technical challenge. Here, we report a rapid size-based extrusion strategy to generate EV-like membranous nanovesicles (NVs) from easily sourced human iPSCs in large quantities (yield 900× natural EVs). NVs isolated using density-gradient separation (buoyant density 1.13 g/mL) are spherical in shape and morphologically intact and readily internalised by human cardiomyocytes, primary cardiac fibroblasts, and endothelial cells. NVs captured the dynamic proteome of parental cells and include pluripotency markers (LIN28A, OCT4) and regulators of cardiac repair processes, including tissue repair (GJA1, HSP20/27/70, HMGB1), wound healing (FLNA, MYH9, ACTC1, ILK), stress response/translation initiation (eIF2S1/S2/S3/B4), hypoxia response (HMOX2, HSP90, GNB1), and extracellular matrix organization (ITGA6, MFGE8, ITGB1). Functionally, NVs significantly promoted tubule formation of endothelial cells (angiogenesis) (p < 0.05) and survival of cardiomyocytes exposed to low oxygen conditions (hypoxia) (p < 0.0001), as well as attenuated TGF-β mediated activation of cardiac fibroblasts (p < 0.0001). Quantitative proteome profiling of target cell proteome following NV treatments revealed upregulation of angiogenic proteins (MFGE8, MYH10, VDAC2) in endothelial cells and pro-survival proteins (CNN2, THBS1, IGF2R) in cardiomyocytes. In contrast, NVs attenuated TGF-β-driven extracellular matrix remodelling capacity in cardiac fibroblasts (ACTN1, COL1A1/2/4A2/12A1, ITGA1/11, THBS1). This study presents a scalable approach to generating functional NVs for cardiac repair.

Funding

This work was supported by the National Heart Foundation of Australia (DG: Vanguard), NHMRC project grant (DG: #1139489, 1057741), Future Fund (DG: MRF1201805), Pankind (DG), Stafford Fox Medical Research Foundation (SYL), and the Victorian Government's Operational Infrastructure Support Program. J.L. and B.C. are supported by an Australian Government Training Program (RTP) scholarship, La Trobe University-Baker Heart and Diabetes Institute joint scholarship.

History

Publication Date

2022-11-18

Journal

International Journal of Molecular Sciences

Volume

23

Issue

22

Article Number

14334

Pagination

25p.

Publisher

Multidisciplinary Digital Publishing Institute (MDPI)

ISSN

1422-0067

Rights Statement

© 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

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