Enrichment, Characterization, and Proteomic Profiling of Small Extracellular Vesicles Derived from Human Limbal Mesenchymal Stromal Cells and Melanocytes
Sebastian Kistenmacher,
Melanie Schwämmle,
Gottfried Martin,
Eva Ulrich,
Stefan Tholen,
Oliver Schilling,
Andreas Gießl,
Ursula Schlötzer-Schrehardt,
Felicitas Bucher,
Günther Schlunck,
Irina Nazarenko,
Thomas Reinhard,
Naresh Polisetti
Affiliations
Sebastian Kistenmacher
Eye Center, Medical Center, Faculty of Medicine, University of Freiburg, Killianstrasse 5, 79106 Freiburg, Germany
Melanie Schwämmle
Eye Center, Medical Center, Faculty of Medicine, University of Freiburg, Killianstrasse 5, 79106 Freiburg, Germany
Gottfried Martin
Eye Center, Medical Center, Faculty of Medicine, University of Freiburg, Killianstrasse 5, 79106 Freiburg, Germany
Eva Ulrich
Eye Center, Medical Center, Faculty of Medicine, University of Freiburg, Killianstrasse 5, 79106 Freiburg, Germany
Stefan Tholen
Institute of Surgical Pathology, Faculty of Medicine, Freiburg, Medical Center, University of Freiburg, 79085 Freiburg im Breisgau, Germany
Oliver Schilling
Institute of Surgical Pathology, Faculty of Medicine, Freiburg, Medical Center, University of Freiburg, 79085 Freiburg im Breisgau, Germany
Andreas Gießl
Department of Ophthalmology, University Hospital Erlangen, Friedrich-Alexander-University of Erlan-gen-Nürnberg, Schwabachanlage 6, 91054 Erlangen, Germany
Ursula Schlötzer-Schrehardt
Department of Ophthalmology, University Hospital Erlangen, Friedrich-Alexander-University of Erlan-gen-Nürnberg, Schwabachanlage 6, 91054 Erlangen, Germany
Felicitas Bucher
Eye Center, Medical Center, Faculty of Medicine, University of Freiburg, Killianstrasse 5, 79106 Freiburg, Germany
Günther Schlunck
Eye Center, Medical Center, Faculty of Medicine, University of Freiburg, Killianstrasse 5, 79106 Freiburg, Germany
Irina Nazarenko
Institute for Infection Prevention and Hospital Epidemiology, Faculty of Medicine, Medical Center, University of Freiburg, 79106 Freiburg, Germany
Thomas Reinhard
Eye Center, Medical Center, Faculty of Medicine, University of Freiburg, Killianstrasse 5, 79106 Freiburg, Germany
Naresh Polisetti
Eye Center, Medical Center, Faculty of Medicine, University of Freiburg, Killianstrasse 5, 79106 Freiburg, Germany
Limbal epithelial progenitor cells (LEPC) rely on their niche environment for proper functionality and self-renewal. While extracellular vesicles (EV), specifically small EVs (sEV), have been proposed to support LEPC homeostasis, data on sEV derived from limbal niche cells like limbal mesenchymal stromal cells (LMSC) remain limited, and there are no studies on sEVs from limbal melanocytes (LM). In this study, we isolated sEV from conditioned media of LMSC and LM using a combination of tangential flow filtration and size exclusion chromatography and characterized them by nanoparticle tracking analysis, transmission electron microscopy, Western blot, multiplex bead arrays, and quantitative mass spectrometry. The internalization of sEV by LEPC was studied using flow cytometry and confocal microscopy. The isolated sEVs exhibited typical EV characteristics, including cell-specific markers such as CD90 for LMSC-sEV and Melan-A for LM-sEV. Bioinformatics analysis of the proteomic data suggested a significant role of sEVs in extracellular matrix deposition, with LMSC-derived sEV containing proteins involved in collagen remodeling and cell matrix adhesion, whereas LM-sEV proteins were implicated in other cellular bioprocesses such as cellular pigmentation and development. Moreover, fluorescently labeled LMSC-sEV and LM-sEV were taken up by LEPC and localized to their perinuclear compartment. These findings provide valuable insights into the complex role of sEV from niche cells in regulating the human limbal stem cell niche.
