Investigative Ophthalmology & Visual Science Cover Image for Volume 65, Issue 7
June 2024
Volume 65, Issue 7
Open Access
ARVO Annual Meeting Abstract  |   June 2024
Effects of limbal niche cells-derived melanosomes and exosomes on human limbal epithelial stem/progenitor cells
Author Affiliations & Notes
  • Andreas Gießl
    Department of Ophthalmology, Friedrich-Alexander-University of Erlangen-Nürnberg, Erlangen, Bavaria, Germany
  • Julia Khorolskaya
    Department of Ophthalmology, Friedrich-Alexander-University of Erlangen-Nürnberg, Erlangen, Bavaria, Germany
  • Günter Lochnit
    Protein Analytics, Institute of Biochemistry, Justus Liebig University Giessen, Faculty of Medicine, Giessen, Hesse, Germany
  • Thomas Timm
    Protein Analytics, Institute of Biochemistry, Justus Liebig University Giessen, Faculty of Medicine, Giessen, Hesse, Germany
  • Jan Van Deun
    Department of Dermatology, Friedrich-Alexander-University of Erlangen-Nürnberg, Erlangen, Bavaria, Germany
  • Matthias Zenkel
    Department of Ophthalmology, Friedrich-Alexander-University of Erlangen-Nürnberg, Erlangen, Bavaria, Germany
  • Andreas Baur
    Department of Dermatology, Friedrich-Alexander-University of Erlangen-Nürnberg, Erlangen, Bavaria, Germany
  • Friedrich E Kruse
    Department of Ophthalmology, Friedrich-Alexander-University of Erlangen-Nürnberg, Erlangen, Bavaria, Germany
  • Ursula Schlötzer-Schrehardt
    Department of Ophthalmology, Friedrich-Alexander-University of Erlangen-Nürnberg, Erlangen, Bavaria, Germany
  • Footnotes
    Commercial Relationships   Andreas Gießl None; Julia Khorolskaya None; Günter Lochnit None; Thomas Timm None; Jan Van Deun None; Matthias Zenkel None; Andreas Baur None; Friedrich Kruse None; Ursula Schlötzer-Schrehardt None
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science June 2024, Vol.65, 1997. doi:
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      Andreas Gießl, Julia Khorolskaya, Günter Lochnit, Thomas Timm, Jan Van Deun, Matthias Zenkel, Andreas Baur, Friedrich E Kruse, Ursula Schlötzer-Schrehardt; Effects of limbal niche cells-derived melanosomes and exosomes on human limbal epithelial stem/progenitor cells. Invest. Ophthalmol. Vis. Sci. 2024;65(7):1997.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : Limbal mesenchymal stromal cells (LMSCs) and limbal melanocytes (LMel) are essential components of the limbal epithelial stem/progenitor cell (LEPC) niche. While it has been demonstrated that both LMSCs and LMel contribute to LEPC maintenance, the molecular mechanisms underlying intercellular communication remain unclear. The aim of this study was to comparatively analyze the protein cargo and functional effects of LMel/LMSC-derived exosomes and LMel-derived melanosomes on LEPC proliferation, migration, stemness and differentiation in vitro.

Methods : Exosomes and melanosomes were isolated from conditioned media and lysates of LMSC, LMel, HEK293 control cells by ultracentrifugation and characterized by electron microscopy, Western blotting, nanoparticle tracking and MALDI-TOF analysis. Proteins were identified by MASCOT peptide mass fingerprint search and classified by using bioinformatic tools. Uptake of fluorescent-labeled exosomes/melanosomes by LEPCs was visualized by confocal microscopy. Functional effects of exosomes/melanosomes on LEPC migration, proliferation and differentiation were analyzed by using appropriate assays and qPCR.

Results : Typical melanosomes and exosomes could be purified from cell lysates (LMel) and conditioned media (LMel, LMSC, HEK293). Particle size measurements revealed a characteristic size range of 30 to 200 nm for exosomes and 100 to 500 nm for melanosomes. Typical marker proteins could be identified in both exosomes (CD9, CD81, PDCD6IP, TSG101) and melanosomes (Melan-A, PMEL). A total of 2365 proteins were identified in melanosomes, but only 401 and 191 proteins in exosomes of LMel and LMSC, respectively, with an overlap of 119 proteins relating to structural, metabolic and immunological functions. Uptake of exosomes and melanosomes by LEPC was confirmed by confocal microscopy. Whereas both LMel and LMSC-derived exosomes stimulated an increased proliferation and migration of LEPC, melanosomes induced a decrease in cell proliferation and migration. Moreover, LMel/LMSC-derived exosomes induced increased expression of progenitor marker (e.g. K15, ABCG2, FZD7) and decreased expression of differentiation marker (e.g. K3, IVL, DSG1) by LEPC.

Conclusions : Based on their potent regulatory properties in niche homeostasis, niche cell-derived exosomes may provide a valuable novel tool in stem cell-based therapies for ocular surface reconstruction.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.

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