Ex vivo 3D human corneal stroma model for schnyder corneal dystrophy - Role of autophagy in its pathogenesis and resolution

Dóra Júlia Szabó, Richárd Nagymihály, Zoltán Veréb, Natasha Josifovska, Agate Noer, Petra Liskova, Andrea Facskó, Morten C. Moe, G. Petrovski

Research output: Contribution to journalArticle

1 Citation (Scopus)

Abstract

Introduction. Multilamellar bodies (MLBs) are concentric cytoplasmic membranes which form through an autophagy-dependent mechanism. In the cornea, the presence of MLBs is associated with Schnyder corneal dystrophy (SCD). Ex vivo 3D modelling of the corneal stroma and SCD can help study pathogenesis and resolution of the disorder. Methods. Corneal stroma explants were isolated from cadavers and cultivated long-term for more than 3 months to achieve spontaneous 3D outgrowth of corneal stroma-derived mesenchymal stem-like cells (CSMSCs). The 3D tissues were then examined by transmission electron microscopy (TEM) for presence of MLBs, and by immunofluorescent labelling against markers for autophagy (p62, LC3). Autophagy was induced by classical serum starvation or rapamycin (RAP) treatment (50nM), and inhibited by the autophagy inhibitor 3-methyladenine (3-MA, 10 mM) for 24 hours. Results. CSMSCs can form spontaneously 3D outgrowths over a 3-4 weeks period, depositing their own extracellular matrix containing collagen I. TEM confirmed the presence of MLBs in the long-term (>3 months) 3D cultures, which became more abundant under starvation and RAP treatment, and decreased in number under autophagy inhibition with 3-MA. The presence of autophagy and its disappearance could be confirmed by an inversely related increase and decrease in the expression of LC3 and p62, respectively. Conclusions. MLB formation in long-standing CSMSC cultures could serve as a potential ex vivo model for studying corneal stroma diseases, including SCD. Inhibition of autophagy can decrease the formation of MLBs, which may lead to a novel treatment of the disease in the future.

Original languageEnglish
Pages (from-to)455-462
Number of pages8
JournalHistology and Histopathology
Volume33
Issue number5
DOIs
Publication statusPublished - May 1 2018

Fingerprint

Corneal Stroma
Autophagy
Mesenchymal Stromal Cells
Sirolimus
Starvation
Transmission Electron Microscopy
Corneal Diseases
Corneal Dystrophy, Crystalline, of Schnyder
Cadaver
Cornea
Extracellular Matrix
Collagen
Cell Culture Techniques
Cell Membrane
Serum

Keywords

  • 3D model
  • Autophagy
  • Corneal stroma-derived mesenchymal stem-like cells (CSMSC)
  • Schnyder corneal dystrophy

ASJC Scopus subject areas

  • Pathology and Forensic Medicine
  • Histology

Cite this

Ex vivo 3D human corneal stroma model for schnyder corneal dystrophy - Role of autophagy in its pathogenesis and resolution. / Szabó, Dóra Júlia; Nagymihály, Richárd; Veréb, Zoltán; Josifovska, Natasha; Noer, Agate; Liskova, Petra; Facskó, Andrea; Moe, Morten C.; Petrovski, G.

In: Histology and Histopathology, Vol. 33, No. 5, 01.05.2018, p. 455-462.

Research output: Contribution to journalArticle

Szabó, DJ, Nagymihály, R, Veréb, Z, Josifovska, N, Noer, A, Liskova, P, Facskó, A, Moe, MC & Petrovski, G 2018, 'Ex vivo 3D human corneal stroma model for schnyder corneal dystrophy - Role of autophagy in its pathogenesis and resolution', Histology and Histopathology, vol. 33, no. 5, pp. 455-462. https://doi.org/10.14670/HH-11-928
Szabó, Dóra Júlia ; Nagymihály, Richárd ; Veréb, Zoltán ; Josifovska, Natasha ; Noer, Agate ; Liskova, Petra ; Facskó, Andrea ; Moe, Morten C. ; Petrovski, G. / Ex vivo 3D human corneal stroma model for schnyder corneal dystrophy - Role of autophagy in its pathogenesis and resolution. In: Histology and Histopathology. 2018 ; Vol. 33, No. 5. pp. 455-462.
@article{35ff4057e332488f9e44762a6354fc5f,
title = "Ex vivo 3D human corneal stroma model for schnyder corneal dystrophy - Role of autophagy in its pathogenesis and resolution",
abstract = "Introduction. Multilamellar bodies (MLBs) are concentric cytoplasmic membranes which form through an autophagy-dependent mechanism. In the cornea, the presence of MLBs is associated with Schnyder corneal dystrophy (SCD). Ex vivo 3D modelling of the corneal stroma and SCD can help study pathogenesis and resolution of the disorder. Methods. Corneal stroma explants were isolated from cadavers and cultivated long-term for more than 3 months to achieve spontaneous 3D outgrowth of corneal stroma-derived mesenchymal stem-like cells (CSMSCs). The 3D tissues were then examined by transmission electron microscopy (TEM) for presence of MLBs, and by immunofluorescent labelling against markers for autophagy (p62, LC3). Autophagy was induced by classical serum starvation or rapamycin (RAP) treatment (50nM), and inhibited by the autophagy inhibitor 3-methyladenine (3-MA, 10 mM) for 24 hours. Results. CSMSCs can form spontaneously 3D outgrowths over a 3-4 weeks period, depositing their own extracellular matrix containing collagen I. TEM confirmed the presence of MLBs in the long-term (>3 months) 3D cultures, which became more abundant under starvation and RAP treatment, and decreased in number under autophagy inhibition with 3-MA. The presence of autophagy and its disappearance could be confirmed by an inversely related increase and decrease in the expression of LC3 and p62, respectively. Conclusions. MLB formation in long-standing CSMSC cultures could serve as a potential ex vivo model for studying corneal stroma diseases, including SCD. Inhibition of autophagy can decrease the formation of MLBs, which may lead to a novel treatment of the disease in the future.",
keywords = "3D model, Autophagy, Corneal stroma-derived mesenchymal stem-like cells (CSMSC), Schnyder corneal dystrophy",
author = "Szab{\'o}, {D{\'o}ra J{\'u}lia} and Rich{\'a}rd Nagymih{\'a}ly and Zolt{\'a}n Ver{\'e}b and Natasha Josifovska and Agate Noer and Petra Liskova and Andrea Facsk{\'o} and Moe, {Morten C.} and G. Petrovski",
year = "2018",
month = "5",
day = "1",
doi = "10.14670/HH-11-928",
language = "English",
volume = "33",
pages = "455--462",
journal = "Histology and Histopathology",
issn = "0213-3911",
publisher = "Histology and Histopathology",
number = "5",

}

TY - JOUR

T1 - Ex vivo 3D human corneal stroma model for schnyder corneal dystrophy - Role of autophagy in its pathogenesis and resolution

AU - Szabó, Dóra Júlia

AU - Nagymihály, Richárd

AU - Veréb, Zoltán

AU - Josifovska, Natasha

AU - Noer, Agate

AU - Liskova, Petra

AU - Facskó, Andrea

AU - Moe, Morten C.

AU - Petrovski, G.

PY - 2018/5/1

Y1 - 2018/5/1

N2 - Introduction. Multilamellar bodies (MLBs) are concentric cytoplasmic membranes which form through an autophagy-dependent mechanism. In the cornea, the presence of MLBs is associated with Schnyder corneal dystrophy (SCD). Ex vivo 3D modelling of the corneal stroma and SCD can help study pathogenesis and resolution of the disorder. Methods. Corneal stroma explants were isolated from cadavers and cultivated long-term for more than 3 months to achieve spontaneous 3D outgrowth of corneal stroma-derived mesenchymal stem-like cells (CSMSCs). The 3D tissues were then examined by transmission electron microscopy (TEM) for presence of MLBs, and by immunofluorescent labelling against markers for autophagy (p62, LC3). Autophagy was induced by classical serum starvation or rapamycin (RAP) treatment (50nM), and inhibited by the autophagy inhibitor 3-methyladenine (3-MA, 10 mM) for 24 hours. Results. CSMSCs can form spontaneously 3D outgrowths over a 3-4 weeks period, depositing their own extracellular matrix containing collagen I. TEM confirmed the presence of MLBs in the long-term (>3 months) 3D cultures, which became more abundant under starvation and RAP treatment, and decreased in number under autophagy inhibition with 3-MA. The presence of autophagy and its disappearance could be confirmed by an inversely related increase and decrease in the expression of LC3 and p62, respectively. Conclusions. MLB formation in long-standing CSMSC cultures could serve as a potential ex vivo model for studying corneal stroma diseases, including SCD. Inhibition of autophagy can decrease the formation of MLBs, which may lead to a novel treatment of the disease in the future.

AB - Introduction. Multilamellar bodies (MLBs) are concentric cytoplasmic membranes which form through an autophagy-dependent mechanism. In the cornea, the presence of MLBs is associated with Schnyder corneal dystrophy (SCD). Ex vivo 3D modelling of the corneal stroma and SCD can help study pathogenesis and resolution of the disorder. Methods. Corneal stroma explants were isolated from cadavers and cultivated long-term for more than 3 months to achieve spontaneous 3D outgrowth of corneal stroma-derived mesenchymal stem-like cells (CSMSCs). The 3D tissues were then examined by transmission electron microscopy (TEM) for presence of MLBs, and by immunofluorescent labelling against markers for autophagy (p62, LC3). Autophagy was induced by classical serum starvation or rapamycin (RAP) treatment (50nM), and inhibited by the autophagy inhibitor 3-methyladenine (3-MA, 10 mM) for 24 hours. Results. CSMSCs can form spontaneously 3D outgrowths over a 3-4 weeks period, depositing their own extracellular matrix containing collagen I. TEM confirmed the presence of MLBs in the long-term (>3 months) 3D cultures, which became more abundant under starvation and RAP treatment, and decreased in number under autophagy inhibition with 3-MA. The presence of autophagy and its disappearance could be confirmed by an inversely related increase and decrease in the expression of LC3 and p62, respectively. Conclusions. MLB formation in long-standing CSMSC cultures could serve as a potential ex vivo model for studying corneal stroma diseases, including SCD. Inhibition of autophagy can decrease the formation of MLBs, which may lead to a novel treatment of the disease in the future.

KW - 3D model

KW - Autophagy

KW - Corneal stroma-derived mesenchymal stem-like cells (CSMSC)

KW - Schnyder corneal dystrophy

UR - http://www.scopus.com/inward/record.url?scp=85044477954&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85044477954&partnerID=8YFLogxK

U2 - 10.14670/HH-11-928

DO - 10.14670/HH-11-928

M3 - Article

C2 - 28872183

AN - SCOPUS:85044477954

VL - 33

SP - 455

EP - 462

JO - Histology and Histopathology

JF - Histology and Histopathology

SN - 0213-3911

IS - 5

ER -