July 2019
Volume 60, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2019
Extracellular matrix regulation of limbal epithelial stem cell function
Author Affiliations & Notes
  • Ursula Schlotzer-Schrehardt
    Department of Ophthalmology, University of Erlangen-Nuernberg, Erlangen, Germany
  • Franziska Kruse
    Department of Ophthalmology, University of Erlangen-Nuernberg, Erlangen, Germany
  • Kerstin Kraus
    Department of Ophthalmology, University of Erlangen-Nuernberg, Erlangen, Germany
  • Matthias Zenkel
    Department of Ophthalmology, University of Erlangen-Nuernberg, Erlangen, Germany
  • Friedrich E Kruse
    Department of Ophthalmology, University of Erlangen-Nuernberg, Erlangen, Germany
  • Footnotes
    Commercial Relationships   Ursula Schlotzer-Schrehardt, None; Franziska Kruse, None; Kerstin Kraus, None; Matthias Zenkel, None; Friedrich Kruse, None
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science July 2019, Vol.60, 4122. doi:
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      Ursula Schlotzer-Schrehardt, Franziska Kruse, Kerstin Kraus, Matthias Zenkel, Friedrich E Kruse; Extracellular matrix regulation of limbal epithelial stem cell function. Invest. Ophthalmol. Vis. Sci. 2019;60(9):4122.

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

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Abstract

Purpose : To dissect the different roles exerted by extracellular matrix (ECM) components establishing a biochemical gradient in the limbal niche in vivo on limbal epithelial stem/progenitor cell (LEPC) behavior in vitro.

Methods : Primary cultures of LEPC were established from human donor corneas and cultivated in 12 well-plates coated with 1-5 µg/cm2 of human recombinant proteins predominantly expressed in the quiescent zone, i.e. laminin (LN-511, LN-521, LN-γ3), nephronectin (NPNT) and junctional adhesion molecule (JAM-C), or in the proliferative zone, i.e. collagen type IV (COL4), LN-111, LN-332, fibronectin (FN), vitronectin (VTN), agrin (AGRN), tenascin-C (TNC), and chondroitin sulfate (CS), of the limbal niche. The effect of specific matrix components on LEPC adhesion, migration, proliferation and differentiation was evaluated by appropriate assays. Expression patterns of differentiation (KRT3, KRT12, Cx-43) and progenitor markers (KRT 15, ABCG2, p63α, N-cadherin) as well of integrin subunits mediating interaction between cells and ECM were analyzed by qPCR and immunocytochemistry.

Results : Coating with COL4, FN, LN-511, LN-521 and LN-γ3 increased, whereas coating with TNC and CS decreased LEPC adhesion significantly over uncoated controls both after 1 and 6 hours. VTN, TNC, NPNT and JAM-C induced a significant increase in cell migration compared to uncoated controls after 3 and 6 hours. Cell proliferation rates were significantly increased in LEPC plated on COL4, FN, LN-111, LN-332, VTN and AGRN, but decreased on LN-511, 24-72 hours after seeding. Expression of differentiation marker KRT3 was downregulated by COL4, LN-111, LN-332, LN-511 and LN-521, whereas expression of progenitor markers KRT15, N-cadherin and ABCG2 was upregulated by LN-511 and LN-γ3; no effect was seen on expression levels of Cx-43 and p63α. These effects were accompanied by substrate-specific alterations in integrin expression patterns involving integrin subunits α1, α2, α3, α4, α5, α6, αv, ß1, ß2, ß4, ß5, ß6 and ß8.

Conclusions : The findings indicate that ECM gradients represent an essential player in the limbal niche modulating the maintenance, proliferation and differentiation of LEPC. In vitro presentation of appropriate ECM cues may represent a useful strategy to influence cell function and to improve LEPC expansion and to maintain stem/progenitor cell function within the graft.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.

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