June 2017
Volume 58, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2017
The mechanical properties of the human corneal limbus and its influence on epithelial stem cell phenotype
Author Affiliations & Notes
  • Che John Connon
    Institute of Genetic Medicine, Newcastle University, Newcastle Upon Tyne, United Kingdom
  • Ricardo Martins Gouveia
    Institute of Genetic Medicine, Newcastle University, Newcastle Upon Tyne, United Kingdom
  • Carl Paterson
    Imperial College London, Loondon, United Kingdom
  • Guillaume Lepert
    Imperial College London, Loondon, United Kingdom
  • Rajiv R Mohan
    University of Missouri, Columbia, Missouri, United States
  • Suneel Gupta
    University of Missouri, Columbia, Missouri, United States
  • Footnotes
    Commercial Relationships   Che Connon, None; Ricardo Martins Gouveia, None; Carl Paterson, None; Guillaume Lepert, None; Rajiv Mohan, None; Suneel Gupta, None
  • Footnotes
    Support  Medical Research Council, UK, MR/K017217/1 Modulation of limbal niche stiffness to regulate stem cell differentiation and University of Missouri Ruth M. Kraeuchi Missouri Endowed Chair Ophthalmology Fund (RRM)
Investigative Ophthalmology & Visual Science June 2017, Vol.58, 4243. doi:
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      Che John Connon, Ricardo Martins Gouveia, Carl Paterson, Guillaume Lepert, Rajiv R Mohan, Suneel Gupta; The mechanical properties of the human corneal limbus and its influence on epithelial stem cell phenotype. Invest. Ophthalmol. Vis. Sci. 2017;58(8):4243.

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

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Abstract

Purpose : The function of the cornea is largely dependent on the maintenance of a healthy stratified epithelium, a process that relies on a population of stem cells residing in the limbus. Limbal epithelial stem cells migrate from the edge towards the center of the cornea, where they undergo differentiation and stratification. However, the mechanisms underpinning this homeostatic process are still unclear. We have hypothesized that the biomechanical properties of the surface supporting the corneal epithelium play a fundamental role in this tissue’s homeostasis. In this study we address this hypothesis by mapping the bulk modulus of fresh human corneas at an unprecedented resolution as well as pharmacologically altering the corneas stiffness to affect epithelial phenotype in vitro and in vivo.

Methods : Brillouin spectro-microscopy was used to accurately measure differences in mechanical response across the human cornea. Limbal stem cell markers were qualitatively and quantitatively assessed by immunohistochemistry and in-cell Westerns across corneal tissue and in vitro models with differing levels of substrate compliance. Central areas of the rabbit cornea were softend by application of collagenase and the effect on epitheial phenotype quantified.

Results : Brillouin spectro-microscopy revealed a significantly different mean frequency shift between central cornea and limbus (6.66±0.04 compared with 6.24±0.09 GHz shifts). These areas corresponded to predictable presence/absence of differentiation or limbal stem call markers i.e. cells on the softer parts expressed limbal stem cell markers wheresas cells on the stiffer parts of the cornea expressed diffferentiation markers.
Collagenase treatment successfully modulated the stiffness of the cornea both in vitro and in vivo and that this treatment subsequently influenced the behavior of human corneal epithelial cells. The results showed that softened tissues supported the growth of ABCG2+/CK15+/α9-integrin+/CK3- corneal epithelial cells capable of depositing a limbus-characteristic basement membrane in the central cornea following collagenase treatment.

Conclusions : Overall, our data supports the existence of a correlation between the mechanical properties of the corneal matrix and the phenotype of corneal epithelial cells. Moreover, it showed that this phenotype can be controlled through the modulation of substrate stiffness, both in situ and in vitro.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.

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