June 2015
Volume 56, Issue 7
Free
ARVO Annual Meeting Abstract  |   June 2015
Low-glucose Enhances Keratocyte-characteristic Phenotype from Corneal Stromal Cells in Serum-free Conditions
Author Affiliations & Notes
  • Che John Connon
    Institute of Genetic Medicine, Newcastle University, Newcastle upon Tyne, United Kingdom
  • James W Foster
    Johns Hopkins School of Medicine, Baltimore, MD
  • Ricardo M Gouveia
    Institute of Genetic Medicine, Newcastle University, Newcastle upon Tyne, United Kingdom
  • Footnotes
    Commercial Relationships Che Connon, None; James Foster, None; Ricardo Gouveia, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2015, Vol.56, 1303. doi:
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    • Get Citation

      Che John Connon, James W Foster, Ricardo M Gouveia; Low-glucose Enhances Keratocyte-characteristic Phenotype from Corneal Stromal Cells in Serum-free Conditions. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):1303.

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

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Abstract
 
Purpose
 

The avascular cornea is a uniquely isolated organ, with its stroma constituting a nutrient-poor environment. Consequently, the availability of metabolites such as glucose to corneal stromal cells is considerably reduced compared with other tissues, or indeed with media commonly used to culture these cells in vitro. However, the role of glucose on the behaviour of human corneal keratocytes has been largely overlooked. As such, we sought to investigate the effects of low-glucose formulations on the phenotype of human corneal stromal cells.

 
Methods
 

Human corneal keratocytes were extracted from normal corneal limbal rings and expanded in DMEM:F12 containing 5% foetal bovine serum for 2 passages to form differentiated fibroblasts. These cells were then maintained in serum free media of different glucose concentrations (low, 2 or high, 4.5 g.L-1) for >3 weeks and their phenotype monitored through immunohistochemistry, quantitative PCR and immuno-blotting.

 
Results
 

Human stromal cells cultured in low-glucose were able to survive for extended periods when compared to high-glucose, serum-free conditions (p≥0.01). Furthermore, low-glucose enhanced their reversal from fibroblast to a keratocyte-characteristic phenotype. Specifically, cells within low-glucose medium assumed dendritic morphologies, with bean-shaped condensed nuclei, absence of alpha-smooth muscle actin or stress fibres, and a corresponding significant reduction in migratory and contractile activities when compared with high-glucose, serum-free conditions (p≥0.01). Moreover, cells within low-glucose recovered the ability to significantly express keratocyte-characteristic markers, CD34 and ALDH1A1 (p≥0.05), above traditional high-glucose serum free media conditions, whereas keratocan, lumican and ALDH3A1 remained comparable.

 
Conclusions
 

Our results indicate that low-glucose enhances keratocyte-characteristic phenotype above and beyond established media formulations and thus has important implications for corneal biology in health and disease.  

 
After 3 weeks in low-glucose (LG) serum-free media corneal stromal cells take on a more normal keratocyte phenotype (dendritic morphology), when compared with the same cells grown in either high-glucose (HG) serum-free or serum-contaning basal media (BM) over the same period.
 
After 3 weeks in low-glucose (LG) serum-free media corneal stromal cells take on a more normal keratocyte phenotype (dendritic morphology), when compared with the same cells grown in either high-glucose (HG) serum-free or serum-contaning basal media (BM) over the same period.

 
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