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James Foster, Ana Maria Ionescu, Roanne Jones, Ricardo Gouveia, Che Connon, ; Integrating human epithelial stem cells and keratocytes into a transparent, collagen based bioengineered cornea for disease modelling. Invest. Ophthalmol. Vis. Sci. 2013;54(15):1006.
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To demonstrate the integration of epithelial stem cells and keratocytes into a single construct using defiend conditions. This will allow the interplay between the two cell types that form the anterior cornea to be investigated. Since the collagen constructs are intended to replace the principal refractive and image forming component of the eye, their optical quality was also studied to ensure the functionality of these bioengineered tissues.
Epithelial stem cells and keratocytes were isolated from cadaverous human tissue and cultivated in vitro. Keratocytes were encapsualted within compressed collagen gels and allowed to proliferate and differentiate for 7 days before addition of limbal epithelial stem cells under differentiation media conditions. Expression levels of specific keratocyte markers ALDH-1, Lumican, Col5a1, and Keratocan were determined through PCR and fluorescent microscopy. The optical quality of resulting constructs was determined by Contrast Transfer Function (CTF), a simple method for the accurate quantification of contrast between adjacent objects. Several bar patterns of alternating white and black lines with increasing frequency (2, 4, 6, 10, 20, 31, and 61 cycles/mm) were displayed on a LCD and images were captured using a N90 Nikon camera. The CTF and transparency levels were calculated using adequate image analysis software (ImageJ). Secretion of ECM components by keratocytes and epithelial cell behaviour were examined over 3 weeks.
The inclusion of cells within the gels increased transparency by 10% to 66%, reaching a value sufficient for a collagen construct to be used as a corneal replacement. The phenotype of the epithelial stem cell was characterized by IHC, qPCR, and flow cytometry as ABCG2- and ΔNP63-positive, and CK3-negative. The epithelial stem cells differentiated to CK3-positive cells with accompanying stratification (>4 cells thick). Keratocytes were shown to secrete col5a1, lumican, and keratocan. Keratocyte organisation showed increased orthogonal alignment within the gels.
Here we have recapitulated some of the complex interplay between human epithelial and stromal cells within a defined culture system. This resulted in corneal constructs with therapeutic levels of transparency and the desirable cell phenotypes.
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