Approximately 4.9 million people worldwide are bilaterally blind due to corneal opacity and corneal blindness is the fifth most common cause of blindness globally. The restoration of a healthy limbal epithelial stem cell fraction is vital to the treatment of corneal blindness associated with the breakdown of corneal epithelial integrity. Corneal limbal stem cell deficiency (LSCD) may be treated using ex vivo limbal epithelial stem cells (LESC) derived from cadaveric donor tissue. However, continuing challenges exist around tissue availability, inflammation and transplant rejection.
The purpose of this study was to optimise current culturing techniques used for LESC transplant tissue, considering expansion and cryopreservation issues surrounding the establishment of a stem cell bank. In addition, a novel anti-inflammatory biomimic was investigated to address issues surrounding amnion and steroid use in LESC transplant, inflammation and transplant rejection.
The project ran from 2009 to 2013.
Key issues remain in the control of inflammation and in the optimisation of cell culture and carrier technology for use in the transport and application of corneal LESC grafts to treat corneal opacity.
The aims of the project were to investigate optimised corneal epithelial extraction and cryopreservation techniques, to synthesise and characterise an IL-1beta receptor antagonist peptide as an anti-inflammatory amnion biomimic, to establish an in vitro model of corneal inflammation and assess the impact of peptide on key markers of corneal keratocyte and epithelial inflammatory response to infection and inflammation. Lipopolysaccharide (LPS) or recombinant human IL-1β stimulated primary human keratocyte and LESC models were used to investigate the anti-inflammatory properties of a short chain, IL-1 receptor antagonist peptide for use in LESC sheet growth to control inflammation.
Current culturing techniques were compared by cell isolation from donor corneal tissue, measuring corneal epithelial cell growth parameters and maintenance of LESC fraction using putative stem cell markers. A decrease in all LESC markers was found post cryopreservation indicating that bio-banking using current sub-culturing techniques is not feasible. An IL-1beta receptor antagonist was synthesised to high purity and was found to produce a significant reduction in rIL-1β stimulated inflammatory cytokine production following LESC and keratocyte incubation with anti-inflammatory peptide and in LPS stimulated IL-6 and IL-8 production following keratocyte incubation with peptide (1mg/ml). LESCs produced no cytokine response to LPS stimulation and showed no TLR4 expression. The peptide supported LESC growth when adhered to a silicone hydrogel contact lens indicating potential use in improved LESC grafting through suppression of inflammation.
Research team
Elsie Fok
Dr Susan Sandeman
Dr Anna Guildford
Output
E Fok*, S Sandeman*, A Guildford, Y Martin (2015). The use of an IL-1 receptor antagonist peptide to control inflammation in the treatment of corneal limbal epithelial stem cell deficiency. Biomed Research International.2015:1-14
Partners
Dr Yella Martin, Blond McIndoe Research Foundation, Queen Victoria Hospital, East Grinstead, West Sussex
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