Cornea 3D culture models, skin and cornea trandifferentiations, epithelilmesenchymal

) To further develop and refine the 3D human cornea model for use in interactive studies and as a tool for cellular and molecular assays.
2) To transdifferentiate human skin/hair epidermis into corneal epithelium using both molecular and direct tissue/cell interaction approaches.
3) To initiate investigations into epigenetic changes occurring during skin to cornea transdifferentiation.
Methodology and Strategy:
Aim 1) Hitherto, the human cells used in the making of our 3D cornea model have been sub-optimum - from stored tissues, and with no selection process. The student will take advantage of recent advances by collaborators for the isolation of specific cell populations from both the stroma and epithelium to produce a model that is robust for up to 3 weeks (the EU standard for tissue models). This will then be used in the trandifferentiation work (Aim 2).
Aim 2) The student will deploy a progressive strategy to transdifferentiate human skin epithelial cells to corneal epithelium. Initially she/he will use transient transfection protocols (electroporation and lipid based) with Pax6 and Wnt7 constructs and test whether this is, of itself, sufficient to elicit key switches in differentiation. As both Pax6 and relevant Wnt signalling components are active in secreted form, the student will also use these molecules, in conjunction with other pathway activators/repressors to promote reprogramming of skin/hair epithelial cells to corneal epithelium. The third approach involves combining skin epithelial cells over a core of corneal stromal cells in our 3D cornea culture model. We envisage that a combination of methods may be required, for example, molecular pre-treatment of cells followed by co-culture with eye stromal cells in the 3D model. Analysis will include PCR, and immunohistochemistry.
Aim 3) Different epigenetic regulators play crucial roles in the execution of lineage-specific gene expression programs in skin keratinocytes. The student will investigate changes to DNA/histone-modifying enzymes, and Polycomb genes during cornea to skin and (when successful) skin to cornea transdifferentiation.
Year 1: Refinement of the human cornea model, corneal transdifferentiation by transfection and molecules.
Year 2:Transdifferentiation by epithelial-mesenchymal interaction. Epigenetics study. Year 3: As year 2 plus combination methods for transdifferentiation if required. Year 4: Completing work/ writing thesis and papers.
Novelty and Outcomes:
In addition to the biological outcomes that are all novel, a new cornea model would be available for testing functionality of cornea cells from other sources (eg iPS cells) and for in vitro testing.
Research Training: In addition to training in specialised cell culture techniques and basic molecular and cell biology, the student will learn specific confocal microscopic skills when studying epigenetic changes, and will benefit from Porfessor Lako's world leading expertise in reprogramming techniques.