Tissue engineering of a limbal graft for personalized limbal stem cell niche reconstruction

• Applicant: Naresh Polisetti, Ph.D.
• Subject Area: Ophthalmology
• Term: since 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 525127340

Epithelial stem and progenitor cells are crucial for the homeostasis of the corneal epithelium of the eye, located at the corneal edge (limbus) where they interact with melanocytes and connective tissue cells in a specially structured extracellular matrix. Damage to this stem cell reservoir or the destruction of the stem cell niche, e.g. through burns or inflammation, leads to limbal stem cell deficiency (LSCD) with corneal opacity, vascularization, and loss of vision. In case of unilateral disease, autologous transfer of limbal tissue or of in vitro expanded limbal epithelial stem and progenitor cells (LEPC) of the partner eye is an established therapeutic strategy to regenerate the damaged corneal surface. However, the long-term outcome of cell transplantation is limited if the tissue structure of the limbal stem cell niche is damaged. In the case of bilateral LSCD, the situation is even more difficult, as the lack of the patient's own cells requires allografting with subsequent immunosuppression and carries a high risk of transplant rejection. Therefore, both the restoration of a healthy niche structure and a source of patient's own cells to colonize the limbal niche are necessary to improve the treatment, especially in patients affected in both eyes. In order to optimize the composition of limbal grafts, it is our goal to further characterize the cell-cell and cell-matrix interactions required for the proper function of the limbal stem cell niche using RNA sequencing, qPCR, Western blot and confocal immunofluorescence microscopy. To investigate the suitability of limbal epithelial cells from human induced pluripotent stem cells (iPSC-LEPC), we will immunohistochemically study iPSC-LEPC grown on decellularized limbal tissue scaffolds. The feasibility and safety of the implantation of repopulated limbal scaffolds are examined in a porcine LSCD model. It is the aim of this project to characterize the contribution of different cells of the limbal niche and the extracellular matrix structure of the limbus to the maintenance of a stem cell phenotype. In addition, the potential suitability of iPSC-LEPC as autologous limbal cells and the feasibility and safety of repopulated limbal constructs will be investigated to further improve the therapeutic options for the treatment of severe LSCD.

• DFG Programme: Research Grants