The research of suitable csenogenic cell source for lacrimal gland tissue engineering

Purpose: Investigation of appropriate xenogenic cell source for in vitro lacrimal gland development by using tissue engineering methods, for use in the treatment of dry eye disease. Clinical relevance: Aqueous deficiency type dry eye disease, which is mostly seen in the setting of autoimmune diseases, leads to serious ocular surface disorders and possible loss of vision. Available options like topical artificial tears, immunosuppressive agents, punctal plugs etc. are insufficient in treatment. Development of an artificial lacrimal gland using stem/progenitor cells localized in patients' lacrimal gland or pluripotent stem cells formed by reprogramming skin fibroblast cells, brings a new perspective to the treatment of dry eye. Method: This study was conducted in two areas: animal experiments and stem cell experiments. Initially, lacrimal gland tissue was dissected and isolated from adult and embryonic mice. Histological, immunohistochemical and flow cytometry analyses were performed. Harvested cells were cultured in a 'lacrimal chip' system that mimics the morphogenesis of the lacrimal gland with its epithelial and mesenchymal compartments as well as a dynamic culture medium, for 14 days. Results were analyzed by immunohistochemistry and qPCR methods. Secondly, induced pluripotent stem cells (IPSCs) were differentiated into the main components of the lacrimal gland, namely conjunctival epithelial cells and periocular mesenchymal cells, by using two different protocols. Results were analyzed by immunohistochemistry. Results: Lacrimal gland cells obtained from adult mice showed low proliferation capacity. Tissues isolated from embryonic mice could be separated into epithelial and mesenchymal tissues under light microscope, verified by the histologic analysis. Cells were mixed with gelatin methacryloyl (GelMA) which acts as tissue scaffold, and then photomasked into the 'lacrimal chip' system that has inner epithelium and outer mesenchymal tissue compartments. Cells were found to be bio-compatible with the gel and were able to maintain their viability for 14 days. They formed appropriate organoid structure. Immunohistochemical analysis revealed lacrimal gland specific Aquaporin-5 (AQP-5) and Zonula occludens-1 (ZO-1) proteins. Tear-specific lysozyme mRNA levels were found to be significantly higher in qPCR analysis than in the control group. IPSCs experiment revealed ocular surface ectodermal cells initially, followed by cells that stained by K13, which is an indicator of conjunctival epithelium, at the end of 42 days. On the 14th day of culture, immunohistochemistry analysis revealed FOXC1; qPCR analysis revealed PITX2 and LMX1B, all of which are mesenchymal markers. Conclusion: Lacrimal gland tissue was produced in in vitro conditions, by tissue engineering methods using embryonic mouse cells. IPSCs were used to obtain epithelial and mesenchymal cells to be used in bio-engineering studies to develop lacrimal gland tissue. In the light of these data, obtaining in vitro lacrimal gland tissue using stem/progenitor cells isolated from either the lacrimal gland or IPSCs derived from skin fibroblasts at the initial stages of dry eye disease and autologous transplantation to the patient might be a sound approach to reduce the risk of serious ocular surface disorders and possible loss of vision in later stages of the disease. Both methods might eliminate ethical problems and risk of immune responses. Furthermore, it is possible that aqueous tear, which has complex ingredients, to be custom-produced individually for topical use in the treatment of dry eye disease, from the lacrimal gland developed in in vitro conditions by using stem cells.