Nucleoporated amniotic fluid cells retain their stemness in in vitro and in vivo experimental models

In regenerative medicine, stem cells are a useful tool for repairing the functionality of damaged tissues and organs. Recently, scientists have given special attention to amniotic derived cells, because these cells have demonstrated the expression of stemness markers and a high degree of plasticity. Other important biological characteristics are low immunogenicity and immunoregulatory features observed in vitro and in vivo, which together allow the use of these cells for allo/xenografts. Amniotic cells have also anti-inflammatory, antimicrobial, antifibroblastic features, do not show any evidence of tumor formation as, in contrast, embryonic stem cells, which could represent, for their high plasticity, alternative therapeutic approaches targeted at different tissues. However, pre-clinical studies are still necessary to demonstrate the regenerative capacity of amniotic stem cells to document the mechanisms and to ensure the long-term safety of the treatment. In this context, it becomes relevant the animal model, which should have morpho-functional characteristics similar to human. For this reason the sheep can be considered an optimal model for studying bone, skeletal muscle or tendon diseases. To assess the regenerative effect of transferred stem cells into a pathological or experimental damaged tissue, it is necessary to have cells which express a marker, such as the Green Fluorescent Protein (GFP), that allows their identification after transplantation. The aim of this study was to verify in vitro and in vivo the stemness properties of ovine amniotic fluid stem cells (ovine amniotic stem cells, oAFSCs), transfected with the eGFP reporter gene using a nucleofection protocol validated for human mesenchymal cells. The oAFSCs after nucleofection showed a high proliferative index, a normal karyotype and stable expression of surface antigens CD49f, CD166 and CD29 and stem cell markers Oct4, Sox2, Nanog and TERT. These cells also retained the capacity to differentiate into osteogenic lineage, as confirmed by Alizarin Red staining and alkaline phosphatase assay. Finally, it was evaluated the in vivo regenerative potential of nucleoporated oAFSCs allotransplanting these cells in a sheep experimentally injured tendon. After 30 days of transplantation, immunohistochemical analysis revealed a major reorganization of the allografted tissue compared to the control one. The treated tissue showed abundant collagen fibers oriented parallel to the longitudinal axis of the tendon, and in particular immature fibers (COLIII), present during tissue regeneration, were completely replaced by mature ones (COLI). Cell transplantation stimulated a rapid vascular reorganization. The GFP+-oAFSCs were found within the injured tissue and some showed a fusiform morphology and were trapped in the newly deposited matrix. Finally, biomechanical tests demonstrated that eGFP+-AFSC treated tendons rapidly improved the recovery of their mechanical properties. Thus, confirming that allotransplanted oAFSCs can be used to improve tendon healing and opens the way for a potential translational use of these cells in regenerative medicine.[...]