Harnessing Amniotic-Derived Cells for Advanced Regenerative Medicine and Tracking Technologies.

Regenerative medicine using cell therapy is emerging as a potential strategy to address several issues in the field of reproduction. Transplantation of different types of adult and fetal mesenchymal stromal cells (MSCs) have been tested with promising results for treating both female (e.g. dysfunctional endometrium, premature ovarian failure, ovarian aging) and male (e.g. azoospermia, aspermia, oligospermia) conditions. Despite recent advances in this field of clinical medicine, the existing challenges are still enormous. Also, immunogenic, and tumorigenic constraints limit the clinical translation of the current research protocols. Amniotic-derived cells, due to their plasticity, immunomodulatory effects, and homing capabilities, present an alternative cell source with great regenerative potential for reproductive medicine. This study has the aim to present a technological advancement of the current protocols of cell tracking to combine with tissue transplantation to offer a straightforward and non-invasive approach to track and drive regenerative processes. The research develops an effective protocol to label amniotic-derived cells tagged with gold nanorods (AuNRs) to be applied for real-time visualization of the cells' engraftment, survival, and integration into the target tissue, thus exploring the pro-regenerative properties of this placental stem cell source widespread used to date for biomedical regenerative applications even if not exploited with tracking technologies yet. The study utilized as cell model, ovine amniotic epithelial cells (AECs) in their native epithelial state or induced mesenchymal phenotype (mAEC) through epithelial-mesenchymal transition (EMT) in vitro. The first aim was to assess the efficacy of various uptake methods validated for MSCs on mAEC. The standardized protocol was then implemented by introducing the innovative biological strategy of mitoception to enhance endocytosis before translating the optimized methods to ovine and human AECs. The findings reveal that the most effective, cost-reducing, and straightforward protocol for AuNRs internalization in living mAEC involved the combination of single-step uptake conditions (cells in suspension) with centrifugation-mediated internalization (G-force), and mitoception (mitochondria isolated from mAEC). This novel protocol resulted in high AuNRs internalization, providing labeled vital mAECs within minutes for a prompt use in both preclinical and clinical trials. The optimized protocol could potentially yield up to 10 million labeled amniotic-derived cells from a single amniotic membrane for biomedical purposes. Similar or even greater efficiency was observed when applying the protocol to both ovine and human AECs, demonstrating its transferability across different cell phenotypes and species. This validates its significant potential for advancing biomedical applications in cell-based therapy and diagnostic imaging providing great opportunity to tackle infertility challenges.