In Vitro derivation of insulin-producing cells from 3D spheroids of human amniotic epithelial cells

Objective: Regenerative medicine and stem cell therapy represent a promising tool for the treatment of non-curable human diseases such as type 1 diabetes. Human amniotic epithelial cells (hAECs) from term placenta have attracted growing interest for their immunological properties, plasticity and availability which make them a promising tool for stem cell-based therapeutic applications. The aim of our study was to culture hAECs in serum-free condition preserving their phenotypic and genetic traits, evaluating their pancreatic differentiative potential in a 3D fashion. Methods: hAECs were isolated and cultured in standard serum-rich medium and serum-free optimized media. Flow Cytometry analysis was performed to evaluate stemness and specific epithelial cells markers. qPCR assessed stem cell and proliferation markers. We established a 3D culture procedure on basement membrane extracts to obtain spheroids mimicking the in vivo morphology and spatial organization of pancreatic islets. Results: The serum free protocol we developed proved to maintain hAECs stemness characteristics and confirmed their immunomodulatory activity on PHA stimulated PBMCs as revealed by proliferation assays. Immunofluorescence revealed the presence of pancreatic endocrine hormones and transmission electron microscopy (TEM) analysis showed a clear membrane-associated organization of secretory granules, consistent with beta cell ultrastructure in vivo. Conclusion: We accordingly propose the outcomes of this study as a novel contribution to the development of a future cell replacement therapy for type 1 diabetes.