Introduction: Liver diseases are associated with increased mortality and morbidity, with organ or hepatocyte transplantations as the sole treatment limited by the shortage of donors. Over the years, several groups/companies have proposed stem cells as an alternative solution, although limited by hepatic maturation capacity. Amnion epithelial stem cells (AESC), isolated from the full-term human placentae, have been proven able to mature into hepatocyte-like cells, rescuing preclinical models of congenital disorders or acute liver failure. Human AESCs represent a promising source of multipotent cells, supporting both regenerative strategies and drug development, if their hepatic maturation can be achieved ex vivo. Decellularized Liver extracellular matrix (dLECM) proteins and 3D architecture play a crucial role in supporting hepatic maturation. Thus, we combined the multipotency ability offered by human AESC seeded into rat dLECM constructs, and evaluated cell differentiation and functional properties in comparison with human fetal and adult hepatocytes.

Methods: Fifty million human AESC were seeded into decellularized rat liver scaffolds and exposed for 40 days to repeated infusions or to endure perfusion of the hepatogenic medium. Initial mitogenic stimuli were replaced by hepatogenic culture conditions, and hepatic metabolism and synthetic activities were monitored at different time points. NMR, ELISA, and fluorescent probes were used to monitor hepatic maturation, and results were compared to primary human adult hepatocytes or fetal hepatoblasts. Transcriptome and proteomic analysis at intermediate and final time points supported functional analysis.

Results: human AESCs were positive for epithelial markers but negative for hepatic enzymes. Once engrafted in rat dLECM, human AESC expressed some level of hepatic maturation within 2 weeks (AFP and Alb). Such hepatic maturation was maintained and enhanced in hepatic maturation medium, resulting in CK18+ hepatocyte-like cells, characterized by secretive and metabolic protein. Albumin and urea production increased in dynamic rather than static conditions, and NMR showed a shift in metabolite production. Transcriptome analysis expanded evaluation for hepatic characteristics in bioengineered liver tissue.

Conclusion: dLECM-scaffold supports the maturation of human AESC in a 3D-whole liver model. Continuous perfusion resulted in remarkable cell distribution and hepato-specific activities compared to static conditions. The bioreactor technology provides enhanced distribution of oxygen and nutrients, leading to a more physiological condition in support of maturation and hepatic metabolism. Encouraged by safety and immune privilege characteristics of perinatal AESC, such technology may represent an effective method to evaluate hepatogenic capacities and provide functional bioartificial tissues instrumental for drug development as well as resolutive clinical applications