Human amnion epithelial stem cells as a therapy for liver disease

Abstract: Placenta-derived stem cells have been proposed as potential new treatments for acute and congenital liver diseases. Of all the different perinatal tissues, amnion membrane and isolated amnion epithelial cells have been shown to be an outstanding readily available source of multipotent stem cells. Human amnion epithelial cells (hAEC) have unique properties, including low immunogenicity and immunomodulatory properties, which may allow the first allogenic stem cell therapy without immunosuppression. Animal studies have shown that hAEC differentiate into hepatocyte-like cells and support missing liver functions commonly responsible for inborn errors of metabolism. In the present thesis, we describe early preclinical steps which will likely be necessary to translate hAEC therapy into clinical practice. These steps include detailed and optimized methods for primary hAEC isolation and preservation, methods to validate the final cell product and investigations of the route of infusion for efficient engraftment in the target organ (liver). The efficacy of hAEC transplants was assessed in preclinical models of liver disease. In Project 1, we have detailed the hAEC isolation procedure with GMP reagents, providing a homogenous amnion epithelial cell suspension. The preclinical validation of hAEC-based therapy was continued in Project 2, where 14 different batches of primary hAEC were characterized by immunocytological and biomolecular techniques. The presented findings indicate this technology results in an enriched suspension of epithelial cells with a minimal contamination with mesenchymal, endothelial or hematopoietic cells. In Project 5, we validated the route of infusion of hAEC to reach high level of engraftment in liver. We investigated the bio-distribution of injected DiR-labelled hAEC administered via tail-vein or intra-splenic, and monitored their localization using in vivo live imaging (IVIS) techniques. Twenty-four hours post-splenic infusion, the majority of hAEC was safely delivered and detected in the liver parenchyma. On the contrary, tail-vein infusion resulted in a wide distribution pattern to multiple organs. In Project 3, we have investigated the in vivo engraftment, long-term survival and hepatic maturation of hAEC. We have injected hAEC into a metabolic liver disease model of Phenylketonuria (PKU). This immune-competent PAH-deficient mouse develops a pathological level of phenylalanine (PHE) in the blood, which is commonly observed in PKU patients. We assessed hAEC engrafted into murine liver parenchyma out to 100 days. Such long-term survival resulted in significant correction of blood PHE levels in blood and a statistical complete correction or PHE levels in the brain. The described xeno-transplantation was carried out without any immunosuppressant regimen, and no signs of rejection were noticed. Problems generating clinically relevant results by extrapolation of data from mouse models was also addressed in Project 4, we successfully generated a liver-humanized mouse model that faithfully reproduces the metabolic liver disease observed in patients. We injected hepatocytes isolated from a CPS1 deficient patient into immune-compromised mice (FRGN), where primary human hepatocytes have been previously reported to engraft and fully repopulate the mouse liver. The resultant chimeric CPS1-Deficient (CPS1-D) model exhibited high blood ammonia levels, elevated disease-correlated amino acids (glutamine and glutamate) and low CPS1 enzymatic activity. In conclusion, during the past 4-year study we have successfully analyzed preclinical data and validated the hypothesis that human amnion epithelial cells are useful for the cellular therapy of liver disease, supporting their potential to become a therapeutic tool to treat and support metabolic liver disease patients.