PEX1 p.G844D +/+ NMRI mouse: a robust pre-clinical model for mild Zellweger spectrum disorder

Zellweger spectrum disorders (ZSD) form a clinical spectrum of diseases due to a peroxisome biogenesis disorder attributable to mutations in PEX genes. ZSD has an incidence of 1:50.000 live-births. Characteristically elevated levels of phytanic, pristanic, pipecolic, and very long-chain fatty acids, and abnormal bile acid intermediates are observed in the plasma of affected children. Clinical presentation ranges from liver failure and death in infancy to chronic liver disease, neurodevelopmental delay, deafness and blindness among those who survive beyond the first decade of life (mild ZSD). No routine treatment has been shown to arrest or reverse the natural history of this terrible disease. Yet, we previously demonstrated the benefit of liver transplantation or hepatocyte transplantation (HT) on biochemical and clinical outcome in 4 patients with mild ZSD. The aim of this study was to validate a mouse model homozygous for the PEX1 p.G844D hypomorphic mutation as a pre-clinical model for mild ZSD treatment evaluation. We started a colony using 6 PEX1 p.G844D +/- C57BL/6N mice from Jackson Laboratory; +/+ mice (ZSD) are sterile and die within one month. We backcrossed three times the mutation in a NMRI background to get larger litters and more robust homozygous mice. Mice growth and food intake were regularly quantified. We measured mice glycemia before and after a 6 hours fasting test. Mice livers collected at 2 months of age were weighted, fixed, paraffin embedded and stained with periodic-acid Schiff (PAS) ± diastase (as a proxy for glycogen content). ZSD mice exhibited (1) a severe growth retardation associated to an increased food intake/g body weight, (2) lower fed blood glucose levels and (3) a worse fasting resistance. PAS staining on liver sections was severely reduced in fed ZSD mice meaning a low glycogen content. This could be one explanation for their reduced fasting resistance. Scarce peroxisomal ghosts where shown in ZSD mice livers by immunofluorescence staining of the 70-kDa peroxisome membrane protein (PMP70). All the above mentioned classical ZSD metabolites were elevated in ZSD mice compared to their WT siblings. In conclusion, PEX1 p.G844D +/+ NMRI mice are a good and robust pre-clinical model recapitulating liver involvement and growth retardation of mild ZSD. We are currently evaluating HT as a potential therapeutic approach in this model

Production and characterization of human liver extracellular matrix hydrogels for in vitro culture of distinct human primary liver cell populations

Background and Aims: The study of human primary liver cell biology is largely hampered by the lack of adequate in vitro culture systems that allow the maintenance of the cells’ viability and physiologic phenotype for prolonged periods of time. In this context, a growing interest for liver decellularization and for the use of liver extracellular matrix (ECM) in cell culture and tissue engineering applications emerged over the past five years. Whereas several publications describe detailed procedures for animal liver decellularization and ECM-based hydrogel production, few is known about the composition and properties of isolated human liver ECM. In this study, we analyzed the composition of human liver ECM and derived hydrogels, and we further addressed the question of the potential benefits of a liver ECM protein-coated surface for the culture of primary human liver cells. Method: A combination of thermic and osmotic shock, enzymatic digestion and detergents has been used to decellularize human liver fragments unsuitable for transplantation. Liver ECM was partially digested with pepsin for coating purposes and characterized by histology and mass spectrometry. Primary human liver cells, including hepatocytes, hepatic stellate cells (HSC) and endothelial cells (EC) were seeded on liver-coated culture dishes. Adhesion (2h), viability (24h), proliferation (7 days) and phenotypic properties were evaluated and compared to a surface of reference (CellBIND or rat tail collagen coating). Results: Our protocol led to a residual amount dsDNA below decellularization standards. Coating derived from liver ECM showed no toxicity for primary human liver cells. Mass spectrometry revealed the abundance of type I and type III fibrillar collagen components (COL1A1, COL1A2, COL3A1) as well as elastin in liver ECM hydrogels used for coating. Preliminary results further suggest the benefits of human liver ECM on human primary liver cell properties in 2D culture as compared to surfaces of reference. Conclusion: Our study provides a first detailed characterization of human liver ECM and derived hydrogels, easily and reproducibly isolated from small human liver fragments. Our results further open the way for the use of human liver ECM as a coating for the culture of primary human liver cells in 2D. In a near future, 3D applications will be developed