POLYMER LIBRARY SCREENING IDENTIFIES AN EXTRACELLULAR MATRIX THAT PROMOTES AND STABILISES HUMAN EMBRYONIC STEM CELL-DERIVED HEPATOCYTE FUNCTION

Background: The generation of functional hepatic endoderm from human embryonic stem cells (hESCs) offers a stable and inexhaustible resource vital for biotechnology, toxicology and clinical applications. Models of hepatocyte differentiation have recently been developed, but long-term hepatocyte function following cell passaging has proven elusive. Aim: In an attempt to identify a defined extracellular matrix that promotes long term hepatocyte function we screened a polymer library for suitable candidates. Results: Human-ESCs were differentiated to hepatocyte like cells in vitro. Upon adopting an hepatic fate (Day 9), HLCs were detached from their biological extracellular matrix and replated onto the polymer library. The array consisted of 380 polymers printed in quadruplicate onto a glass microscope slide. Primary screening identified 6 polymers (three polyacrylates 2AE7, 7G7, 3AA7 and three polyurethanes 134, 212, 223) that supported HLC attachment and identity. We further characterised hepatocytic function in detail on these six matrices. Using this strategy, polymer 134 was identified as the most effective cellular support associated with enhanced expression of Fibrinogen, transthyretin (TTR) and soluble fibronectin. Key cytochrome p450 (CYP) activities on the different extracellular matrices were also characterised. CYP1A2 activity was increased ~6 fold and CYP3A4 ~2 fold on polymer 134 as compared to standard matrigel conditions or the other polymers tested. Conclusion: In conclusion, the high through-put screening of a polymer microarray allowed the identification of a new polymer matrix that promotes long-term hepatocellular differentiated function before and after passaging. These attributes bypass current limitations associated adult uman hepatocytes, can be manufactured to GMP standards and will play important roles in developing in vitro models of drug toxicicology, provide a resource for the construction of extra-corporeal devices and facilitate novel studies of human liver development and disease.