Abstract
The liver is one of the most vital organs in the human body. Whole-organ transplantation is the only established treatment for liver failure. Thousands die before a suitable organ is found. Hepatocytes comprise over 70% of the liver’s mass and carry out most of its specialized functions. The liver’s ability to regenerate itself from as little as a third of its undamaged mass through proliferation of its remaining hepatocytes has spurred interest in hepatocyte transplantation to failing livers, but hepatocyte acquisition competes with whole-organ procurement. Mature hepatocytes do not proliferate in vitro, and tend to lose their liver-specific characteristics. Human embryonic stem cells (hESCs), capable of self-renewal and able to give rise to most other cell types, could potentially provide an unlimited supply of hepatocytes. Through careful monitoring and passaging, a number of hESC lines have been generated. Early hESC differentiation methods led to the formation of embryoid bodies (EB), containing a low percentage of cells expressing hepatic proteins. Differentiating hESCs first to definitive endoderm before progressing them towards hepatocytes has resulted in the generation of hepatocyte-like populations with over 90% of the cells expressing mature hepatocyte properties. Cultured cells are often visually evaluated for morphology, or by immunohistochemistry (IHC) for stage- or tissue-specific markers, such as Oct4 in hESCs or albumin for hepatocytes. But the ability to function equivocally to in vivo counterparts or functionality of vital cellular structures like mitochondria, which produce ATP, may also be measured. hESCs for use in human cell therapy, must be of high quality to ensure genetic stability, and derived under xeno-free culture conditions to eliminate risk of transmission of animal pathogens or immune reactions to animal proteins in human patients. Commonly, hESCs are maintained in culture on a layer of mouse embryonic fibroblasts (MEFs). Alternatives to MEFs may still contain animal products or are more expensive. Six clinical-grade hESC lines have recently been developed and are available to researchers. In this study, lines ESI-017 and EST-035 were found to proliferate well on MEFs and Matrigel at early passage, similar to H9, but acquired genetic mutation beyond passage 60 and demonstrated decreased ability to differentiate towards hepatocytes with increasing passage number and do not appear to be able to match the hepatic functionality of H9-derived-hepatocytes nor primary hepatocytes.