The establishment and optimization of functional human hepatocytes derived from postnatal progenitor/stem cells: a novel in vitro model for early preclinical testing during drug development.

1) AIMS
At present, risk assessment of new chemical entities (NCEs) for human use is usually accomplished by performing in vivo experiments on living animals. Extrapolation problems from animals to humans, time and resources consumption, ethical considerations, the increasing number of safety criteria imposed, as well as the progressive number of newly designed molecules, have prompted the development of in vitro assays for high throughput preclinical screening purposes. Utilizing these in vitro models early during development enable pharmaceutical companies to reduce clinical failure rates. As the liver is the principal organ for xenobiotic biotransformation and failures of lead candidates are predominantly due to induced liver toxicity (1), in vitro models to predict drug metabolism are preferably derived from healthy human liver tissues. Several in vitro models based on primary hepatocytes are available. However, their use is currently limited by the progressive occurrence of dedifferentiation, including limited survival and loss of liver-specific functions. (http://minf.ac.be/~fafy). In addition, the availability of fresh and healthy human tissue is scarce. Given the self renewing and multipotent capacity of postnatal stem cells, including the production of functional hepatocyte-like cells, human stem cell based strategies might provide a potential alternative. In this context, the group FAFY is not only specialized in the development of in vitro models based on primary hepatocytes but has also built great expertise in the establishment of culture systems derived of postnatal stem cells (http://minf.ac.be/~fafy). In previous research, performed by the applicant in the framework of obtaining a PhD degree in pharmaceutical sciences, hepatic differentiation of both rat liver epithelial cells of biliary origin (RLEC) and bone marrow stem cells, in specific rat multipotent adult progenitor cells (MAPCs) and human mesenchymal stem cells (MSC), was obtained upon sequential induction of the differentiation process reflecting the embryonic liver development in vivo. In particular, it was shown that sequential exposure of the bone marrow stem cells to fibroblast growth factor 4 (FGF4), hepatocyte growth factor (HGF) and finally HGF, insulin and dexamethasone significantly promoted the mesenchymal to hepatic conversion and eventually resulted in a more homogenous population of hepatic cells in comparison to traditionally applied simultaneous exposure to a mixture of these factors. Histone remodelling by the histone deacetylase (HDAC) inhibitor trichostatin A (TSA) synergistically stimulated hepatic differentiation and maturation as assessed by improved expression of hepatic phenotype and functional maturation. In the postdoc project, more easily accessible and readily available sources than bone marrow (such as human adipose tissue and skin) will also be explored. These sources have the additional advantage that they may be obtained of volunteers of all ages.
Still, current strategies to isolate, select, purify, expand and differentiate stem cells lack standardisation. In particular, the imprecise definition of starting cell preparations remains a key obstacle for in vitro purposes of stem cells. As such, haematopoietic, mesenchymal and liver stem cells lack well defined characterization, hampering easy and reproducible isolation. The heterogeneity of the initial population for most of the transdifferentiation experiments hinders accurate interpretation and reciprocal comparison of results between different groups. Variations in procedures might in some cases even explain nonreproducibility or discrepancies between results, reported thusfar. Standardisation is thus no doubt a conditio sine qua non for future in vitro toxicity testing and clinical application. In a first part of the postdoc project, we will search for suitable markers to isolate, select and purify defined populations of plastic MSC, preferably predestined to differentiate along the hepatic lineage. In addition, the molecular signalling pathways, committing the transition of stem cells into hepatocytes, are at present not elucidated. Therefore, the second part of the project will be focused on unravelling the mechanism underlying the transdifferentiation processes. Based on the results obtained, stem cells will be transfected with selected transcription factor genes in order to initiate the cellular signalling pathway for hepatic differentiation and improve the hepatic differentiation efficacy and competence in vitro. As final end-point of the project, it will be investigated whether transplanted human hepatic cells, derived from MSC, differentiate in vivo into functional resident liver cells. The obtained in vivo data might deliver valuable information with regard to the clinical potential of stem cell based hepatic cells.

2) OBJECTIVES
The development and mechanistic characterization of a novel standardized in vitro model of human hepatocytes, based on postnatal progenitor/stem cells, for application in early preclinical safety studies of drugs.