Hepatic and Biliary Differentiation of Somatic Cells

In the human body, the liver plays an important role in a variety of functions from protein and bile production to carbohydrate, cholesterol and mineral metabolism, detoxification and storage. Although already recognized by the Greeks in the ancient myth of Prometheus that the liver has powerful capacity to regenerate itself, in diseased conditions this regeneration can be severely hampered or even completely abolished. For end-stage liver disease, organ transplantation is still the most effective treatment so far. However, due to the lack of donor livers many patients die while on the waiting list. An alternative to donor organs is therefore urgently needed. The principal aim of this thesis is to generate functional cells of the liver, which can ultimately provide the possibility to fabricate the whole organ in future. As the largest internal organ in the human body, the liver possesses a complex architecture to support its massive metabolism capacity. The smallest functional units in the liver are hepatic lobules, each of which consists of a central vein surrounded by plates of cells. These cells are generally derived from two sources. During early liver development, embryonic pluripotent stem cells generate an endoderm germ layer followed by foregut formation and then differentiate into hepatoblasts. These hepatoblasts are bi-potent somatic stem cells which can further differentiate towards either hepatocytes or cholangiocytes. Hepatocytes and cholangiocytes are the two main epithelial cell types performing most liver functions. Other supporting cell types in the liver are mainly derived from mesoderm. In light of the complexity of the liver, in Chapter 1, we systematically review the key factors directing the differentiation of somatic (liver) stem cells and the maintenance of mature functions of the liver. We propose possible strategies applying these key factors to generate functionally mature hepatocytes in vitro and discuss how to characterize the resulting cells. In Chapter 2, we describe a method to generate hepatocyte-like cells from somatic cells through a technique called direct reprogramming. We use native extracellular matrix to induce the drug metabolism capacity of hepatocyte-like cells. Although attempts have been made, the resulting hepatocyte-like cells still exhibit limited hepatic maturation. Therefore, in Chapter 3, in a dedifferentiating hepatocyte model, we describe a screening based on epigenetic modification and transcriptional analysis to identify novel transcription factors affecting this procedure. To make sure we had fully matured hepatocytes, we started with freshly isolated hepatocytes that dedifferentiate while in culture. We further apply this finding in inducing the terminal maturation of hepatocyte-like cells. Cholangiocytes are the epithelial cells forming the intra- and extra-hepatic bile ducts. Biliary excretion is the main route to eliminate waste products and toxins from the liver. In Chapter 4, we establish a protocol to generate functional cholangiocyte-like cells from somatic liver stem cells. We also prove that these cholangiocyte-like cells can form artificial bile ducts which possess transportation capacity. The findings and overall conclusion of these studies are summarized and discussed in Chapter 5 and 6