The hepatic progenitor cell niche: under experimental conditions and in human liver disease

The hepatic progenitor cells (HPCs) are the stem cell-like cells of the liver. They are small, situated in the canals of Hering, and are characterized by a relative large oval nucleus and scanty cytoplasm. HPCs express both markers of (immature) hepatocytes (e.g. alpha-fetoprotein) and markers of cholangiocytes (e.g. cytokeratin K7 and K19) and can differentiate into cholangiocytes or hepatocytes. Nevertheless, the mechanisms behind proliferation and differentiation of human HPCs are still poorly understood. More knowledge is needed in order to identify the therapeutic potential of HPCs in chronic liver disease and liver cancer.

The aim of this project was to study the HPCs and their niche in human liver diseases with the focus on understanding the activation and differentiation of hepatic progenitor cells in human liver failure and their role in carcinogenesis.

The major part of this dissertation comprised the isolation of HPC-enriched cell populations from adult human liver tissue using different isolation methods and the comparison of these populations on both protein and RNA level. We collected fresh human liver tissue from alcoholic steatohepatitis (ASH) explant livers and isolated the HPC enriched populations via three different methods. The Side Population (SP) method is based on the efflux capacities of the progenitor cells of the fluorescent DNA-binding dye Hoechst-33342. The expression of EpCAM and TROP-2, two membrane markers, were evaluated in the alcoholic liver samples and tested to be used to isolate HPCs. Comparison of TROP-2 and EpCAM expression with that of K19, a known and validated HPC marker, revealed a similar pattern and thus expression on the membranes of HPCs. Via fluorescent activated cell sorting (FACS) method we isolated the SP, EpCAM+ and TROP-2+ populations. Marker analysis showed the enrichment of HPCs in the isolated populations and the relative absence of other cell types. Comparing these HPC-enriched populations with whole liver extracts and performing pathway analysis after high-throughput RNA sequencing, revealed the enrichment and activation of known HPC pathways mainly from animal models like Wnt/beta-catenin, HGF, FGF and TWEAK signalling. Signals, like TWEAK and FGF7, triggering these pathways in HPCs could be found in their close neighbourhood when we isolated HPCs with their niche via laser capture microdissection (LMD). Moreover, analysing the different HPC-enriched groups revealed potential new upstream regulators like the cytokines and growth factors TNFα, PDGFB, VEGFA, MIF and IGF-1. These factors have not been described yet to be linked with HPCs but have been found to be involved among others in maintaining stemness, cell survival and self-renewal of mesenchymal stem cells. Further pathway analysis indicated the possible importance of the IL-17A signalling for HPCs and liver disease. Together with the HPC expression of immune cell chemoattractant genes, these data suggest that HPCs could actively contribute to the liver inflammation.

In another part of this dissertation, we aimed to characterize the cancer stem cell (CSC) niche in different subtypes of hepatic carcinomas. We isolated the SP of K19-negative, K19-positive and mixed phenotypes of human hepatocellular carcinomas. The size of the SP correlated with the degree of HPC features of the tumours and showed an elevated expression of HPC markers. The detected elevated expression of the extracellular matrix marker LAMC2, coding for the laminin γ2-chain, could be linked with increased K19-expression, elevated chemo-resistance and reduced tumour growth. This was demonstrated in vitro as well as in vivo.

Thus, We provided the first RNA-sequencing-based, comparative transcriptome analysis of isolated human HPCs from ASH patients and revealed active signalling between HPCs and their surrounding niche cells, suggesting that HPCs could actively contribute to the liver inflammation. Still in vitro/in vivo functional assays are required to sustain this hypothesis. We also identified a prominent role for laminin-332 as part of the specialized CSC niche in maintaining and supporting cell stemness, which leads to chemoresistance and quiescence of liver tumours.