Cultivation and characterisation of human adult liver progenitor cells from patient samples

Aims: Our work centres around human adult liver progenitor cells isolated from primary human hepatocytes received from our clinical cooperation partner at MHH. Hepatocyte samples were isolated by a two-step collagenase perfusion treatment of liver biopsies obtained during liver surgery and cultivated on Collagen-coated culture plates.

Here we describe in more detail our first progenitor cell population obtained from one of these patient samples. Currently we are in the process of repeating the successful progenitor enrichment protocol with various independent patient samples.

Results: After successful enrichment and purification of progenitor cells using a highly modified „plate-and-wait“ method, cells were kept in culture for almost two months before freezing for Nitrogen-storage. They can be passaged at low ratios and show no visible signs of senescence. In particular during the first days after passaging before reaching confluency, the cells exhibit stunning morphologic plasticity and sensitivity to the slightest changes of culture conditions, strongly indicating an undifferentiated stem cell phenotype. While their growth is significantly slower after thawing, they are able to recover to confluency when cultured under appropriate conditions.

We use a specific panel of numerous calibrated RT-qPCR primers that provide a reliable overall phenotypic picture by showing the expression of genes specific to hepatocyte-related metabolism, transcription, secretion, or maturation state. Thanks to this panel, we are able to thoroughly compare liver cell types. When comparing our progenitor like cell population to primary hepatocytes kept in culture for up to 168 hours, the most striking differences arise in the strong expression of EpCAM, which is upregulated in the progenitor cell population, while on the other hand, the expression of Albumin and most liver metabolic markers is greatly decreased but measurable in this cell type. Also, AFP expression is barely detectable. Wnt2, Wnt10b and CK19 are also upregulated in progenitor cells, whereas in particular basal levels of liver enriched transcription factors are largely similar to primary hepatocyte isolates.

Outlook: Isolating progenitor cell populations from more independent patient samples is our main current goal. Simultaneously the thorough characterisation of the existing populations under specific culture conditions by the means of RT-qPCR and Immunohistochemistry are ongoing. The evalulation and exploitaion of their differentiation/maturation potential, mainly towards hepatic and cholangiocytic phenotypes, are also being carried out. Functional competence will also be verified by cell transplantation into our immunodeficient mouse model (ongoing).