Liver Stem Cell Niche

Abstract

Liver progenitor cells are able to differentiate into either hepatocytic or cholangiocytic cell types. Recreating the environment found in developing fetal liver allows us to expand these cells in vivo. 16 weeks human fetal liver were treated with standard collagenase digestion. Cells were sorted using anti-EpCAM-conjugated magnetic beads. Cultures were performed on plates comparing various extracellular matrices. Cultures were passaged at confluence and immunophenotyped using RT-PCR and immunofluorescent staining. Proliferation was measured with Alamar Blue. Fetal epithelial progenitor cells are EpCAM+ and CD44+. They were isolated with 94% purity and maintained in culture beyond 50 days. Immunophenotyping confirmed maintenance of phenotype and cell markers. Testing of extracellular matrix showed that combination of collagen, laminin and hyaluronic acid were most optimal in maintaining adhesion, proliferation and phenotype. Fibroblast growth factor and Epidermal growth factor were critical to these cells. Expanded cells differentiated into albumin-producing hepatocytes when cultured on collagen and bile ducts when cultured on Hyaluronic acid. In contrast, cells deteriorated in Heparan sulphate, favouring selection of mesenchymal cells. A 3-D culture system with cell-cell contact and polarity enhanced the culture of these cells. In-vivo transplantation of these cells showed engraftment and survival. Extracellular matrix plays a critical role in driving liver progenitor cell fate and proliferation. EpCAM+ sorted cells are able to expand more than 100-fold while still maintaining their EpCAM and CD44 markers. These cells therefore still maintained bipotentiality and are a promising cell source for therapies in liver failure.