Investigating the liver progenitor cell niche in the developing human liver

Abstract

Liver cirrhosis places an increasing burden on healthcare worldwide. Currently the only treatment is liver transplantation. Whilst liver transplant has a relatively good five-year survival, donor organ shortage costs many lives every year and results in lifelong immunosuppression. Alternative treatments are thus urgently needed. It is with this background that there is understandable interest for the development of stem cell therapies for liver regeneration. The identification of putative liver stem cells has brought closer the previously separate fields of liver ontology, regeneration, and carcinogenesis. Significant overlaps in the regulation of these processes are now being described. For example, studies in embryonic liver development have already provided the basis for directed differentiation of human embryonic stem cells and induced pluripotent stem cells into hepatocyte-like cells. As a result, the understanding of the cell biology of proliferation and differentiation in the liver has been improved. This knowledge can be used to improve the function of hepatocyte-like cells for drug testing, bio-artificial livers, and transplantation. In parallel, the mechanisms regulating cancer cell biology are now clearer, providing fertile soil for novel therapeutic approaches. Recognition of the relationships between development, regeneration, and carcinogenesis, and the increasing evidence for the role of stem cells in all of these areas, has sparked fresh enthusiasm in understanding the underlying molecular mechanisms and has led to new targeted therapies for liver cirrhosis and primary liver cancers. Human liver progenitor cells (LPCs) have therapeutic potential but their in vitro culture results in inadequate differentiation, function, and phenotypic instability reflecting an incomplete understanding of in vivo processes. LPCs can be robustly isolated from second trimester human foetal livers by immunoselection for EpCAM+/CD29+/CD49d+/CD49e–/CD235a–/CD45– cells. Expression profiling of mRNA and microRNA in human foetal LPCs was performed and compared with mature human hepatocytes and human embryonic stem cells undergoing hepatocytic differentiation. Foetal LPCs exhibit a distinct transcriptome profile consistent with a stem cell signature, cell division, and some liver-specific functions. Bioinformatic integration of microRNA and mRNA datasets revealed that microRNAs up-regulated in LPCs targeted genes involved in metabolic processes implying repression of the mature hepatocyte phenotype. Control of LPC gene expression therefore occurs at both transcriptional and, via microRNAs, post-transcriptional levels. Furthermore, transcription factor binding site analyses revealed enriched E2F1 motif in gene and microRNA promoters suggesting feedback control in determining LPC fate. Foetal LPCs were capable of differentiation to a hepatocytic phenotype in the presence of appropriate paracrine signals provided by EpCAM– non-parenchymal cells (NPCs), which consist mainly of endothelial cells and hepatic stellate cells. Fibronectin, despite being produced in abundance by EpCAM– NPCs, had no effect on LPC synthetic function in vitro. The expression of fibronectin in the perisinusoidal space suggests its potential role of modulating cross-talk between hepatoblasts/hepatocytes, liver sinusoidal endothelial cells, and hepatic stellate cells. Fibronectin expression in the portal vein mesenchyme and laminin α5 expression along the ductal plate suggest that both matrix molecules, located in close proximity to LPCs, may be important in supporting the LPC niche. Findings in this work provide insight into the regulation of the human foetal LPC functional phenotype, bringing stem cell-based therapies for liver disease one step closer.