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Introduction During chronic liver disease the liver can be regenerated from a population of endogenous hepatic progenitor cells (HPCs). These cells are known to be bipotent and can regenerate both hepatocytes and cholangiocytes, however the mechanisms by which these lineages are delineated from HPCs is currently unknown. Here we describe how the Notch and Wnt signalling pathways are differentially regulated and interact via NUMB to define cholangiocytes and hepatocytes from HPCs. Results We have found that during biliary regeneration in primary sclerosing cholangitis and primary biliary cirrhosis, as well as mouse models of such, the Notch pathway is activated as demonstrated through nuclear Notch-1 in HPCs. This Notch mechanism is mediated through interaction of Jagged-1 supplied by a transient myofibroblast niche in mouse, or autonomous cell signalling in established human disease. In vitro when murine HPCs are co-cultured with myofibroblasts y-secretase inhibition significantly reduced expression of the Notch effectors HeyL (p<0.001 3.133±0.32 vs 0.98±0.09) and Hey1 (p<0.001 1.67±0.34 vs 0.08±0.01); associated with this reduction we found a significant reduction in expression of biliary genes HNF6 (p<0.001 77.94±12.52 vs 14.97±4.57) and GGT (p<0.01 6082±757.70 vs 3169±449.0). In vivo administration of DAPT during murine biliary regeneration demonstrates a significant reduction of cholangiocyte numbers (p<0.05 957.7±114.2 vs 1787±266) and also a reduction in pro-biliary genes including HNF6 and HNF1β. NUMB, a negative regulator of Notch signalling is lost during murine biliary regeneration (p<0.01; 1.19±0.07 vs 0.10±0.02) as well as during PSC/PBC (p<0.01; 8748±3090 vs 1814±277). During regeneration of hepatocytes from HPCs in HCV and in murine models NUMB levels remain high and Notch signalling is suppressed. During murine hepatocyte regeneration NUMB is maintained via macrophage derived Wnt. In vitro activation of the Wnt pathway in HPCs resulted in a twofold induction of NUMB and 35-fold induction of HNF4, without significantly affecting HNF1β. We ablated macrophages using liposomal clodronate this resulted in a conversion of hepatocyte HPCs into biliary HPCs which formed luminal structures with membranous b-catenin, lost expression of Wnt pathway target Axin2, NUMB and HNF4 as well as inducing expression of Hes-1, HNF1β and HNF6. Conclusion During regeneration of the adult liver, Notch signalling is required for biliary specification however is actively sequestered via NUMB during regeneration of hepatocytes. This hepatocyte phenotype is induced through signalling via the Wnt pathway, removal of which results in a biliary phenotype during hepatocyte regeneration. We also describe how this mechanism is conserved from mouse models to human disease, where during biliary diseases such as PSC and PBC the Notch pathway is activated, however is actively restricted during regeneration of in hepatocytes during HCV. [ABSTRACT FROM PUBLISHER]

Introduction Autologous bone marrow cell (BMC) therapy for liver disease has shown increased liver regeneration in animal studies and in phase 1 clinical studies. As yet no clear mechanism accounts for these observations. BMCs do not directly form into hepatocytes but can improve liver regeneration and function. In chronic liver disease hepatic progenitor cells (HPCs) are a potential source of parenchymal regeneration. Whilst the transplantation of human HPCs is not currently a practical therapeutic option, manipulating endogenous HPCs in vivo represents a potential approach. HPCs exist in a specialised niche of leukocytes and mesenchymal cells, which secrete cytokines that are capable of regulating HPC behaviour. We therefore hypothesised that infusion of BMC into the liver may induce HPC expansion via paracrine signalling. Aim To investigate the effect of autologous BMC infusion upon regeneration by hepatic progenitor cells. Method 107 syngenic BMCs were infused into healthy mice by tail vein, we examined intrahepatic donor cell engraftment, cytokine expression, liver function tests, and HPC activation. Cell tracking utilised either GFP+ or male cells delivered into female wild type mice. Whole liver and specific cell fractions were analysed by immunocytochemistry, in-situ hybridisation, FACS, and qRT-PCR. Results Following BMC transfer, a progressive and sustained expansion of HPCs was observed (mean±SEM 41.9±2.1 cells per field vs PBS control 23.5±4.1 at 21 days post infusion, p=0.003) with associated increase in liver/body weight ratio (BMC 0.0513±0.001 vs PBS control 0.0470±0.001, p=0.022). BMCs engrafted the liver adjacent to HPCs for up to 3 weeks and were mostly F4/80+ macrophages (81%). Transfer of F4/80+ macrophages alone into healthy mice recapitulated the HPC expansion. Cytokine gene expression analysis revealed one soluble signal in particular, TWEAK, increased in the recipient's liver following donor BMC engraftment (5.20-fold induction vs control at Day 3). Extracted donor derived BMCs expressed TWEAK, as do macrophages. TWEAK is known to be a direct mitogen to HPCs via the Fn14 receptor. When Fn14-/- mice were used as recipients to wild type BMC infusions the expansion of HPCs was entirely lost (mean±SEM 27.2±1.4 vs negative PBS control 30.6±2.4, p=0.128 vs positive wild-type control 42.7±2.3, p=0.0002), demonstrating that the HPC expansion is dependent on TWEAK-Fn14 paracrine signalling between BMCs and HPCs. Conclusion These data describe a hitherto unknown mechanism by which infused autologous macrophages signal in a paracrine manner to HPCs via TWEAK. These observations suggest potential for the development of novel therapies to promote human liver regeneration. [ABSTRACT FROM PUBLISHER]