Liver injury stimulates hepatocyte proliferation, regenerating the liver through self-replication. In cases where there is severe, repetitive, parenchymal damage, as seen in human chronic liver disease, hepatocyte mediated regeneration becomes impaired. In this setting it is currently unclear whether endogenous biliary epithelial cells can repopulate the hepatocyte compartment. This thesis therefore aimed to address this point by lineage tracing the main two liver epithelia populations on a background of impaired hepatocyte regeneration. To impair regeneration, an Itgb1 transgene was specifically deleted, conditionally, from the hepatocyte epithelium. Long-term loss of β1-Integrin alone or with additional injury caused an epithelial ductular reaction of biliary origin. Alongside β1-Integrin ablation, the hepatocyte epithelium was also labelled with a heritable ROSA26LSLtdTomato reporter. Impaired hepatocyte regeneration mediated by β1- integrin ablation resulted in 25% of hepatocytes becoming tdTomato negative (non-hepatocyte derived). To verify that the non-hepatocyte mediated regeneration was originating from the biliary epithelium, anti-Itgb1 RNAi was administrated to K19CreERT LSLtdTomato mice. Resulting in tdTomato positive hepatocytes that had differentiated from the labelled tdTomato positive biliary epithelial cells. In summary, this thesis demonstrates that hepatocyte β1-Integrin ablation combined with toxic damage causes marked ductular reactions and results in a substantial regeneration of functional hepatocytes from the biliary epithelium.

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Chronic Liver Disease (CLD) and Acute Liver Failure (ALF) are serious medical syndromes. Current therapeutic options consist of managing complications, and liver transplant. Even if liver transplantation is thought to be suitable for CLD or ALF patients, there are not enough organs available and thus increasingly more deaths occur on the transplant waiting list. Therefore, there is a pressing need to develop additional therapies. This thesis firstly systematically reviews trials in autologous cell therapies as possible treatments for patients with cirrhosis. The published literature is imperfect and the difference in trial design means it has not been possible to conduct a meta-analysis. Regardless of these shortcomings, cell therapy is a potentially positive prospect. In ALF and CLD monocyte-macrophages have diverse roles within the liver. Monocyte and immune cell changes in ALF are investigated and it is demonstrated for the first time that patients with paracetamol induced ALF have a significantly altered blood compartment and that these changes correlate with patient outcome. It is possible that these results may help stratify which patients may spontaneously survive and which patients may require an emergency liver transplant. Furthermore, modulation of these changes may improve outcomes for patients. The thesis also examines monocyte-macrophages in cirrhotic patients and demonstrates the feasibility of differentiating cirrhotic patients’ monocytes into functional macrophages, comparable to healthy volunteers in a Good Manufacturing Practice (GMP) environment. A first in-man trial using macrophages infused to patients with cirrhosis as a potential new treatment is also detailed. Finally, this thesis outlines developmental work for cell therapy in patients with cirrhosis in the multi-centre REALISTIC trial. Patients were randomly assigned to receive; standard medical care, Granulocyte Stimulating Factor (GCSF) injections alone or GCSF combined with repeated stem cell infusion.

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Chronic liver disease remains a significant cause of morbidity and mortality globally. Transplantation is the only effective treatment for end-stage disease but is limited by organ availability, surgical complications and risks of long term immunosuppression. Novel therapies for advanced disease are therefore required. The liver has a remarkable capacity to regenerate through division of mature hepatocytes, however in chronic or severe disease hepatocyte replication fails, senescence occurs and liver failure ensues. Ductular reactions (DRs), containing hepatic progenitor cells capable of repopulating the parenchyma, arise in chronic liver injury when hepatocyte regeneration is impaired. Enhancing this endogenous repair mechanism is a key therapeutic goal. Notch and Wnt are key signals required for liver regeneration, however to date they have principally been characterised in end-point disease and the temporal kinetics of these signalling pathways not known. I sought to identify if these signals control expansion of DRs after hepatocyte injury and whether they can be therapeutically manipulated. I examined the dynamics of Notch and Wnt activity using a genetic model of hepatocellular injury and ductular-mediated regeneration whereby induction of injury could be timed, synchronising the regenerative response. Using lineage tracing, small molecules, blocking antibodies and genetic loss of function experiments I defined distinct time-sensitive Notch and Wnt signatures where early regeneration is driven by Notch and the later response by Wnt. I demonstrated that inhibition of Notch1 and Notch3 but not Notch2 reduces the generation of DRs. I identified that DRs were a source of potent growth hormone IGF1 and this production was Wnt driven. Notch driven expression of IGF1-receptor within DRs identified this axis as a node for cooperation between Notch and Wnt signals. Blocking the IGF1 axis prevented DR expansion, which conversely could be enhanced by administration of recombinant IGF1. Here, I functionally defined complex temporal dynamics controlling of DRs and identified therapeutic pathways to enhance liver regeneration.

The liver has the ability to regenerate rapidly during acute liver injury by activating mature hepatocytes to divide and restore the damaged liver mass. In contrast, the liver relies on hepatic progenitor cells (HPCs) which have the ability to differentiate into both hepatocytes and biliary cells for regeneration during chronic liver injuries. Whole organ transplant is the most effective treatment for end stage liver diseases. However, there is a constant shortage of donor organs causing the death of many patients while waiting for suitable donor organs. HPC transplant is a potential alternative for whole organ transplant. However the isolation of HPC which is scarce in the liver and the expansion of these cells to a number that is suitable for transplant have been challenging. To investigate the plausibility of using HPCs as an alternative for liver transplant, I developed a protocol to isolate and expand HPC in vitro. Using this system, I investigated the complex hierarchy of HPCs in aid to select a defined population of HPC that is suitable for transplant. I found the EpCAM+ CD24+ population marks a naïve population of HPC that might be suitable for cell therapy. I further investigate the liver repopulating capacities of these cells by isolating EpCAM+CD24+ HPC population by Fluorescence Activated Cell Sorting (FACS) from a hepatocellular injury model. Surprisingly, a subpopulation of the EpCAM+ CD24+ HPCs which are also CD133+ possesses a higher colony forming capacities has been identified. Most importantly, this population can be expanded to a large scale in vitro and able to repopulate the injured liver after transplant. This defined population of HPCs can also be isolated from a mouse model of fatty liver disease and the isolated HPCs can be expanded in vitro. These cells are able to repopulate the liver after cell transplantation. The presence of HPCs that are capable of being isolated from the fatty liver proved the potential of using HPCs for transplant in a clinical setting by using cells isolated human fatty liver that are from rejected for transplant to overcome the shortage of donor organs.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1960. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

During hepatic disease the liver has the unrivalled ability to regenerate, by activating mature hepatocytes which can divide and thereby reconstitute the functional liver mass. However in the context of chronic hepatocellular disease the liver can regenerate from an endogenous population of hepatic progenitor cells (HPCs). The mechanisms which are involved in the activation and differentiation of these HPCs is not fully understood. To investigate whether there is a differential signalling requirement in HPCs acquiring a biliary versus hepatocellular fate we established in the laboratory two models of chronic liver damage and regeneration, one of which causes hepatocellular death, and results in infiltrating HPCs regenerating hepatocytes, and a second which causes biliary blockage and death, resulting in biliary regeneration. Here we describe how during biliary regeneration the Notch signalling pathway is highly expressed and activated. HPCs cells are consistently associated with a myofibroblast niche which expressed the ligand Jagged-1 at high levels. We have modulated the Notch signalling pathway in both a co-culture system and our models in vivo to demonstrate that Notch signalling is important in the specification of biliary cells, and that inhibition of this pathway both in vitro and in vivo results in the abrogation of biliary commitment. During hepatocellular regeneration we have found that the negative repressor of Notch signalling Numb is highly expressed in tandem with a low expression of the Notch pathway. We suggest that Wnt signalling maintains Numb within these HPCs at a high level and that this, along with stimulation of a hepatocellular programme allows HPCs to exit from a biliary fate and assume a hepatocellular phenotype. Finally we have found that macrophage ingestion of debris promotes the expression of Wnt, and that ablation of these cells results in a phenotypic switch between HPCs assuming a hepatocellular fate and a biliary one.

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