Myofibroblasts and stem/progenitor cell function

Recent research is highlighting the important role of mesenchymal cells in tissue differentiation [1]. These cells generate the extracellular matrix environment and secrete chemokines and cytokines that direct and maintain the differentiation of other cells. Thus, mesenchymal cells are critical to the development and maintenance of tissue phenotype at all stages, from controlling stem cells (both embryonic and adult) to controlling normal tissue homeostasis. Accordingly, inappropriate mesenchymal activity contributes to the major disease states such as fibrogenesis and cancer [1]. This project will examine the role of a mesenchymal cell within the liver - termed the myofibroblast - on the growth and differentiation of a liver progenitor cell. The C1-3 antibody reagent - developed by the Academic Supervisor [2] (and licensed to the Industrial Collaborator following a previously successful BBSRC CASE award) - will be used to deplete myofibroblasts from the liver in vivo [3,4] in order to examine their role on progenitor cell proliferation and differentiation. The major functional cell of the liver is the hepatocyte. Recent work in the academic lab includes the use of an in vitro model of hepatocyte differentiation from a progenitor cell [5,6]. This precursor has now been stably transfected with selectable vectors that encode a gene for visualising these cells in vitro or in vivo (green fluorescent protein and luciferase). The role of liver myofibroblast interactions on the activity of the labelled hepatocyte pre-cursor will initially be investigated in co-cultures in vitro. Culture vessels will be seeded with myofibroblasts with or without pre-treatment with a range of factors suspected to affect myofibroblasts and/or progenitor cell activity (e.g. fibrogenic factors TGFb, TNFa; growth factors such as EGF, HGF; cytokines; anti-fibrogenic factors such as NF-kB inhibitors, PXR activators, anti-TGFb, anti-TIMP1). GFP-labelled progenitor cells will then be cultured alone or with myofibroblasts (with or without direct physical contact) and the effect of myofibroblasts on progenitor cell proliferation, response to growth factors and promoters of apoptosis examined. Confocal microscopy will be used to distinguish between myofibroblasts and progenitor cells and to co-localise expression of selected genes. FACS analysis will also be used to separate myofibroblasts and progenitor cells for quantitative analysis of any effects. To examine the effect of myofibroblasts on progenitor cells in vivo, luciferase-labelled progenitor cells will be injected (via the hepatic portal vein but other potential routes, such as the spleen will be examined) into SCID mice. Location and growth of progenitor cells and derived mature cells, will be monitored using a CCD (1.4M pixels) camera [Lightools Inc, USA] with close focus zoom lens. Myofibroblast activity will be modulated using a variety of established protocols (carbon tetrachloride treatment, bile duct ligation, concanavalin A, methionine and choline-deficient diet) and the effects on progenitor cell growth monitored. Myofibroblast depletion and its effects on progenitor cell function will be achieved using the C1-3 antibody technology [3,4]. At appropriate timepoints, dependent on imaging data, tissue will be harvested and the interactions of myofibroblasts and progenitor cells examined by histological and immunocytochemical analyses. This project will establish how myofibroblast activity impacts on liver progenitor cell activity and will determine whether therapeutics designed to modulate myofibroblast activity may impact on normal tissue regenerative potential. 1 Wallace K et al Biochem J 2008;411:1-18 2 Elrick LJ et al J Hepatol 2005;42:888-96 3 Douglass A et al J Hepatol 2008;49:88-98 4 Douglass A. et al Hepatol Int. 2008 in press 5 Marek CJ et al Biochem J 2003; 6 Wallace K et al 2008 submitted.