Expansion Of Hepatic Progenitor Cells Research Articles

Fibroblast Growth Factor Receptor 2 Expression Is Associated with Hepatic Progenitor Cell Expansion in the DDC Model

Introduction: Liver transplantation, the only curative treatment for end-stage liver failure, is limited by the availability of donor organs; therefore, other potential methods of hepatocyte replacement, such as hepatic progenitor cell (HPC) transplantation, require investigation. Fibroblast Growth Factors (FGFs) are required for induction of the hepatic bud from the foregut endoderm at the initial stages of hepatogenesis. We recently showed that FGF10 promotes HPC survival and proliferation via β-catenin activation through receptor FGFR2IIIb activation.

Postnatally Induced Over-Expression of FGF10 Promotes Murine Hepatic Progenitor Cell Expansion Similar to the DDC Diet Model

Introduction: Liver cell replacement therapy is an area of active investigation given the lack of organ donors. Greater understanding of what regulates HPC expansion during hepatogenesis may lead to effective stem cell therapies for liver diseases as well as other system pathologies. Fibroblast Growth Factors (FGFs), a family of 23 ligands and 4 FGFR tyrosine kinases, regulate many aspects of development including hepatogenesis. We previously showed that FGF10 promotes the proliferation and survival of fetal hepatic progenitor cells (HPC) called hepatoblasts during early hepatogenesis in mice (Berg et al, Hepatology, 2007).

New school in liver development: Lessons from zebrafish #

There is significant overlap in the genes and pathways that control liver development and those that regulate liver regeneration, hepatic progenitor cell expansion, response to injury, and cancer. Additionally, defects in liver development may underlie some congenital and perinatal liver diseases. Thus, studying hepatogenesis is important for understanding not only how the liver forms, but also how it functions. Elegant work in mice has uncovered a host of transcription factors and signaling molecules that govern the early steps of hepatic specification; however, the inherent difficulty of studying embryogenesis in utero has driven developmental biologists to seek new systems. The rapidly developing vertebrate zebrafish is a favorite model for embryology. The power of forward genetic screens combined with live real-time imaging of development in transparent zebrafish embryos has highlighted conserved processes essential for hepatogenesis and has uncovered some exciting new players. This review presents the advantages of zebrafish for studying liver development, underscoring how studies in zebrafish and mice complement each other. In addition to their value for studying development, zebrafish models of hepatic and biliary diseases are expanding, and using these small, inexpensive embryos for drug screening has become de rigueur. Zebrafish provide a shared platform for developmental biology and translational research, offering innovative methods for studying liver development and disease. The story of hepatogenesis has something for everyone. It involves transcriptional regulation, cell-cell interaction, signaling pathways, control of cell proliferation and apoptosis, plus morphogenic processes that sculpt vasculature, parenchymal cells, and mesenchyme to form the multifaceted liver. Decades of research on liver development in mice and other vertebrates offer valuable lessons in how the multipotent endoderm is programmed to form a functional liver. Of equal importance are insights that have illuminated the mechanisms by which hepatic progenitors are activated in a damaged liver, how the adult liver regenerates, and, possibly, the basis for engineering liver cells in vitro for cell transplantation to sustain patients with liver failure. Moreover, processes that are key to liver development are often co-opted during pathogenesis. Therefore, reviewing hepatogenesis is informative for both basic and translational researchers. In this review, we bring to light the many advantages offered by the tropical freshwater vertebrate zebrafish (Danio rerio) in studying hepatogenesis. By comparing zebrafish and mice, we highlight how work in each system complements the other and emphasize novel paradigms that have been uncovered using zebrafish. Finally, we highlight exciting efforts using zebrafish to model hepatobiliary diseases.

2 A NOVEL INDUCIBLE GENETIC MURINE MODEL OF HEPATOCYTE SENESCENCE CAUSES A MASSIVE HEPATIC OVAL CELL RESPONSE AND COMPLETE HEPATOCYTE REPLACEMENT FROM ENDOGENOUS PROGENITOR CELLS

Background and aims: In severe advanced liver disease it is increasingly recognised that hepatic progenitor cells (HPCs) are a major source of parenchymal regeneration but may also be a cellular source of liver cancers. Murine liver injury models eliciting HPC activation frequently demonstrate relatively small and heterogeneous activation of HPCs, together with simultaneous hepatocyte proliferation. While purified HPCs from such models have been shown in transplant models to repopulate injured liver the full repopulation potential of endogenous HPC remains unclear. In order to investigate the role of HPCs directly it is necessary to specifically inhibit hepatocyte proliferation. Mdm2 is a key inhibitor of the tumour suppressor gene p53. P53 activation results in cell cycle arrest, therefore hepatocyte specific Mdm2 knockout using Cre Lox technology provides an opportunity to investigate HPC mediated liver regeneration. Methods: Hepatocyte specific loss of Mdm2 was achieved by i.p. administration of β-naphthoflavone to AhCre+ Mdm2fl/fl mice. Immunohistochemical analysis was performed for p21, p53, and the HPC marker panCK. Cellular proliferation was assessed using Ki67 and BrdU label incorporation. Serum was analysed for LFTs. Results: Hepatocytes, but not other non parenchymal hepatic cells, are p53hi and p21hi following induction of Mdm2 loss by recombination. Loss of hepatocyte proliferation and massive expansion of p53low HPCs rapidly occurs. This results in the production of phenotypically normal p53low/p21low hepatocytes and the restoration of normal liver function. PanCK+ progenitor cell expansion is over three times greater than is seen in CDE diet induced HPC expansion (Mean±SEM; Mdm2 212± 27.48 versus CDE 60.89 ± 17.59 versus control 13.85± 0.82). No cancers or significant fibrosis are observed following complete replacement of the hepatic parenchyma during long term follow up. Conclusions: Mdm2 knockout induces hepatocyte senescence and induces massive expansion of p53low HPCs resulting in complete regeneration of the liver parenchyma in the absence of long term sequelae. This novel model permits detailed examination of HPC mediated regeneration and demonstrates the physiological potential of HPCs and highlights their therapeutic potential.

Pathobiology of biliary epithelia and cholangiocarcinoma: Proceedings of the Henry M. and Lillian Stratton basic research single-topic conference

In June 2008, the American Association for the Study of Liver Diseases (AASLD) sponsored the Henry M. and Lillian Stratton Basic Research Single-Topic Conference on the Pathobiology of Biliary Epithelia and Cholangiocarcinoma, which was held in Atlanta, GA. Attendees from 12 different countries participated in this conference, making it a truly international scientific event. Both oral and poster presentations were given by multidisciplinary experts, who highlighted important areas of current basic and translational research on biliary epithelial cell biology and pathophysiology, and on the etiology, cellular and molecular pathogenesis, and target-based therapy of cholangiocarcinoma. The specific goals and objectives of the conference were: (1) to advance knowledge of basic and molecular mechanisms underlying developmental and proliferative disorders of the biliary tract; (2) to foster a better and more comprehensive understanding of mechanisms regulating biliary epithelial (cholangiocyte) growth and transport, signaling, cell survival, and abnormalities that result in disease; and (3) to understand basic mechanisms of cholangiocarcinoma development and progression, with the added goal of identifying and exploiting potentially critical molecular pathways that may be targeted therapeutically. A number of interrelated themes emerged from the oral and poster sessions that affected current understandings of the complex organization of transcriptional and signaling mechanisms that regulate bile duct development, hepatic progenitor cell expansion, cholangiocyte secretory functions and proliferation, and mechanisms of cholangiocarcinogenesis and malignant cholangiocyte progression. Most notable were the critical questions raised as to how best to exploit aberrant signaling pathways associated with biliary disease as potential targets for therapy.

Human hepatoblast phenotype maintained by hyaluronan hydrogels

Human hepatoblasts and hepatic stem cells, pluripotent hepatic progenitors that give rise to hepatocytes and biliary cells, were isolated from fetal livers and found to express hyaluronan receptors (CD44) in both the freshly isolated cells and after culture. This implicates an in vivo connection to hyaluronan (HA), an embryonic matrix component, as a candidate 3-dimensional (3-D) scaffold for hepatic progenitor cell expansion and/or differentiation. To assess HAs as scaffolds, hepatoblasts and hepatic stem cells were seeded into HA hydrogels with a serum-free, hormonally defined medium tailored for expansion of hepatic progenitors. Cell aggregates formed within the HA hydrogels and remained viable, proliferative, and demonstrated a stable phenotype intermediate between that of hepatic stem cells and hepatoblasts throughout more than 4 weeks of culturing, with little evidence of lineage restriction towards either hepatocytic or biliary pathways. The phenotype consisted of stable co-expression of both hepatocytic and biliary markers such as biliary-specific cytokeratin, CK19, low levels of expression of albumin, and urea synthesis. HA hydrogels are ideal as 3-D scaffolds for pluripotent hepatic progenitors and should be useful for generating cells to be used in bioartificial livers or tissue engineered liver grafts.

Progressive Fibrosis in Nonalcoholic Steatohepatitis: Association With Altered Regeneration and a Ductular Reaction

Portal fibrosis and linkage is a key feature of progressive disease in nonalcoholic steatohepatitis (NASH), but not simple steatosis. It is underappreciated and poorly understood. Fatty liver has impaired regeneration that induces a secondary replicative pathway using bipotential, periportal, hepatic progenitor cells (HPCs). We propose that activation of this pathway, with increased cell injury in NASH, also induces a periportal ductular reaction (DR) that could produce a profibrogenic stimulus. Biopsy specimens from 107 patients with nonalcoholic fatty liver disease and 11 controls were immunostained with cytokeratin-7 to quantify the DR and HPCs, and with p21 to assess hepatocyte replicative arrest. These results were correlated with clinicopathologic variables. Patients with nonalcoholic fatty liver disease had expansion of HPCs, with a strong association between HPCs and the DR (r(s) = 0.582, P < .0001). In those with NASH (n = 69) there was an increased DR compared with simple steatosis, which correlated with the stage of fibrosis (r(s) = 0.510, P < .0001). The DR increased with the grade of NASH activity (r(s) = 0.478, P < .0001), grade of portal inflammation (r(s) = 0.445, P < .0001), and extent of hepatocyte replicative arrest (r(s) = 0.446, P < .0001). Replicative arrest was in turn associated with insulin resistance (r(s) = 0.450, P < .0001) and NASH activity (r(s) = 0.452, P < .0001). By multivariate analysis, the extent of DR (odds ratio [OR] = 17.9, P = .016), hepatocyte ballooning (OR = 8.1, P < .0001), and portal inflammation (OR = 3.3, P = .005) were associated independently with fibrosis. These findings suggest that an altered replication pathway in active NASH promotes a periportal DR, which in turn may provoke progressive periportal fibrogenesis.

Fibrosis correlates with a ductular reaction in hepatitis C

The mechanisms for progressive fibrosis and exacerbation by steatosis in patients with chronic hepatitis C (HCV) are still unknown. We hypothesized that proliferative blockade in HCV-infected and steatotic hepatocytes results in the default activation of hepatic progenitor cells (HPC), capable of differentiating into both biliary and hepatocyte lineages, and that the resultant ductular reaction promotes portal fibrosis. To study this concept, 115 liver biopsy specimens from subjects with HCV were scored for steatosis, inflammation, and fibrosis. Biliary epithelium and HPC were decorated by cytokeratin 7 immunoperoxidase, and the replicative state of hepatocytes was assessed by p21 and Ki-67 immunohistochemistry. A ductular reaction at the portal interface was common. There was a highly significant correlation between the area of ductular reaction and fibrosis stage (r = 0.453, P < .0001), which remained independently associated after multivariate analysis. HPC numbers also correlated with fibrosis (r = 0.544, P < .0001) and the ductular area (r = 0.624, P < .0001). Moreover, steatosis correlated with greater HPC proliferation (r = 0.372, P = .0004) and ductular reaction (r = 0.374, P < .0001) but was not an obligate feature. Impaired hepatocyte replication by p21 expression was independently associated with HPC expansion (P = .002) and increased with the body mass index (P < .001) and lobular inflammation (P = .005). In conclusion, the strong correlation between portal fibrosis and a periportal ductular reaction with HPC expansion, the exacerbation by steatosis, and the associations with impaired hepatocyte replication suggest that an altered regeneration pathway drives the ductular reaction. We believe this triggers fibrosis at the portal tract interface. This may be a stereotyped response of importance in other chronic liver diseases.