Joe W. Grisham

Molecular pathogenesis of human hepatocellular carcinoma.

Hepatocarcinogenesis is a slow process during which genomic changes progressively alter the hepatocellular phenotype to produce cellular intermediates that evolve into hepatocellular carcinoma. During the long preneoplastic stage, in which the liver is often the site of chronic hepatitis, cirrhosis, or both, hepatocyte cycling is accelerated by upregulation of mitogenic pathways, in part through epigenetic mechanisms. This leads to the production of monoclonal populations of aberrant and dysplastic hepatocytes that have telomere erosion and telomerase re-expression, sometimes microsatellite instability, and occasionally structural aberrations in genes and chromosomes. Development of dysplastic hepatocytes in foci and nodules and emergence of hepatocellular carcinoma are associated with the accumulation of irreversible structural alterations in genes and chromosomes, but the genomic basis of the malignant phenotype is heterogeneous. The malignant hepatocyte phenotype may be produced by the disruption of a number of genes that function in different regulatory pathways, producing several molecular variants of hepatocellular carcinoma. New strategies should enable these variants to be characterized.

Hematopoietic cells as hepatocyte stem cells: A critical review of the evidence†

The authors reviewed 77 published reports available before August 1, 2005 that examined the ability of hematopoietic cells to generate hepatocytes in the liver. A list of these publications and a synopsis of each are available on‐line. We interpret the evidence provided by this data set to suggest that one or more types of hematopoietic cells may rarely acquire the hepatocyte phenotype in the liver (frequency ≤10−4), although the nature of the hematopoietic cells involved and the mechanisms responsible for acquisition of a hepatocyte phenotype are still controversial. Hematopoietic stem cells do not appear to be direct precursors of hepatocytes, which, instead, can be generated from cells of the macrophage–monocyte lineage. Fusion between hepatocytes and transplanted hematopoietic cells has been substantiated as a mechanism by which hepatocytes that carry a bone marrow tag are generated, but direct transdifferentiation of hematopoietic cells has not been demonstrated. In conclusion, hematopoietic cells contribute little to hepatocyte formation under either physiological or pathological conditions, although they may provide cytokines and growth factors that promote hepatocyte functions by paracrine mechanisms. Cells of the endodermal hepatocyte lineage are far more potent generators of hepatocytes than are hematopoietic cells. (HEPATOLOGY 2006;43:2–8.)

Liver Regeneration in Rats with Retrorsine-Induced Hepatocellular Injury Proceeds through a Novel Cellular Response

The adult rodent liver contains at least two recognized populations of cells with stem-like properties that contribute to liver repair/regeneration under different pathophysiological circumstances: (i) unipotential committed progenitor cells (differentiated hepatocytes and biliary epithelial cells) and (ii) multipotential nonparenchymal progenitor cells (oval cells). In retrorsine-induced hepatocellular injury the capacity of fully differentiated rat hepatocytes to replicate is severely impaired and massive proliferation of oval cells does not occur. Nevertheless, retrorsine-exposed rats can replace their entire liver mass after 2/3 surgical partial hepatectomy through the emergence and expansion of a population of small hepatocyte-like progenitor cells that expresses phenotypic characteristics of fetal hepatoblasts, oval cells, and fully differentiated hepatocytes, but differ distinctly from each type of cell. The activation, proliferation, and complete regeneration of normal liver structure from small hepatocyte-like progenitor cells have not been recognized in other models of liver injury characterized by impaired hepatocyte replication. We suggest that the selective emergence and expansion of small hepatocyte-like progenitor cells observed in the retrorsine model reflect a novel mechanism of complete liver regeneration in the adult rat. Furthermore, we suggest that these cells may represent a novel progenitor cell population that (i) responds to liver deficit when the replication capacity of differentiated hepatocytes is impaired, (ii) expresses an extensive proliferative capacity, (iii) can give rise to large numbers of progeny hepatocytes, and (iv) can restore tissue mass.

Adult-Derived Stem Cells from the Liver Become Myocytes in the Heart in Vivo

Recent evidence suggests that adult-derived stem cells, like their embryonic counterparts, are pluripotent. These simple, undifferentiated and uncommitted cells are able to respond to signals from their host tissue microenvironment and differentiate, producing progeny that display a phenotype characteristic of the mature cells of that tissue. We used a clonal stem cell line (termed WB-F344) that was derived from an adult male rat liver to investigate the possibility that uncommitted stem cells from a nonmyogenic tissue source would respond to the tissue microenvironment of the heart in vivo and differentiate into cardiac myocytes. Male WB-F344 cells that carry the Escherichia coli beta-galactosidase gene were identified in the left ventricular myocardium of adult female nude mice 6 weeks after transplantation. We confirmed the presence of a rat Y-chromosome-specific repetitive DNA sequence exclusively in the beta-galactosidase-positive myocytes by polymerase chain reaction and fluorescence in situ hybridization. Immunohistochemistry, using a cardiac troponin T-specific monoclonal antibody, and ultrastructural analysis confirmed a cardiac myocyte phenotype of the stem cell-derived myocytes. The beta-galactosidase-positive myocytes ranged from < 20 microm to 110 microm in length. The longer of these cells contained well-organized sarcomeres and myofibrils, and formed intercalated disks and gap junctions with endogenous (host-derived) myocytes, suggesting that WB-F344-derived myocytes participate in the function of the cardiac syncytium. These results demonstrate that adult liver-derived stem cells respond to the tissue microenvironment of the adult heart in vivo and differentiate into mature cardiac myocytes.

Temporal Analysis of Hepatocyte Differentiation by Small Hepatocyte-Like Progenitor Cells during Liver Regeneration in Retrorsine-Exposed Rats

Liver regeneration after two-thirds surgical partial hepatectomy (PH) in rats treated with the pyrrolizidine alkaloid retrorsine is accomplished through the activation, expansion, and differentiation of a population of small hepatocyte-like progenitor cells (SHPCs). We have examined expression of the major liver-enriched transcription factors, cytochrome P450 (CYP) enzymes, and other markers of hepatocytic differentiation in SHPCs during the protracted period of liver regeneration after PH in retrorsine-exposed rats. Early-appearing SHPCs (at 3-7 days after PH) express mRNAs for all of the major liver-enriched transcription factors at varying levels compared to fully differentiated hepatocytes. In addition, SHPCs lack (or have significantly reduced) expression of mRNA for hepatocyte markers tyrosine aminotransferase and alpha-1 antitrypsin, but their expression levels of mRNA and/or protein for WT1 and alpha-fetoprotein (AFP) are increased. With the exception of AFP expression, SHPCs resembled fully differentiated hepatocytes by 14 days after PH. Expression of AFP was maintained by most SHPCs through 14 days after PH, gradually declined through 23 days after PH, and was essentially absent from SHPC progeny by 30 days after PH. Furthermore, early appearing SHPCs lack (or have reduced expression) of hepatic CYP proteins known to be induced in rat livers after retrorsine exposure. The resistance of SHPCs to the mitoinhibitory effects of retrorsine may be directly related to a lack of CYP enzymes required to metabolize retrorsine to its toxic derivatives. These results suggest that SHPCs represent a unique parenchymal (less differentiated) progenitor cell population of adult rodent liver that is phenotypically distinct from fully differentiated hepatocytes, biliary epithelial cells, and (ductular) oval cells.

Functional genomics of hepatocellular carcinoma

The majority of DNA‐microarray based gene expression profiling studies on human hepatocellular carcinoma (HCC) has focused on identifying genes associated with clinicopathological features of HCC patients. Although notable success has been achieved, this approach still faces significant challenges due to the heterogeneous nature of HCC (and other cancers) as well as the many confounding factors embedded in gene expression profile data. However, these limitations are being overcome by improved bioinformatics and sophisticated analyses. Also, application of cross comparison of multiple gene expression data sets from human tumors and animal models are facilitating the identification of critical regulatory modules in the expression profiles. The success of this new experimental approach, comparative functional genomics, suggests that integration of independent data sets will enhance our ability to identify key regulatory elements in tumor development. Furthermore, integrating gene expression profiles with data from DNA sequence information in promoters, array‐based CGH, and expression of non‐coding genes (i.e., microRNAs) will further increase the reliability and significance of the biological and clinical inferences drawn from the data. The pace of current progress in the cancer profiling field, combined with the advances in high‐throughput technologies in genomics and proteomics, as well as in bioinformatics, promises to yield unprecedented biological insights from the integrative (or systems) analysis of the combined cancer genomics database. The predicted beneficial impact of this “new integrative biology” on diagnosis, treatment and prevention of liver cancer and indeed cancer in general is enormous. (Hepatology 2006;43:S145–S150.)

Isolation, culture, and transplantation of rat hepatocytic precursor (stem-like) cells.

Abstract From a review of past studies and the report of new studies from our laboratory, this article provides strong evidence to show that WB-F344 (WB) rat liver epithelial cells are stem-like precursor cells for hepatocytes. WB cells are structurally and phenotypically simple epithelial cells that were isolated from the liver of an adult male Fischer 344 rat, under conditions that excluded their origin from hepatocytes in vivo. WB cells express a phenotypic repertory that overlaps, but is distinct from, that of both hepatocytes and bile duct epithelial cells. The complex phenotype of WB cells is compatible with their being embryonic or undifferentiated variants of either hepatocytes or bile duct epithelial cells. When WB cells are tagged genetically with genes for bacterial β-galactosidase and neomycin resistance (BAG2-WB), they and their progeny can be distinguished from parental WB cells and hepatocytes by the expression of these gene products. Progeny of BAG2-WB cells that were transplanted into the liver parenchyma of syngeneic rats integrated into hepatic plates and acquired the morphological and functional attributes of adjacent host hepatocytes; the progeny of BAG2-WB cells in the liver express albumin, tyrosine aminotransferase, α-1-antitrypsin, and transferrin. We also demonstrate that progeny of BAG2-WB cells can be recovered from livers into which they have been transplanted, which may allow the elucidation of alterations in gene expression that accompany their differentiation.

Inhibition of proliferation of cultured rat liver epithelial cells at specific cell cycle stages by transforming growth factor-β

Proliferation of early-passage propagable cultured rat liver epithelial cells derived from normal adult rats is markedly inhibited by transforming growth factor-beta (TGF-beta). Inhibition, which is completely reversible, is effected at two distinct points of the cell cycle, the G1/S border and the G0 or early G1 phase. With increasing passages in culture, hepatic epithelial cells progressively become less sensitive to the inhibitory effect of TGF-beta.

Isolation, short-term culture, and transplantation of small hepatocyte-like progenitor cells from retrorsine-exposed rats.

Background. Complete liver regeneration after partial hepatectomy (PH) in rats treated with the pyrrolizidine alkaloid retrorsine can be accomplished through the activation, expansion, and differentiation of a novel population of small hepatocyte-like progenitor cells (SHPCs). These cells have not been isolated in pure form, established in primary culture, or transplanted into syngeneic rats to examine their differentiation potential. Methods. Primary liver cells enriched for SHPCs were prepared by differential centrifugation of primary liver cell dispersions from retrorsine-exposed rats 6–8 days and 13–15 days after PH. Isolated SHPCs were characterized for cell size, morphology, and expression of cell type-specific markers (including hepatocyte and bile duct–oval cell markers), and established in short-term primary culture. Isolated SHPCs were transplanted into the livers of syngeneic rats to evaluate their ability to engraft and differentiate into mature hepatocytes. Results. SHPCs obtained from retrorsine-exposed rats 6–8 days and 13–15 days after PH were small (10–12 &mgr;m in diameter), morphologically resembled hepatocytes, and were predominately H.4 antigen-positive, &agr;-fetoprotein-positive, and OV6-negative. SHPCs did not proliferate in culture and could not be passaged, but short-term cultures were established using protein substrates (collagen or laminin) and defined medium containing epidermal growth factor and nicotinamide. After transplantation into the livers of syngeneic hosts, SHPCs insert into hepatic plates and give rise to differentiated hepatocyte progeny. The SHPC-derived hepatocyte progeny express a differentiated phenotype (albumin-positive, transferrin-positive, &agr;-fetoprotein-negative) and are able to proliferate in vivo in response to the growth stimulus provided by PH. Conclusions. The results demonstrate that enriched SHPC populations can be isolated from retrorsine-exposed rats and established in short-term culture, and they can engraft and differentiate after transplantation into the livers of syngeneic rats.

Scanning electron microscopy of mouse intrahepatic structures

Abstract Livers of normal mice were prepared for scanning electron microscopic (SEM) study by fracturing or slicing lobes fixed in situ by perfusion with paraformaldehyde. Fracturing fixed liver exposes surfaces of hepatocytes and sinusoidal endothelial cells, whereas slicing the tissue reveals the internal structures of portal tracts. Earlier studies have delineated the major surface characteristics of hepatocytes and sinusoidal endothelial cells of rats. Surfaces of hepatocytes in the mouse differ from those in the rat by having larger and more numerous peg and hole complexes on the flat intercellular surface and less dense populations of perisinusoidal microvilli. Sinusoidal endothelial cells in the mouse have fewer large fenestrations than do similar cells in the rat; clusters of small fenestrations appear similarly distributed in both species. The surfaces of capsular mesothelial cells, Kupffer cells, bile duct epithelial cells, and endothelial cells of major vessels are similar in rat and mouse. The methods described for preparing liver for SEM examination are simple, rapid, and reproducible. The SEM is a useful tool with which to study intrahepatic surface structures, and its use may allow further correlations to be made between hepatic structure and function in both health and disease.