The Liver Is a Peculiar Organ When It Comes to Stem Cells

Tissue biology has emerged as an interesting field of study. Differentiation of cells in different tissues follows similar rules, although it involves different tissue-specific genes, proteins, and extracellular matrix components. In tissues such as skin, hematopoietic cells, intestinal mucosa, and the glands of prostate and bronchus, cell renewal is dependent on the presence and continual proliferation of tissue-specific stem cells, which reside in specific histological locations, clearly visible under microscopic examination. Bone marrow hematopoietic stem cells, although not easily identifiable in tissue sections, can be easily separated and studied using cell-sorting methods. The sole role of tissue-specific stem cells is the maintenance of the needed numbers of the fully differentiated epithelial cells, which are the hallmark of the organ’s identity and function. Cells derived from such tissue-specific stem cells, or progenitor cells, are not yet fully differentiated but continue to proliferate toward the terminal differentiated phenotype. Studies in liver growth biology have been tackling such issues, and much new knowledge has emerged. The article by Sekiya and Suzuki1Sekiya S. Suzuki A. Hepatocytes, rather than cholangiocytes, can be the major source of primitive ductules in the chronically injured mouse liver.Am J Pathol. 2014; 184: 1468-1478Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar in this issue of The American Journal of Pathology highlights the complexity of hepatic biology in the context of liver growth. Despite several discussions concerning the existence and nature of liver-specific stem cells2Sell S. Is there a liver stem cell?.Cancer Res. 1990; 50: 3811-3815PubMed Google Scholar and identification of isolated candidate cells with aberrant biomarkers by electron microscopy or immunohistochemistry,3Novikoff P.M. Yam A. Stem cells and rat liver carcinogenesis: contributions of confocal and electron microscopy.J Histochem Cytochem. 1998; 46: 613-626Crossref PubMed Scopus (22) Google Scholar there is no concrete evidence of a histologically demonstrable location for stem cells in hepatic lobules. Moreover, studies have shown that all mature and fully differentiated liver cell types participate in liver regeneration after partial hepatectomy, without the intervention of stem cells,4Stöcker E. Wullstein H.K. Bräu G. Capacity of regeneration in liver epithelia of juvenile, repeated partially hepatectomized rats: autoradiographic studies after continous infusion of 3H-thymidine (author’s transl) [German].Virchows Arch B Cell Pathol. 1973; 14: 93-103PubMed Google Scholar, 5Michalopoulos G.K. Principles of liver regeneration and growth homeostasis.Compr Physiol. 2013; 3: 485-513Crossref PubMed Scopus (176) Google Scholar raising questions about their existence. In view of the failure of classic histological approaches to identify a specific and easily demonstrable lobular location for hepatic stem cells, other molecular approaches have been used. For example, tissue-specific stem cells typically proliferate by generating another tissue-specific stem cell and a progenitor cell, which continue to proliferate and derive the finally differentiated epithelial cells of the organ. Thus, administration of the DNA-labeling agent, bromodeoxyuridine (BrdU), results in long-term labeling of the tissue-specific stem cells, whereas the label is diluted from the multiple sequential proliferation cycles of the progenitor cells. By using this approach, Theise and colleagues6Kuwahara R. Kofman A.V. Landis C.S. Swenson E.S. Barendswaard E. Theise N.D. The hepatic stem cell niche: identification by label-retaining cell assay.Hepatology. 2008; 47: 1994-2002Crossref PubMed Scopus (223) Google Scholar demonstrated BrdU-retaining cells in biliary ductules and the immediate proximate periportal hepatocytes. Although these cells retain BrdU for a long period, they cannot be viewed as tissue-specific stem cells because they perform routine differentiated functions for the organ (eg, proteins specific to hepatocyte differentiation and lining of mature bile ductules). Hepatocytes play an essential role in liver regeneration after partial hepatectomy. Hepatocytes undergo the necessary number of proliferation cycles, as well as produce paracrine signals, which stimulate proliferation of other liver cells, which, in turn, use growth factors and cytokines to further enhance hepatocyte proliferation.5Michalopoulos G.K. Principles of liver regeneration and growth homeostasis.Compr Physiol. 2013; 3: 485-513Crossref PubMed Scopus (176) Google Scholar, 7Michalopoulos G.K. Liver regeneration.J Cell Physiol. 2007; 213: 286-300Crossref PubMed Scopus (1177) Google Scholar Inhibition of hepatocyte proliferation after partial hepatectomy results in the appearance of a new population of cells, called oval cells because of the shape of their nucleus.8Tatematsu M. Ho R.H. Kaku T. Ekem J.K. Farber E. Studies on the proliferation and fate of oval cells in the liver of rats treated with 2-acetylaminofluorene and partial hepatectomy.Am J Pathol. 1984; 114: 418-430PubMed Google Scholar Pioneering studies by Thorgeirsson and his collaborators, using thymidine pulse labeling, identified biliary epithelial cells as the origin of oval cells. Soon after their appearance, oval cells continue to express biliary biomarkers, while at the same time initiating albumin and α-fetoprotein production. This is associated with the expression of hepatocyte-specific transcription factors in the biliary epithelial cells.9Evarts R.P. Hu Z. Omori N. Omori M. Marsden E.R. Thorgeirsson S.S. Precursor-product relationship between oval cells and hepatocytes: comparison between tritiated thymidine and bromodeoxyuridine as tracers.Carcinogenesis. 1996; 17: 2143-2151Crossref PubMed Scopus (67) Google Scholar, 10Hu Z. Evarts R.P. Fujio K. Marsden E.R. Thorgeirsson S.S. Expression of fibroblast growth factor receptors flg and bek during hepatic ontogenesis and regeneration in the rat.Cell Growth Differ. 1995; 6: 1019-1025PubMed Google Scholar, 11Nagy P. Bisgaard H.C. Thorgeirsson S.S. Expression of hepatic transcription factors during liver development and oval cell differentiation.J Cell Biol. 1994; 126: 223-233Crossref PubMed Scopus (196) Google Scholar Toxic damage to the biliary cells before the initiation of the oval cell process eliminates the appearance of the oval cells.12Petersen B.E. Zajac V.F. Michalopoulos G.K. Bile ductular damage induced by methylene dianiline inhibits oval cell activation.Am J Pathol. 1997; 151: 905-909PubMed Google Scholar Oval cells gradually increase in size, convert to small hepatocytes, and eventually become regular hepatocytes, thus providing the necessary number of hepatocytes needed to complete regeneration. Several elegant studies have demonstrated the supporting role of hepatic stellate cells, growth factors, such as hepatocyte growth factor (HGF), and epidermal growth factor receptor (EGFR) ligands, and changes in extracellular matrix during the conversion of biliary epithelial cells to hepatocytes via the oval cell pathway.9Evarts R.P. Hu Z. Omori N. Omori M. Marsden E.R. Thorgeirsson S.S. Precursor-product relationship between oval cells and hepatocytes: comparison between tritiated thymidine and bromodeoxyuridine as tracers.Carcinogenesis. 1996; 17: 2143-2151Crossref PubMed Scopus (67) Google Scholar, 10Hu Z. Evarts R.P. Fujio K. Marsden E.R. Thorgeirsson S.S. Expression of fibroblast growth factor receptors flg and bek during hepatic ontogenesis and regeneration in the rat.Cell Growth Differ. 1995; 6: 1019-1025PubMed Google Scholar, 11Nagy P. Bisgaard H.C. Thorgeirsson S.S. Expression of hepatic transcription factors during liver development and oval cell differentiation.J Cell Biol. 1994; 126: 223-233Crossref PubMed Scopus (196) Google Scholar Although oval cells are not derived from a tissue-specific stem cell, they are classified as progenitor cells. The progression of biliary cells to progenitor cells is a complex process in which specific receptors and tissue-based growth factors appear. The Wnt-related protein LGR5, a receptor for Rspo1, is expressed in intestinal stem cells. Although it is not expressed in mature adult liver, it is expressed after damage of the biliary epithelium. Organoid cultures derived from Lgr5-expressing cells and under the influence of Rspo1-generated progenitor cells that were transdifferentiating into hepatocytes in the Fah−/− mouse liver recolonization model.13Huch M. Dorrell C. Boj S.F. van Es J.H. Li V.S. van de Wetering M. Sato T. Hamer K. Sasaki N. Finegold M.J. Haft A. Vries R.G. Grompe M. Clevers H. In vitro expansion of single Lgr5+ liver stem cells induced by Wnt-driven regeneration.Nature. 2013; 494: 247-250Crossref PubMed Scopus (996) Google Scholar It would be interesting to examine if Lgr5 is expressed in the biliary cells early in their transformation to progenitor cells. This discovery suggests that the progenitor cells, although having a biliary origin and a hepatocyte destination, can proceed through gene expression and cell proliferation rules of their own until they achieve their final objective. Most of the early studies on progenitor cells were conducted in rats, using the model of suppressed liver regeneration after partial hepatectomy with simultaneous administration of the carcinogenic chemical 2-acetylaminofluorene (AAF).9Evarts R.P. Hu Z. Omori N. Omori M. Marsden E.R. Thorgeirsson S.S. Precursor-product relationship between oval cells and hepatocytes: comparison between tritiated thymidine and bromodeoxyuridine as tracers.Carcinogenesis. 1996; 17: 2143-2151Crossref PubMed Scopus (67) Google Scholar, 11Nagy P. Bisgaard H.C. Thorgeirsson S.S. Expression of hepatic transcription factors during liver development and oval cell differentiation.J Cell Biol. 1994; 126: 223-233Crossref PubMed Scopus (196) Google Scholar The administration of AAF causes DNA adduct formation and activation of Cdkn1a-dependent (alias p21) and Trp53-dependent pathways, thus blocking hepatocyte proliferation. AAF induces DNA adducts in rat hepatocytes because of the presence of the enzyme N-sulfotransferase, which generates sulfate salts with the activated form of AAF, the N-OH AAF radical, thus allowing it to migrate to the nucleus before becoming inactivated by glutathione in the cytoplasm.14DeBaun J.R. Rowley J.Y. Miller E.C. Miller J.A. Sulfotransferase activation of N-hydroxy-2-acetylaminofluorene in rodent livers susceptible and resistant to this carcinogen.Proc Soc Exp Biol Med. 1968; 129: 268-273Crossref PubMed Scopus (117) Google Scholar However, N-sulfotransferase does not exist in the mouse14DeBaun J.R. Rowley J.Y. Miller E.C. Miller J.A. Sulfotransferase activation of N-hydroxy-2-acetylaminofluorene in rodent livers susceptible and resistant to this carcinogen.Proc Soc Exp Biol Med. 1968; 129: 268-273Crossref PubMed Scopus (117) Google Scholar and AAF is a weak mouse carcinogen and it does not block liver regeneration. Therefore, many investigators, including Sekiya and Suzuki,1Sekiya S. Suzuki A. Hepatocytes, rather than cholangiocytes, can be the major source of primitive ductules in the chronically injured mouse liver.Am J Pathol. 2014; 184: 1468-1478Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar use the 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet in mouse, which induces cell death and continuing proliferation of biliary epithelial cells associated with biliary repair. However, DDC also induces porphyria and toxicity to hepatocytes.15Magnus I.A. Roe D.A. Bhutani L.K. Factors affecting the induction of porphyria in the laboratory rat: biochemical and photobiological studies using diethyl 1,4-dihydro-2,4,6-trimethyl-pyridine-3,5-dicarboxylate (DDC) as a porphyrogenic agent.J Invest Dermatol. 1969; 53: 400-413Abstract Full Text PDF PubMed Scopus (20) Google Scholar The mechanisms causing toxicity, death, and proliferation of biliary epithelial cells in the DDC diet are not well understood. Although oval cells in the AAF-rat and the proliferating biliary cells in the DDC-mice have the same origin, it is not clear whether they both represent a progenitor cell population with the same features and characteristics. Also, the purpose of proliferating biliary cells in the DDC diet is debatable, other than a local response aimed to repair biliary epithelial cells in response to injury. Such injury to biliary epithelial cells does not occur in the rat AAF/partial hepatectomy model. Although there is little doubt about the origin of the progenitor cells and (at least in the rat) their transition from biliary cells to hepatocytes, the opposite pathway has been the subject of much recent debate. In human pathology, proliferation of biliary epithelial cells yielding well-formed ductules is a common feature in obstructive cholangiopathies. Atypical biliary pseudoductules appear in primary biliary cirrhosis, an autoimmune disease associated with destruction of the intrahepatic biliary ductules. In all these situations, it is assumed that the cells of origin of the proliferating ductules are surviving biliary epithelial cells. There is also little doubt that proliferating biliary epithelial cells are the origin of new ductules formed after bile duct ligation in experimental models. In view of this, previous suggestions that hepatocytes may contribute to the formation of biliary ductules have met with skepticism. Such studies have persisted, however, to understand how the biliary epithelium can be repaired under conditions of chronic toxic or autoimmune injury (eg, primary biliary cirrhosis). Studies were conducted in rats using a chimeric rat liver in which hepatocytes of a strain of Fisher rats have a mutated and nonfunctional enzyme—dipeptidylpeptidase 4 (DPP IV). A simple histochemical assay stains DPP hepatocytes of the chemical to DPP rats causes DNA and hepatocytes from proliferation after partial hepatectomy. via the of DPP hepatocytes into the liver of DPP rats to partial hepatectomy results in a chimeric liver in which the DPP hepatocytes proliferate and liver E. R. E. S. P. D.A. liver by of isolated hepatocytes in rats treated with J Pathol. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar DPP stains hepatocyte A.V. Michalopoulos G.K. of hepatocyte growth and epidermal growth in of rat hepatocytes to biliary 2008; 47: PubMed Scopus Google Scholar and bile ductules. In the chimeric however, hepatocytes derived from the DPP cells whereas bile ductules were such chimeric livers were to bile duct a small of the proliferating bile ductules were DPP that it been derived from hepatocytes. However, this process after administration of the chemical methylene a specific to biliary epithelial cells, proliferating bile ductules of the chimeric livers to bile duct ligation a much of DPP bile G.K. of rat hepatocytes into biliary cells after bile duct ligation and toxic biliary PubMed Scopus Google Scholar The under regular in which biliary epithelium can repair the of hepatocytes to the formation of biliary ductules is the other under conditions in which the biliary epithelium cannot repair because of toxic the of hepatocytes is much and essential for the biliary repair. The same conversion of hepatocytes to biliary epithelial cells demonstrated in cultures derived from the same DPP chimeric The of DPP biliary epithelial cells the same as the of DPP hepatocytes into the Cell studies it to use conditions and identify growth factors and cytokines that this many growth factors and the were to the of hepatocytes to biliary cells, via dependent on and with and all of the other growth factors were subject to by gene and types of gene expression with all other growth factors and are essential for this The studies in rats were by a of strain thus cell to the of chimeric studies were also in of the of mouse and the of hepatocyte-specific previous studies are by Sekiya and S. Suzuki A. Hepatocytes, rather than cholangiocytes, can be the major source of primitive ductules in the chronically injured mouse liver.Am J Pathol. 2014; 184: 1468-1478Abstract Full Text Full Text PDF PubMed Scopus (94) Google have been to in which hepatocytes and biliary epithelial cells be clearly by different This on the of induced by expression of under the of specific to hepatocytes and biliary cells, but different for study. Studies by and S. N. R. H. of mature hepatocytes in mouse liver and regeneration.J PubMed Scopus Google Scholar hepatocyte-specific There no evidence of of hepatocytes to of biliary epithelial cells. However, ductules hepatocyte were studies were in in which there no injury to the biliary epithelium it to repair without needed by hepatocytes, as with the DPP chimeric rat model G.K. of rat hepatocytes into biliary cells after bile duct ligation and toxic biliary PubMed Scopus Google Scholar also that hepatocytes are a major source of a hepatic of biliary S. N. S. M. H. can from hepatocytes in PubMed Scopus Google Scholar that this is to to biliary cells of numbers that can become the source of typically a small of cell chronic to become results that the conversion of hepatocytes to biliary epithelial cells is much than in their study. By using the DDC and K. R. R.G. during liver 2013; PubMed Scopus Google Scholar that of the biliary cells were derived from hepatocytes. and K. R. R.G. during liver 2013; PubMed Scopus Google Scholar also that were expressed in hepatocytes in the DDC thus allowing for the that the origin of the biliary ductules with hepatocyte derived from hepatocytes. However, the by and K. R. R.G. during liver 2013; PubMed Scopus Google Scholar are fully with previous from human pathology, in which biliary biomarkers are expressed in human hepatocytes in several G. Michalopoulos G.K. E.R. Expression of specific hepatocyte and transcription factors in human liver disease and 2008; PubMed Scopus (67) Google Scholar Sekiya and Suzuki1Sekiya S. Suzuki A. Hepatocytes, rather than cholangiocytes, can be the major source of primitive ductules in the chronically injured mouse liver.Am J Pathol. 2014; 184: 1468-1478Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar approaches to previous by hepatocytes and biliary cells many before the DDC results that biliary epithelial is the DDC many of the new ductules derive from hepatocytes. This not in regular liver regeneration after partial hepatectomy or simple chemical injury by The many approaches to the that albumin expression by the activation of the and a of from S. Suzuki A. Hepatocytes, rather than cholangiocytes, can be the major source of primitive ductules in the chronically injured mouse liver.Am J Pathol. 2014; 184: 1468-1478Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar However, results that the DDC injury the of biliary cells that these ductules are as different from the ductules derived from biliary epithelial cells. However, the The of this from a of human disease is because in human that the conversion of hepatocytes to biliary cells, the not The also that and are in this that as no the to the formation of the biliary during S.S. formation of the of intrahepatic bile in PubMed Scopus Google Scholar Despite in and in vitro of hepatocytes to biliary cells, the studies in the mouse have different of view and which may a and expressed about the of cell studies in Although the studies in rats on a and cell of an studies in on expression of for the The functions and of of these are not fully understood. By using of biliary cells, K. H. M. S. T. S. A. T. M. T. M. K. R. E. R. T. S. cell from a progenitor in adult liver, and PubMed Scopus Google Scholar demonstrated during the of a there is of hepatocytes by other hepatocytes derived from biliary epithelial cells. that the same in the By using a similar approach, and R. van N. S. A. P. S. P. I.A. M. cells to cholangiocytes, periportal hepatocytes, and adult liver progenitor Full Text Full Text PDF PubMed Scopus Google Scholar demonstrated that there no such with the of immediate periportal S. N. R. H. of mature hepatocytes in mouse liver and regeneration.J PubMed Scopus Google Scholar It is to such results on the of these approaches on a new in the mouse it is that the location of the in the not may be proximate to other is causing results not to the of the or the In the by K. H. M. S. T. S. A. T. M. T. M. K. R. E. R. T. S. cell from a progenitor in adult liver, and PubMed Scopus Google Scholar it is that hepatocytes may have a block that inhibits the of cell proliferation for simple maintenance of hepatocyte numbers for the maintenance of the organ. this were to be the the biliary cells would be activated to hepatocytes, as in the AAF/partial hepatectomy model in the The existence of between biliary cells and hepatocytes as no of the cell types derive in development from The cells a gene expression The between hepatocytes and biliary cells in development in the R. van N. S. A. P. S. P. I.A. M. cells to cholangiocytes, periportal hepatocytes, and adult liver progenitor Full Text Full Text PDF PubMed Scopus Google Scholar Thus, it be that of be rather This is hepatocytes are in primary to of hepatocytes biomarkers of biliary epithelial H.C. expression by adult rat hepatocytes on A. PubMed Scopus Google Scholar studies have also shown that at the the of into with hepatocytes in the there are cells transcription factors and biomarkers common to both cell K. A. K. S. M. Michalopoulos G. epithelial in human a in epithelial 2013; PubMed Scopus Google Scholar Thus, such cells to but it is not clear whether they exist in numbers that would be a source to all or a of the progenitor cells. In biliary cells of mature intrahepatic ductules hepatocyte-specific transcription factors after initiation of the AAF/partial hepatectomy model. However, as Sekiya and Suzuki1Sekiya S. Suzuki A. Hepatocytes, rather than cholangiocytes, can be the major source of primitive ductules in the chronically injured mouse liver.Am J Pathol. 2014; 184: 1468-1478Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar progenitor cells in the as as on the causing the hepatic It is that the biliary cells of the ductules are the source of oval cells in periportal locations, whereas progenitor cells in of the may derive from cells of differentiation in the of The in also have for human liver Expression of biliary transcription factors in hepatocytes is in human cholangiopathies. expression of transcription factors in biliary cells is in chronic associated with continual hepatocyte G. Michalopoulos G.K. E.R. Expression of specific hepatocyte and transcription factors in human liver disease and 2008; PubMed Scopus (67) Google Scholar are liver histological in human hepatic It many of cells with and biliary A. E. A. Michalopoulos G.K. Expression of hepatocyte epidermal growth factor and in of hepatocytes, cholangiocytes, and progenitor cells in human liver Pathol. 2013; Full Text Full Text PDF PubMed Scopus Google Scholar It is not clear which the is in these The number of proliferating biliary epithelial cells transcription factors suggests that the in the proliferating biliary epithelium an to repair a organ. A of of hepatic as much as There is no evidence that the of are associated with these It is that little histological from such because of the of with all of the and the associated Most of knowledge about these of from livers in the of liver It is of in these the receptors for and are but their activation is in the hepatocyte of the cell proliferation is that the of these receptors is not aimed at cell proliferation but rather toward of cell This is of the role that these receptors play in of in as The expression of receptor and the growth S. J.H. 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