The American Journal of Pathology, Vol. 184, No. 5, May 2014
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COMMENTARY The Liver Is a Peculiar Organ When It Comes to Stem Cells George K. Michalopoulos From the Department of Pathology, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania
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 tissuespecific 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 Suzuki1 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 cells2 and identification of isolated candidate cells with aberrant biomarkers by electron microscopy or immunohistochemistry,3 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,4,5 raising questions about their existence.
Progenitor Cells and Their Origins from Biliary Epithelium 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 Copyright ª 2014 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ajpath.2014.02.020
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 tissuespecific stem cells, whereas the label is diluted from the multiple sequential proliferation cycles of the progenitor cells. By using this approach, Theise and colleagues6 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 tissuespecific 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.5,7 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.8 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 a-fetoprotein production. This is associated with the expression of hepatocyte-specific transcription factors in the biliary epithelial cells.9e11 Toxic damage to Supported by NIH grant CA103958. Accepted for publication February 24, 2014. Disclosures: None declared. Address correspondence to George K. Michalopoulos, M.D., Ph.D., Department of Pathology, School of Medicine, University of Pittsburgh, S-410 Biomedical Science Tower, Pittsburgh, PA 15261. E-mail: [email protected]
Michalopoulos the biliary cells before the initiation of the oval cell process eliminates the appearance of the oval cells.12 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.9e11 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 tissuebased 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.13 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).9,11 The administration of AAF causes DNA adduct formation and activation of Cdkn1a-dependent (alias p21) and Trp53dependent 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.14 However, N-sulfotransferase does not exist in the mouse14 and AAF is a weak mouse carcinogen and it does not block liver regeneration. Therefore, many investigators, including Sekiya and Suzuki,1 use the 3,5-diethoxycarbonyl-1,4dihydrocollidine (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.15 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
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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.
Hepatocytes as Facultative Stem Cells for Biliary Epithelium 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 enzymeddipeptidylpeptidase 4 (DPP IV). A simple histochemical assay stains DPP IVe positive hepatocytes orange. Administration of the chemical retrorsine to DPP IVenegative rats causes DNA crosslinking and prevents hepatocytes from proliferation after partial hepatectomy. Direct injection via the portal vein of DPP IVepositive hepatocytes into the liver of retrorsinetreated DPP IVenegative rats subjected to partial hepatectomy results in a chimeric liver in which the DPP IVepositive hepatocytes proliferate and restore liver mass.16 DPP IV stains hepatocyte bile,17 canaliculi, and bile ductules. In the chimeric livers, however, only hepatocytes derived from the injected DPP IVepositive cells appeared positive, whereas bile ductules were uniformly negative. When such chimeric livers were subjected to bile duct ligation, a small percentage (1.46%) of the newly proliferating bile ductules were DPP IV positive, suggesting that it had been derived from hepatocytes. However, when this process was performed after administration of the chemical methylene dianiline, a specific toxin to biliary epithelial cells, proliferating bile ductules of the chimeric livers subjected to bile duct ligation had a much higher percentage (53.02%) of DPP IVepositive bile ductules.18 The study showed that, under regular conditions, in which biliary epithelium can repair itself, the contribution of hepatocytes to the formation of biliary ductules is small. On the other hand, under
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Commentary conditions in which the biliary epithelium cannot repair itself because of toxic injury, the contribution of hepatocytes is much more expanded and essential for the biliary repair. The same conversion of hepatocytes to biliary epithelial cells was demonstrated in ordinary cultures derived from the same DPP IV chimeric livers. The percentage of DPP IVepositive biliary epithelial cells was the same as the percentage of DPP IVepositive hepatocytes introduced into the cultures. Cell culture studies made it possible to use selective conditions and identify growth factors and cytokines that support this conversion. From many growth factors studied, only HGF and the EGFR ligand EGF were able to facilitate the transdifferentiation of hepatocytes to biliary cells, via pathways dependent on phosphoinositide 3-kinase and Akt. Cultures with HGF, EGF, and all of the other growth factors were subject to analysis by gene array. HGF and EGF could induce distinct types of gene expression patterns compared with all other growth factors and are probably essential for facilitating this transdifferentiation. The studies in rats were facilitated by a naturally occurring advantageous situation of strain tagging (and thus cell tagging), leading to the generation of chimeric livers. Multiple subsequent studies were also initiated in mice, taking advantage of the tools of mouse genetics and the availability of well-characterized, hepatocyte-specific vectors. (These previous studies are extensively discussed by Sekiya and Suzuki.1) Various tools have been used to generate lineage tagging, in which hepatocytes and biliary epithelial cells could be clearly distinguished by different immunofluorescence colors. This was on the basis of genetic manipulations induced by expression of Cre recombinase under the control of promoters specific to hepatocytes and biliary cells, but different for each study. Studies by Willenbring and colleagues19 used hepatocyte-specific vectors. There was no evidence of transdifferentiation of hepatocytes to ducts composed of biliary epithelial cells. However, atypical ductules carrying hepatocyte markers were occasionally seen. These studies were performed in situations in which there was no injury to the biliary epithelium and, thus, it was probably able to repair itself without needed contribution by hepatocytes, as with the DPP IV chimeric rat model previously described.18 They also reported that hepatocytes are a major source of cholangiocarcinoma, a hepatic malignancy composed of neoplastic biliary cells,20 suggesting that this transdifferentiation is frequent enough to lead to biliary cells of sufficient numbers that can stochastically become the source of cholangiocarcinomas. Because typically only a small percentage of any cell pool accumulates sufficient chronic alterations to become malignant, results suggest that the conversion of hepatocytes to biliary epithelial cells is much more frequent than envisioned in their earlier study. By using the DDC model, Stanger and collueages21 found that approximately 14% of the biliary cells were derived from hepatocytes. Stanger and colleagues21 also found that biliary-associated markers were expressed in hepatocytes in
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the DDC diet, thus allowing for the possibility that the origin of the biliary ductules with hepatocyte markers was derived from altered hepatocytes. However, the observations by Stanger and colleagues21 are fully consistent with previous observations from human pathology, in which biliary biomarkers are expressed in human hepatocytes in several cholangiopathies (and vice versa).22 Sekiya and Suzuki1 used approaches intended to overcome previous concerns, by tagging hepatocytes and biliary cells many days before administering the DDC diet. Their results unambiguously demonstrate that when biliary epithelial viability is impaired (by the DDC diet), many of the emerging new ductules derive from hepatocytes. This was not seen in regular liver regeneration after partial hepatectomy or simple chemical injury by CCl4. The authors used many approaches to eliminate the possibility that transient albumin expression by the cholangiocytes could trigger activation of the Cre recombinase and generate a false impression of derivation from hepatocytes.1 However, results show that when the DDC injury was withdrawn, the percentage of hepatocyte-derived biliary cells dramatically decreased, suggesting that these ductules are seen as different from the native ductules derived from biliary epithelial cells. However, the reasons remain unclear. The significance of this finding from a perspective of human disease is probably small, because in most human diseases that trigger the conversion of hepatocytes to biliary cells, the injurious stimuli do not go away. The authors also found that Notch and Hes-1 are involved in this transdifferentiation, something that comes as no surprise given the extensive literature linking Notch to the formation of the biliary tree during embryonic development.23
Clarity and Controversies: Genetic Lineage Tagging Despite established in vivo and in vitro transdifferentiation of hepatocytes to biliary cells, the studies in the mouse have generated different points of view and considerable controversy, which may reflect a justified and often expressed skepticism about the apparent clarity of cell lineage tagging studies in mouse. Although the studies done in rats depend on a naturally occurring and stable cell tagging mutation of an enzyme, lineage tagging studies in mice depend on expression of promoters used for the genetic manipulation. The functions and cellular specificities of most of these promoters are not fully understood. By using lineage tagging of biliary cells, Furuyama et al24 demonstrated that, during the course of a mouse’s life, there is progressive replacement of hepatocytes by other hepatocytes derived from biliary epithelial cells. They showed that the same phenomenon occurs in the pancreas. By using a similar lineage tagging approach, Lemaigre and colleagues25 demonstrated that there was no such replacement, with the exception of immediate periportal hepatocytes.19 It is difficult to reconcile such opposing results
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Michalopoulos purely on the basis of promoter function per se. Because these approaches often depend on inserting a new genetic element in the mouse DNA, it is conceivable that the location of the transgene in the genome (usually not identified) may be proximate to other genes whose function is inadvertently modified, causing results not related to the innate function of the promoter or the transgene. In the study by Furuyama et al,24 it is possible that hepatocytes may have acquired a block that inhibits the amount of cell proliferation required for simple maintenance of hepatocyte numbers required for the maintenance of the organ. If this were to be the case, then the biliary cells would be activated to slowly replace hepatocytes, as in the AAF/partial hepatectomy model in the rat. The existence of transdifferentiation patterns between biliary cells and hepatocytes should come as no surprise. Both of the cell types derive in embryonic development from hepatoblasts. The latter cells carry a predominantly hepatocytic gene expression pattern. The lineage split between hepatocytes and biliary cells occurs late in embryonic development in the rodents.25 Thus, it should be expected that interconversions of phenotype should be rather easy. This is seen when hepatocytes are placed in primary culture. Within 2 to 3 days of culture, hepatocytes begin expressing biomarkers characteristic of biliary epithelial cells.26 Recent studies have also shown that at the point where the canals of Hering come into contact with hepatocytes deep in the lobule, there are cells expressing transcription factors and biomarkers common to both cell types.27 Thus, such cells seem to pre-exist, but it is not clear whether they exist in numbers that would be a sufficient source to generate all or a portion of the progenitor cells. In addition, biliary cells of mature intrahepatic ductules do acquire hepatocyte-specific transcription factors soon after initiation of the AAF/partial hepatectomy model. However, as Sekiya and Suzuki1 point out, progenitor cells often arise deep in the lobule, as far as pericentral regions, depending on the toxin causing the hepatic damage. It is possible that the biliary cells of the portal ductules are the source of oval cells arising in periportal locations, whereas progenitor cells arising in pericentral regions of the lobule may derive from pre-existing cells of mixed differentiation deep in the canals of Hering.
Implications for Regenerative Mechanisms in Liver Failure The findings in animal models also have implications for human liver disease. Expression of biliary transcription factors in hepatocytes is seen in human cholangiopathies. Conversely, expression of hepatocyte-associated transcription factors in biliary cells is seen in chronic diseases associated with continual hepatocyte death.22 Even more revealing are liver histological characteristics in human fulminant hepatic failure. It shows many regenerative clusters composed of cells with mixed hepatocytic and biliary differentiation.28 It is not clear which way the transdifferentiation is proceeding in these
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clusters. The overall huge number of proliferating biliary epithelial cells rampantly expressing hepatocyte-associated transcription factors suggests that the regenerative clusters in the proliferating biliary epithelium support an SOS mechanism to repair a rapidly declining organ. A fraction of cases of fulminant hepatic failure, as much as 30%, spontaneously recover. There is no evidence that the cases of spontaneous recovery are associated with these regenerative clusters. It is unfortunate that little histological material exists from such cases because of the difficulty of performing biopsies, with all of the coagulopathies and the associated high morbidity. Most of our knowledge about these clusters of transdifferentiation comes from livers removed in the course of orthotopic liver transplantation. It is of interest that, in these clusters, the receptors for HGF and EGF are widely expressed, but their activation is only seen in the hepatocyte component of the clusters. Little cell proliferation is seen, suggesting that the effect of these mitogenic receptors is not aimed at cell proliferation but rather toward control of cell differentiation. This finding is reminiscent of the critical role that these two receptors play in control of transdifferentiation seen in organoid cultures, as previously mentioned. The high expression of receptor FAS and the growth inhibitor GPC329 in these clusters may play a regulatory role.
Summary and General Considerations It is now clear that when it comes to stem cell biology, liver is a bipolar organ. Committed, liver-specific stem cells, with no other function than their stemness, do not appear to exist in liver, as in intestine and skin. Biliary cells and hepatocytes can function as facultative stem cells for each other if one of the two cell systems is incapable of repairing itself in situations that are critical for the survival of the organ. Although seemingly paradoxical, it does have advantages related to organ survival. The existence of many facultative stem cells allows for quick recovery because only a few proliferative events are needed to provide sufficient numbers or progenitor cells for effective repair. This is not the case for tissue-specific stem cells, each one of which needs to proliferate multiple times to generate sufficient numbers of progenitor cells. All liver-specific regenerative pathways, most of them extreme compared with other organs, have been honed by evolution to allow for quick recovery of an organ vital for the survival of the organism.
References 1. Sekiya 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, 184:1468e1478 2. Sell S: Is there a liver stem cell? Cancer Res 1990, 50:3811e3815 3. Novikoff PM, Yam A: Stem cells and rat liver carcinogenesis: contributions of confocal and electron microscopy. J Histochem Cytochem 1998, 46:613e626 4. Stöcker E, Wullstein HK, Bräu G: Capacity of regeneration in liver epithelia of juvenile, repeated partially hepatectomized rats:
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autoradiographic studies after continous infusion of 3H-thymidine (author’s transl) [German]. Virchows Arch B Cell Pathol 1973, 14: 93e103 Michalopoulos GK: Principles of liver regeneration and growth homeostasis. Compr Physiol 2013, 3:485e513 Kuwahara R, Kofman AV, Landis CS, Swenson ES, Barendswaard E, Theise ND: The hepatic stem cell niche: identification by labelretaining cell assay. Hepatology 2008, 47:1994e2002 Michalopoulos GK: Liver regeneration. J Cell Physiol 2007, 213: 286e300 Tatematsu M, Ho RH, Kaku T, Ekem JK, Farber E: Studies on the proliferation and fate of oval cells in the liver of rats treated with 2acetylaminofluorene and partial hepatectomy. Am J Pathol 1984, 114:418e430 Evarts RP, Hu Z, Omori N, Omori M, Marsden ER, Thorgeirsson SS: Precursor-product relationship between oval cells and hepatocytes: comparison between tritiated thymidine and bromodeoxyuridine as tracers. Carcinogenesis 1996, 17:2143e2151 Hu Z, Evarts RP, Fujio K, Marsden ER, Thorgeirsson SS: Expression of fibroblast growth factor receptors flg and bek during hepatic ontogenesis and regeneration in the rat. Cell Growth Differ 1995, 6: 1019e1025 Nagy P, Bisgaard HC, Thorgeirsson SS: Expression of hepatic transcription factors during liver development and oval cell differentiation. J Cell Biol 1994, 126:223e233 Petersen BE, Zajac VF, Michalopoulos GK: Bile ductular damage induced by methylene dianiline inhibits oval cell activation. Am J Pathol 1997, 151:905e909 Huch M, Dorrell C, Boj SF, van Es JH, Li VS, van de Wetering M, Sato T, Hamer K, Sasaki N, Finegold MJ, Haft A, Vries RG, Grompe M, Clevers H: In vitro expansion of single Lgr5þ liver stem cells induced by Wnt-driven regeneration. Nature 2013, 494:247e250 DeBaun JR, Rowley JY, Miller EC, Miller JA: Sulfotransferase activation of N-hydroxy-2-acetylaminofluorene in rodent livers susceptible and resistant to this carcinogen. Proc Soc Exp Biol Med 1968, 129: 268e273 Magnus IA, Roe DA, Bhutani LK: 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, 53:400e413 Laconi E, Oren R, Mukhopadhyay DK, Hurston E, Laconi S, Pani P, Dabeva MD, Shafritz DA: Long-term, near-total liver replacement by transplantation of isolated hepatocytes in rats treated with retrorsine. Am J Pathol 1998, 153:319e329 Limaye PB, Bowen WC, Orr AV, Luo J, Tseng GC, Michalopoulos GK: Mechanisms of hepatocyte growth factor-mediated and epidermal growth factor-mediated signaling in transdifferentiation of rat hepatocytes to biliary epithelium. Hepatology 2008, 47:1702e1713
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18. Michalopoulos GK, Barua L, Bowen WC: Transdifferentiation of rat hepatocytes into biliary cells after bile duct ligation and toxic biliary injury. Hepatology 2005, 41:535e544 19. Malato Y, Naqvi S, Schurmann N, Ng R, Wang B, Zape J, Kay MA, Grimm D, Willenbring H: Fate tracing of mature hepatocytes in mouse liver homeostasis and regeneration. J Clin Invest 2011, 121:4850e4860 20. Fan B, Malato Y, Calvisi DF, Naqvi S, Razumilava N, Ribback S, Gores GJ, Dombrowski F, Evert M, Chen X, Willenbring H: Cholangiocarcinomas can originate from hepatocytes in mice. J Clin Invest 2012, 122:2911e2915 21. Yanger K, Zong Y, Maggs LR, Shapira SN, Maddipati R, Aiello NM, Thung SN, Wells RG, Greenbaum LE, Stanger BZ: Robust cellular reprogramming occurs spontaneously during liver regeneration. Genes Dev 2013, 27:719e724 22. Limaye PB, Alarcon G, Walls AL, Nalesnik MA, Michalopoulos GK, Demetris AJ, Ochoa ER: Expression of specific hepatocyte and cholangiocyte transcription factors in human liver disease and embryonic development. Lab Invest 2008, 88:865e872 23. Sparks EE, Huppert KA, Brown MA, Washington MK, Huppert SS: Notch signaling regulates formation of the three-dimensional architecture of intrahepatic bile ducts in mice. Hepatology 2010, 51:1391e1400 24. Furuyama K, Kawaguchi Y, Akiyama H, Horiguchi M, Kodama S, Kuhara T, Hosokawa S, Elbahrawy A, Soeda T, Koizumi M, Masui T, Kawaguchi M, Takaori K, Doi R, Nishi E, Kakinoki R, Deng JM, Behringer RR, Nakamura T, Uemoto S: Continuous cell supply from a Sox9-expressing progenitor zone in adult liver, exocrine pancreas and intestine. Nat Genet 2011, 43:34e41 25. Carpentier R, Suner RE, van Hul N, Kopp JL, Beaudry JB, Cordi S, Antoniou A, Raynaud P, Lepreux S, Jacquemin P, Leclercq IA, Sander M, Lemaigre FP: Embryonic ductal plate cells give rise to cholangiocytes, periportal hepatocytes, and adult liver progenitor cells. Gastroenterology 2011, 141:1432e1438. 1438.e1e4 26. Sirica AE, Richards W, Tsukada Y, Sattler CA, Pitot HC: Fetal phenotypic expression by adult rat hepatocytes on collagen gel/nylon meshes. Proc Natl Acad Sci U S A 1979, 76:283e287 27. Isse K, Lesniak A, Grama K, Maier J, Specht S, Castillo-Rama M, Lunz J, Roysam B, Michalopoulos G, Demetris AJ: Preexisting epithelial diversity in normal human livers: a tissue-tethered cytometric analysis in portal/periportal epithelial cells. Hepatology 2013, 57:1632e1643 28. Hattoum A, Rubin E, Orr A, Michalopoulos GK: Expression of hepatocyte epidermal growth factor receptor, FAS and glypican 3 in EpCAM-positive regenerative clusters of hepatocytes, cholangiocytes, and progenitor cells in human liver failure. Hum Pathol 2013, 44: 743e749 29. Liu B, Bell AW, Paranjpe S, Bowen WC, Khillan JS, Luo JH, Mars WM, Michalopoulos GK: Suppression of liver regeneration and hepatocyte proliferation in hepatocyte-targeted glypican 3 transgenic mice. Hepatology 2010, 52:1060e1067
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See related article on page 1468.
COMMENTARY The Liver Is a Peculiar Organ When It Comes to Stem Cells George K. Michalopoulos From the Department of Pathology, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania
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 tissuespecific 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 Suzuki1 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 cells2 and identification of isolated candidate cells with aberrant biomarkers by electron microscopy or immunohistochemistry,3 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,4,5 raising questions about their existence.
Progenitor Cells and Their Origins from Biliary Epithelium 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 Copyright ª 2014 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ajpath.2014.02.020
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 tissuespecific stem cells, whereas the label is diluted from the multiple sequential proliferation cycles of the progenitor cells. By using this approach, Theise and colleagues6 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 tissuespecific 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.5,7 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.8 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 a-fetoprotein production. This is associated with the expression of hepatocyte-specific transcription factors in the biliary epithelial cells.9e11 Toxic damage to Supported by NIH grant CA103958. Accepted for publication February 24, 2014. Disclosures: None declared. Address correspondence to George K. Michalopoulos, M.D., Ph.D., Department of Pathology, School of Medicine, University of Pittsburgh, S-410 Biomedical Science Tower, Pittsburgh, PA 15261. E-mail: [email protected]
Michalopoulos the biliary cells before the initiation of the oval cell process eliminates the appearance of the oval cells.12 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.9e11 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 tissuebased 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.13 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).9,11 The administration of AAF causes DNA adduct formation and activation of Cdkn1a-dependent (alias p21) and Trp53dependent 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.14 However, N-sulfotransferase does not exist in the mouse14 and AAF is a weak mouse carcinogen and it does not block liver regeneration. Therefore, many investigators, including Sekiya and Suzuki,1 use the 3,5-diethoxycarbonyl-1,4dihydrocollidine (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.15 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
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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.
Hepatocytes as Facultative Stem Cells for Biliary Epithelium 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 enzymeddipeptidylpeptidase 4 (DPP IV). A simple histochemical assay stains DPP IVe positive hepatocytes orange. Administration of the chemical retrorsine to DPP IVenegative rats causes DNA crosslinking and prevents hepatocytes from proliferation after partial hepatectomy. Direct injection via the portal vein of DPP IVepositive hepatocytes into the liver of retrorsinetreated DPP IVenegative rats subjected to partial hepatectomy results in a chimeric liver in which the DPP IVepositive hepatocytes proliferate and restore liver mass.16 DPP IV stains hepatocyte bile,17 canaliculi, and bile ductules. In the chimeric livers, however, only hepatocytes derived from the injected DPP IVepositive cells appeared positive, whereas bile ductules were uniformly negative. When such chimeric livers were subjected to bile duct ligation, a small percentage (1.46%) of the newly proliferating bile ductules were DPP IV positive, suggesting that it had been derived from hepatocytes. However, when this process was performed after administration of the chemical methylene dianiline, a specific toxin to biliary epithelial cells, proliferating bile ductules of the chimeric livers subjected to bile duct ligation had a much higher percentage (53.02%) of DPP IVepositive bile ductules.18 The study showed that, under regular conditions, in which biliary epithelium can repair itself, the contribution of hepatocytes to the formation of biliary ductules is small. On the other hand, under
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Commentary conditions in which the biliary epithelium cannot repair itself because of toxic injury, the contribution of hepatocytes is much more expanded and essential for the biliary repair. The same conversion of hepatocytes to biliary epithelial cells was demonstrated in ordinary cultures derived from the same DPP IV chimeric livers. The percentage of DPP IVepositive biliary epithelial cells was the same as the percentage of DPP IVepositive hepatocytes introduced into the cultures. Cell culture studies made it possible to use selective conditions and identify growth factors and cytokines that support this conversion. From many growth factors studied, only HGF and the EGFR ligand EGF were able to facilitate the transdifferentiation of hepatocytes to biliary cells, via pathways dependent on phosphoinositide 3-kinase and Akt. Cultures with HGF, EGF, and all of the other growth factors were subject to analysis by gene array. HGF and EGF could induce distinct types of gene expression patterns compared with all other growth factors and are probably essential for facilitating this transdifferentiation. The studies in rats were facilitated by a naturally occurring advantageous situation of strain tagging (and thus cell tagging), leading to the generation of chimeric livers. Multiple subsequent studies were also initiated in mice, taking advantage of the tools of mouse genetics and the availability of well-characterized, hepatocyte-specific vectors. (These previous studies are extensively discussed by Sekiya and Suzuki.1) Various tools have been used to generate lineage tagging, in which hepatocytes and biliary epithelial cells could be clearly distinguished by different immunofluorescence colors. This was on the basis of genetic manipulations induced by expression of Cre recombinase under the control of promoters specific to hepatocytes and biliary cells, but different for each study. Studies by Willenbring and colleagues19 used hepatocyte-specific vectors. There was no evidence of transdifferentiation of hepatocytes to ducts composed of biliary epithelial cells. However, atypical ductules carrying hepatocyte markers were occasionally seen. These studies were performed in situations in which there was no injury to the biliary epithelium and, thus, it was probably able to repair itself without needed contribution by hepatocytes, as with the DPP IV chimeric rat model previously described.18 They also reported that hepatocytes are a major source of cholangiocarcinoma, a hepatic malignancy composed of neoplastic biliary cells,20 suggesting that this transdifferentiation is frequent enough to lead to biliary cells of sufficient numbers that can stochastically become the source of cholangiocarcinomas. Because typically only a small percentage of any cell pool accumulates sufficient chronic alterations to become malignant, results suggest that the conversion of hepatocytes to biliary epithelial cells is much more frequent than envisioned in their earlier study. By using the DDC model, Stanger and collueages21 found that approximately 14% of the biliary cells were derived from hepatocytes. Stanger and colleagues21 also found that biliary-associated markers were expressed in hepatocytes in
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the DDC diet, thus allowing for the possibility that the origin of the biliary ductules with hepatocyte markers was derived from altered hepatocytes. However, the observations by Stanger and colleagues21 are fully consistent with previous observations from human pathology, in which biliary biomarkers are expressed in human hepatocytes in several cholangiopathies (and vice versa).22 Sekiya and Suzuki1 used approaches intended to overcome previous concerns, by tagging hepatocytes and biliary cells many days before administering the DDC diet. Their results unambiguously demonstrate that when biliary epithelial viability is impaired (by the DDC diet), many of the emerging new ductules derive from hepatocytes. This was not seen in regular liver regeneration after partial hepatectomy or simple chemical injury by CCl4. The authors used many approaches to eliminate the possibility that transient albumin expression by the cholangiocytes could trigger activation of the Cre recombinase and generate a false impression of derivation from hepatocytes.1 However, results show that when the DDC injury was withdrawn, the percentage of hepatocyte-derived biliary cells dramatically decreased, suggesting that these ductules are seen as different from the native ductules derived from biliary epithelial cells. However, the reasons remain unclear. The significance of this finding from a perspective of human disease is probably small, because in most human diseases that trigger the conversion of hepatocytes to biliary cells, the injurious stimuli do not go away. The authors also found that Notch and Hes-1 are involved in this transdifferentiation, something that comes as no surprise given the extensive literature linking Notch to the formation of the biliary tree during embryonic development.23
Clarity and Controversies: Genetic Lineage Tagging Despite established in vivo and in vitro transdifferentiation of hepatocytes to biliary cells, the studies in the mouse have generated different points of view and considerable controversy, which may reflect a justified and often expressed skepticism about the apparent clarity of cell lineage tagging studies in mouse. Although the studies done in rats depend on a naturally occurring and stable cell tagging mutation of an enzyme, lineage tagging studies in mice depend on expression of promoters used for the genetic manipulation. The functions and cellular specificities of most of these promoters are not fully understood. By using lineage tagging of biliary cells, Furuyama et al24 demonstrated that, during the course of a mouse’s life, there is progressive replacement of hepatocytes by other hepatocytes derived from biliary epithelial cells. They showed that the same phenomenon occurs in the pancreas. By using a similar lineage tagging approach, Lemaigre and colleagues25 demonstrated that there was no such replacement, with the exception of immediate periportal hepatocytes.19 It is difficult to reconcile such opposing results
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Michalopoulos purely on the basis of promoter function per se. Because these approaches often depend on inserting a new genetic element in the mouse DNA, it is conceivable that the location of the transgene in the genome (usually not identified) may be proximate to other genes whose function is inadvertently modified, causing results not related to the innate function of the promoter or the transgene. In the study by Furuyama et al,24 it is possible that hepatocytes may have acquired a block that inhibits the amount of cell proliferation required for simple maintenance of hepatocyte numbers required for the maintenance of the organ. If this were to be the case, then the biliary cells would be activated to slowly replace hepatocytes, as in the AAF/partial hepatectomy model in the rat. The existence of transdifferentiation patterns between biliary cells and hepatocytes should come as no surprise. Both of the cell types derive in embryonic development from hepatoblasts. The latter cells carry a predominantly hepatocytic gene expression pattern. The lineage split between hepatocytes and biliary cells occurs late in embryonic development in the rodents.25 Thus, it should be expected that interconversions of phenotype should be rather easy. This is seen when hepatocytes are placed in primary culture. Within 2 to 3 days of culture, hepatocytes begin expressing biomarkers characteristic of biliary epithelial cells.26 Recent studies have also shown that at the point where the canals of Hering come into contact with hepatocytes deep in the lobule, there are cells expressing transcription factors and biomarkers common to both cell types.27 Thus, such cells seem to pre-exist, but it is not clear whether they exist in numbers that would be a sufficient source to generate all or a portion of the progenitor cells. In addition, biliary cells of mature intrahepatic ductules do acquire hepatocyte-specific transcription factors soon after initiation of the AAF/partial hepatectomy model. However, as Sekiya and Suzuki1 point out, progenitor cells often arise deep in the lobule, as far as pericentral regions, depending on the toxin causing the hepatic damage. It is possible that the biliary cells of the portal ductules are the source of oval cells arising in periportal locations, whereas progenitor cells arising in pericentral regions of the lobule may derive from pre-existing cells of mixed differentiation deep in the canals of Hering.
Implications for Regenerative Mechanisms in Liver Failure The findings in animal models also have implications for human liver disease. Expression of biliary transcription factors in hepatocytes is seen in human cholangiopathies. Conversely, expression of hepatocyte-associated transcription factors in biliary cells is seen in chronic diseases associated with continual hepatocyte death.22 Even more revealing are liver histological characteristics in human fulminant hepatic failure. It shows many regenerative clusters composed of cells with mixed hepatocytic and biliary differentiation.28 It is not clear which way the transdifferentiation is proceeding in these
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clusters. The overall huge number of proliferating biliary epithelial cells rampantly expressing hepatocyte-associated transcription factors suggests that the regenerative clusters in the proliferating biliary epithelium support an SOS mechanism to repair a rapidly declining organ. A fraction of cases of fulminant hepatic failure, as much as 30%, spontaneously recover. There is no evidence that the cases of spontaneous recovery are associated with these regenerative clusters. It is unfortunate that little histological material exists from such cases because of the difficulty of performing biopsies, with all of the coagulopathies and the associated high morbidity. Most of our knowledge about these clusters of transdifferentiation comes from livers removed in the course of orthotopic liver transplantation. It is of interest that, in these clusters, the receptors for HGF and EGF are widely expressed, but their activation is only seen in the hepatocyte component of the clusters. Little cell proliferation is seen, suggesting that the effect of these mitogenic receptors is not aimed at cell proliferation but rather toward control of cell differentiation. This finding is reminiscent of the critical role that these two receptors play in control of transdifferentiation seen in organoid cultures, as previously mentioned. The high expression of receptor FAS and the growth inhibitor GPC329 in these clusters may play a regulatory role.
Summary and General Considerations It is now clear that when it comes to stem cell biology, liver is a bipolar organ. Committed, liver-specific stem cells, with no other function than their stemness, do not appear to exist in liver, as in intestine and skin. Biliary cells and hepatocytes can function as facultative stem cells for each other if one of the two cell systems is incapable of repairing itself in situations that are critical for the survival of the organ. Although seemingly paradoxical, it does have advantages related to organ survival. The existence of many facultative stem cells allows for quick recovery because only a few proliferative events are needed to provide sufficient numbers or progenitor cells for effective repair. This is not the case for tissue-specific stem cells, each one of which needs to proliferate multiple times to generate sufficient numbers of progenitor cells. All liver-specific regenerative pathways, most of them extreme compared with other organs, have been honed by evolution to allow for quick recovery of an organ vital for the survival of the organism.
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