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The quest for liver progenitor cells: A practical point of view

Open AccessPublished:October 28, 2009DOI:https://doi.org/10.1016/j.jhep.2009.10.009
      Many chronic liver diseases can lead to hepatic dysfunction with organ failure. At present, orthotopic liver transplantation represents the benchmark therapy of terminal liver disease. However this practice is limited by shortage of donor grafts, the need for lifelong immunosuppression and very demanding state-of-the-art surgery. For this reason, new therapies have been developed to restore liver function, primarily in the form of hepatocyte transplantation and artificial liver support devices. While already offered in very specialized centers, both of these modalities still remain experimental. Recently, liver progenitor cells have shown great promise for cell therapy, and consequently they have attracted a lot of attention as an alternative or supportive tool for liver transplantation. These liver progenitor cells are quiescent in the healthy liver and become activated in certain liver diseases in which the regenerative capacity of mature hepatocytes and/or cholangiocytes is impaired. Although reports describing liver progenitor cells are numerous, they have not led to a consensus on the identity of the liver progenitor cell. In this review, we will discuss some of the characteristics of these cells and the different ways that have been used to obtain these from rodents. We will also highlight the challenges that researchers are facing in their quest to identify and use liver progenitor cells.

      Keywords

      Abbreviations:

      DNA (deoxyribonucleic acid), LPC (liver progenitor cell), CCl4 (carbotetrachloride), AAF (2-acetylaminofluorene), APAP (N-acetyl-p-aminophenol), SCF (stem cell factor), SDF1 (stromalcell-derived factor-1), CXCR4 (CXC chemokine receptor4), TWEAK (tumor necrosis factor-like weak inducer of apoptosis), KT (cytokeratin), ALB (albumin), AAT (alpha anti-trypsine), CD (cluster of differentiation), AFP (alpha fetoprotein), N-CAM (neural-cell adhesion molecule), Thy-1 (thymus antigen 1), Sca-1 (stem cell antigen 1), BM (basal membrane), ECM (extracellular matrix), IL (interleukin), TNF (tumor necrosis factor), CDE (choline-deficient, ethionine-supplemented diet), AA (allyl alcohol), PH (partial hepatectomy), EpCAM (epithelial cell adhesion molecule), ABCG2 (ATP-binding-cassette transporter-G2), PF (parenchymal fraction), NPF (non-parenchymal fraction), MACS (magnetic activated cell sorting), FACS (fluorescence-activated cell sorting), SP (side population), LCM (laser capture micro-dissection), FAH (fumarylacetoacetate hydrolase), GFP (green fluorescent protein), DDC (3-diethoxycarbonyl-1,4-dihydrocollidine), DIPIN (1,4-bis[N,N′-di(ethylene)phosphamide]piperazine), DEN (diethylnitrosamine), CCRP (core circadian regulatory protein), HNF (hepatocyte nuclear factor), Cx (Connexin), MPK (muscle pyruvate kinase), GST (glutathione S transferase), GGT (gamma glutamyl transpeptidase), Dlk (delta-like protein), Chrom-A (Chromogranin A)

      Introduction

      At present orthotopic liver transplantation is the standard treatment for several acute (e.g. intoxication, fulminant viral hepatitis), chronic inborn (e.g. urea cycle disorders, glycogenosis type I, Crigler Najjar) or acquired (e.g. non-alcoholic fatty liver disease, chronic viral hepatitis) end stage liver diseases. As a consequence of the worldwide shortage of donor organs, allocation of liver grafts in a fair and balanced manner has given rise to controversial ethical discussions [
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      • Samuel D.
      Hepatic transplantation.
      ] (for instance, what criteria make a patient eligible to receive a donor organ?). Additionally, the technically demanding “state-of-the-art” surgery [
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      • Davis C.L.
      Renal dysfunction in the perioperative liver transplant period.
      ], and especially the cost and risks of a life long immunosuppression [
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      • Botha J.F.
      • Mukherjee U.
      Immunosuppression in liver transplantation.
      ] have prompted the search for alternative treatments. Transplantation of isolated hepatocytes represents a treatment option for inborn errors of liver metabolism, to bridge unstable patients to transplantation or allows a patient to recover from fulminant liver failure [
      • Sokal E.M.
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      • Bourgois A.
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      • Buts J.P.
      • Reding R.
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      Hepatocyte transplantation in a 4-year-old girl with peroxisomal biogenesis disease: technique, safety, and metabolic follow-up.
      ]. However, the low liver-engraftment rate and survival of transplanted hepatocytes hamper this procedure [
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      • Chowdhury N.R.
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      Treatment of the Crigler-Najjar syndrome type I with hepatocyte transplantation.
      ]. In general, isolated hepatocytes are only available from cadaveric donor livers, which means that the cells largely lack transplantation quality and quantity, if they are available at all [
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      Robust expansion of human hepatocytes in Fah−/−/Rag2−/−/Il2rg−/− mice.
      ]. Moreover, cells are generally cryopreserved before use, and this leads to an additional substantial loss of viability and function. Hence, for these reasons, research is also aiming to obtain transplantable cells from other sources, such as embryonic, induced or adult stem cells, or liver progenitor cells that can be expanded in vitro[
      • Cantz T.
      • Manns M.P.
      • Ott M.
      Stem cells in liver regeneration and therapy.
      ]. In addition, the use of autologous stem cells (mesenchymal or induced) would abolish the need for life long immunosuppression.
      Hepatocytes are not exclusively responsible for the regenerative effect of an injured liver. There has been increasing evidence of transit-amplifying cells contributing to liver regeneration [
      • Alison M.
      • Golding M.
      • Lalani E.N.
      • Nagy P.
      • Thorgeirsson S.
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      Wholesale hepatocytic differentiation in the rat from ductular oval cells, the progeny of biliary stem cells.
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      Oval cells and liver carcinogenesis: an analysis of cell lineages in hepatic tumors using oncogene transfection techniques.
      ,
      • Sell S.
      Is there a liver stem cell?.
      ,
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      Hepatic stem cells in liver regeneration.
      ]. As soon as hepatocyte growth is severely impaired or blocked during chronic injury, other cells will take over. In rodents, they emerge from the portal or periportal zone and they are referred to as “oval cells” due to their oval shaped nucleus. Once activated, they proliferate (i.e. transit-amplifying cell), infiltrate along the liver plate towards the central vein, and differentiate into hepatocytes and cholangiocytes to restore liver function and cell mass [
      • Alison M.R.
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      Hepatic stem cells: from inside and outside the liver?.
      ,
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      Similarities in the sequence of early histological changes induced in the liver of the rat by ethionine, 2-acetylamino-fluorene, and 3′-methyl-4-dimethylaminoazobenzene.
      ,
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      The role of hepatocytes and oval cells in liver regeneration and repopulation.
      ,
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      • Oertel M.
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      Liver stem cells and prospects for liver reconstitution by transplanted cells.
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      • Suzuki A.
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      • Onodera M.
      • Fukao K.
      • Nakauchi H.
      • et al.
      Clonal identification and characterization of self-renewing pluripotent stem cells in the developing liver.
      ]. Recent progress in the isolation and characterization of these bipotential cells has raised expectations that cell therapy may be possible by transplanting these stem/progenitor cells. However, several issues have to be addressed to keep the promise of cell therapy.
      In this review, we will discuss the difficulties associated with the isolation of liver progenitor cells from rodents and point out the challenges that researchers are facing in their pursuit of liver progenitor cells. This review does not address the controversial issue of the hepatocytic potential of bone marrow-derived stem cells, nor discusses the challenges encountered in cell culture; these topics have been covered extensively in other recent reviews [
      • Alison M.R.
      Stem cells in pathobiology and regenerative medicine.
      ,
      • Grompe M.
      The role of bone marrow stem cells in liver regeneration.
      ,
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      Stem cells, cell transplantation and liver repopulation.
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      Role of epigenetics in liver-specific gene transcription, hepatocyte differentiation and stem cell reprogrammation.
      ].

      Liver regeneration and turnover: heterogeneity and diversity of proliferating cells

      Hepatocyte turnover always occurs

      Under normal circumstances the liver shows a slow rate of hepatocyte renewal. Accordingly, the liver needs at least one year for complete renewal. This is in contrast to other rapidly renewing organs like the skin and gut that need less than two weeks to renew. It has been postulated that this normal liver turnover relies on the lineage progression of hepatocytes originating from the portal tract and migrating towards the central vein. This ‘streaming liver’ theory, which offers an explanation for the maintenance of the liver via cell division of hepatocytes, has found both proponents [
      • Fellous T.G.
      • Islam S.
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      • Elia G.
      • Kocher H.M.
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      • et al.
      Locating the stem cell niche and tracing hepatocyte lineages in human liver.
      ,
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      A methodological approach to tracing cell lineage in human epithelial tissues.
      ,
      • Zajicek G.
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      The streaming liver.
      ] and opponents [
      • Bralet M.P.
      • Branchereau S.
      • Brechot C.
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      Cell lineage study in the liver using retroviral mediated gene transfer. Evidence against the streaming of hepatocytes in normal liver.
      ,
      • Shiojiri N.
      • Sano M.
      • Inujima S.
      • Nitou M.
      • Kanazawa M.
      • Mori M.
      Quantitative analysis of cell allocation during liver development, using the spf(ash)-heterozygous female mouse.
      ]. Despite the low replication rate of hepatocytes in the normal liver, these highly differentiated cells replicate in a regulated manner after loss of tissue mass. Little is known about the turnover of other cell types that constitute the liver e.g. cholangiocytes and other non-parenchymal cells. For instance, cholangiocytes also have low basal DNA synthesis but they proliferate in a number of experimental models of cholestasis [
      • Alpini G.
      • Lenzi R.
      • Sarkozi L.
      • Tavoloni N.
      Biliary physiology in rats with bile ductular cell hyperplasia. Evidence for a secretory function of proliferated bile ductules.
      ,
      • Alpini G.
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      • Phillips J.O.
      • Pham L.D.
      • Miller L.J.
      • LaRusso N.F.
      Upregulation of secretin receptor gene expression in rat cholangiocytes after bile duct ligation.
      ].
      It is only upon extensive and chronic liver injury that another cell type is activated: the liver progenitor cells (LPCs) [
      • Bird T.G.
      • Lorenzini S.
      • Forbes S.J.
      Activation of stem cells in hepatic diseases.
      ,
      • Duncan A.W.
      • Dorrell C.
      • Grompe M.
      Stem cells and liver regeneration.
      ,
      • Erker L.
      • Grompe M.
      Signaling networks in hepatic oval cell activation.
      ]. These cells probably do not participate in the usual maintenance of the liver mass, but they are activated when an extensive injury occurs that overwhelms the regenerative capacity of hepatocytes. Nevertheless, the two regenerative modes are not entirely mutually exclusive, as LPC and hepatocyte replication can be observed simultaneously in some injury models [
      • Paku S.
      • Schnur J.
      • Nagy P.
      • Thorgeirsson S.S.
      Origin and structural evolution of the early proliferating oval cells in rat liver.
      ,
      • Rosenberg D.
      • Ilic Z.
      • Yin L.
      • Sell S.
      Proliferation of hepatic lineage cells of normal C57BL and interleukin-6 knockout mice after cocaine-induced periportal injury.
      ,
      • Wang X.
      • Foster M.
      • Al-Dhalimy M.
      • Lagasse E.
      • Finegold M.
      • Grompe M.
      The origin and liver repopulating capacity of murine oval cells.
      ].

      Hepatocyte mediated liver regeneration

      Although the Greek myth of Prometheus outbid the restorative capacity of the liver, it appears that this organ indeed does have an amazing ability for self repair following partial resection (or hepatectomy) [
      • Grompe M.
      Liver repopulation for the treatment of metabolic diseases.
      ,
      • Michalopoulos G.K.
      • Bowen W.C.
      • Mule K.
      • Stolz D.B.
      Histological organization in hepatocyte organoid cultures.
      ,
      • Michalopoulos G.K.
      • DeFrances M.C.
      Liver regeneration.
      ,
      • Taub R.
      Liver regeneration: from myth to mechanism.
      ,
      • Thorgeirsson S.S.
      Hepatic stem cells in liver regeneration.
      ]. The research on the potential therapeutic use of LPCs has accelerated significantly in recent years giving rise to a vast amount of data on the power of regeneration of the liver driven by hepatocytes and LPCs [
      • Alison M.R.
      Stem cells in pathobiology and regenerative medicine.
      ,
      • Bird T.G.
      • Lorenzini S.
      • Forbes S.J.
      Activation of stem cells in hepatic diseases.
      ,
      • Duncan A.W.
      • Dorrell C.
      • Grompe M.
      Stem cells and liver regeneration.
      ,
      • Lemaigre F.P.
      Mechanisms of liver development: concepts for understanding liver disorders and design of novel therapies.
      ] (Fig. 1). Following 70% partial hepatectomy, rat liver completely recovers its initial volume at day 20 [
      • Higgins G.M.
      • Anderson R.M.
      Experimental pathology of the liver. I. Restoration of the liver of the white rat following partial surgical removal.
      ], while after right lobe transplantation in humans, donor and recipient livers reached their original weight by 60 days after surgery [
      • Marcos A.
      • Fisher R.A.
      • Ham J.M.
      • Shiffman M.L.
      • Sanyal A.J.
      • Luketic V.A.
      • et al.
      Liver regeneration and function in donor and recipient after right lobe adult to adult living donor liver transplantation.
      ]. Following different types of injury, repair is mainly accomplished by mature hepatocytes, which are highly differentiated cells with a long lifespan that can re-enter the cell cycle and restore the liver mass in response to parenchymal loss [
      • Fausto N.
      • Campbell J.S.
      • Riehle K.J.
      Liver regeneration.
      ,
      • Michalopoulos G.K.
      • DeFrances M.C.
      Liver regeneration.
      ,
      • Sell S.
      Heterogeneity and plasticity of hepatocyte lineage cells.
      ] (Fig. 1). It has been shown that hepatocytes are capable of at least 69 cell divisions and can restore normal architecture and impaired function in the injured liver [
      • Overturf K.
      • al-Dhalimy M.
      • Ou C.N.
      • Finegold M.
      • Grompe M.
      Serial transplantation reveals the stem-cell-like regenerative potential of adult mouse hepatocytes.
      ,
      • Rhim J.A.
      • Sandgren E.P.
      • Degen J.L.
      • Palmiter R.D.
      • Brinster R.L.
      Replacement of diseased mouse liver by hepatic cell transplantation.
      ]. Grompe’s group has demonstrated that adult hepatocytes expand clonally and may be serially transplanted [
      • Overturf K.
      • Al-Dhalimy M.
      • Finegold M.
      • Grompe M.
      The repopulation potential of hepatocyte populations differing in size and prior mitotic expansion.
      ,
      • Overturf K.
      • al-Dhalimy M.
      • Ou C.N.
      • Finegold M.
      • Grompe M.
      Serial transplantation reveals the stem-cell-like regenerative potential of adult mouse hepatocytes.
      ]. These studies raise the possibility that hepatocytes may display multipotentiality, one of the defining characteristics of stem cells.
      Figure thumbnail gr1
      Fig. 1Schematic representation of the two regenerative pathways involved in liver repair. In normal circumstances, the regeneration/recovery of the liver is driven by the fully differentiated hepatocyte compartment (left side). After a short period of time parenchymal and non-parenchymal cells have restored the hepatic mass and functions. When hepatocytes are impaired, blocked or the growth is overwhelmed by severe injury (right side), the liver progenitor cell compartment (LPC) (light blue) will take over. Once activated, these cells proliferate (yellow arrows) and give rise to bipotential transit-amplifying cells or oval cells and their progeny (dark blue). In rodents, these oval cells emerge from the periportal zone (close to bile ducts, BD), give rise to cords of oval cells that infiltrate along the liver plate, and then differentiate into hepatocytes and cholangiocytes to rescue the liver.
      By definition, regeneration is the reconstitution of a lost or damaged organ. However, in liver regeneration the excised or damaged liver part never grows back. In a sense, the process has all the characteristics of a compensatory growth accompanied by hypertrophy, and that is governed by functional constraints rather than anatomical needs [
      • Bucher N.L.
      Regeneration of mammalian liver.
      ,
      • Fausto N.
      Liver regeneration.
      ,
      • Fausto N.
      Liver regeneration: from laboratory to clinic.
      ]. Following hepatectomy, other liver cells undergo a wave of mitosis to restore the organ. A plethora of cytokines, growth factors and enzymes fulfil this well-orchestrated regeneration [
      • Campbell J.S.
      • Prichard L.
      • Schaper F.
      • Schmitz J.
      • Stephenson-Famy A.
      • Rosenfeld M.E.
      • et al.
      Expression of suppressors of cytokine signaling during liver regeneration.
      ,
      • Fausto N.
      Hepatocyte differentiation and liver progenitor cells.
      ,
      • Taub R.
      Liver regeneration: from myth to mechanism.
      ]. Much less is known about how liver regeneration is terminated once the appropriate liver mass is restored. Although the liver functions are restored, the anatomical structures are not reconstituted.

      LPC-mediated regeneration

      Some liver diseases (alcoholic liver disease, chronic cholestatic diseases, or hepatitis) significantly impair the ability of the hepatocytes to replenish the organ, thus promoting the activation of a secondary intra-hepatic regenerative compartment [
      • Alison M.
      • Golding M.
      • Lalani el N.
      • Sarraf C.
      Wound healing in the liver with particular reference to stem cells.
      ,
      • Fausto N.
      Liver regeneration: from laboratory to clinic.
      ,
      • Fausto N.
      • Campbell J.S.
      The role of hepatocytes and oval cells in liver regeneration and repopulation.
      ,
      • Sell S.
      The role of progenitor cells in repair of liver injury and in liver transplantation.
      ,
      • Shafritz D.A.
      • Dabeva M.D.
      Liver stem cells and model systems for liver repopulation.
      ,
      • Shinozuka H.
      • Lombardi B.
      • Sell S.
      • Iammarino R.M.
      Early histological and functional alterations of ethionine liver carcinogenesis in rats fed a choline-deficient diet.
      ] (Fig. 1). This so-called “oval cells compartment” consists of ‘small ovoid cells with scant lightly basophilic cytoplasm and pale blue-staining nuclei’ [
      • Farber E.
      Similarities in the sequence of early histological changes induced in the liver of the rat by ethionine, 2-acetylamino-fluorene, and 3′-methyl-4-dimethylaminoazobenzene.
      ]. While the term oval cell is widely used to describe liver progenitors, it is important to note that investigators do not agree on the phenotype and molecular signature of these cells. The terminal bile ductular system (also known as the canal of Hering) is thought to be the main source of oval cells [
      • Paku S.
      • Schnur J.
      • Nagy P.
      • Thorgeirsson S.S.
      Origin and structural evolution of the early proliferating oval cells in rat liver.
      ,
      • Roskams T.A.
      • Theise N.D.
      • Balabaud C.
      • Bhagat G.
      • Bhathal P.S.
      • Bioulac-Sage P.
      • et al.
      Nomenclature of the finer branches of the biliary tree: canals, ductules, and ductular reactions in human livers.
      ,
      • Saxena R.
      • Theise N.D.
      • Crawford J.M.
      Microanatomy of the human liver-exploring the hidden interfaces.
      ,
      • Theise N.D.
      • Saxena R.
      • Portmann B.C.
      • Thung S.N.
      • Yee H.
      • Chiriboga L.
      • et al.
      The canals of Hering and hepatic stem cells in humans.
      ]. The oval cell compartment can probably not be attributed to a single cell type [
      • Zheng Y.W.
      • Taniguchi H.
      Diversity of hepatic stem cells in the fetal and adult liver.
      ]. In order to avoid misunderstandings, the term oval cell activation (or response) is used to describe the heterogeneous cellular changes that accompany the appearance of progenitor cells, whereas the term oval cells refer to the progenitors themselves. It is generally accepted that oval cells are bipotential transit-amplifying cells derived from normally quiescent ‘true stem cells’ that reside in the biliary tree and are absent in healthy liver [
      • Shafritz D.A.
      • Oertel M.
      • Menthena A.
      • Nierhoff D.
      • Dabeva M.D.
      Liver stem cells and prospects for liver reconstitution by transplanted cells.
      ]. Proliferating oval cells constitute a heterogeneous population justifying the different names used to describe them: ductular progenitor cells [
      • Paku S.
      • Nagy P.
      • Kopper L.
      • Thorgeirsson S.S.
      2-Acetylaminofluorene dose-dependent differentiation of rat oval cells into hepatocytes: confocal and electron microscopic studies.
      ,
      • Paku S.
      • Schnur J.
      • Nagy P.
      • Thorgeirsson S.S.
      Origin and structural evolution of the early proliferating oval cells in rat liver.
      ], atypical ductular cells [
      • Jensen C.H.
      • Jauho E.I.
      • Santoni-Rugiu E.
      • Holmskov U.
      • Teisner B.
      • Tygstrup N.
      • et al.
      Transit-amplifying ductular (oval) cells and their hepatocytic progeny are characterized by a novel and distinctive expression of delta-like protein/preadipocyte factor 1/fetal antigen 1.
      ], peri-ductular liver progenitor cells [
      • Sell S.
      Mouse models to study the interaction of risk factors for human liver cancer.
      ,
      • Sell S.
      The role of progenitor cells in repair of liver injury and in liver transplantation.
      ] or individual progenies [
      • Yoon B.I.
      • Choi Y.K.
      • Kim D.Y.
      Differentiation processes of oval cells into hepatocytes: proposals based on morphological and phenotypical traits in carcinogen-treated hamster liver.
      ].

      Experimentally induced LPC-mediated regeneration

      In general, two strategies have been adopted for the experimental induction of LPC-mediated liver regeneration; one relies on surgical resection and the other on an injury by toxins (reviewed in [
      • Alison M.
      • Golding M.
      • Lalani el N.
      • Sarraf C.
      Wound healing in the liver with particular reference to stem cells.
      ,
      • Duncan A.W.
      • Dorrell C.
      • Grompe M.
      Stem cells and liver regeneration.
      ,
      • Palmes D.
      • Spiegel H.U.
      Animal models of liver regeneration.
      ,
      • Sancho-Bru P.
      • Najimi M.
      • Caruso M.
      • Pauwelyn K.
      • Cantz T.
      • Forbes S.
      • et al.
      Stem and progenitor cells for liver repopulation: can we standardise the process from bench to bedside?.
      ,
      • Santoni-Rugiu E.
      • Jelnes P.
      • Thorgeirsson S.S.
      • Bisgaard H.C.
      Progenitor cells in liver regeneration: molecular responses controlling their activation and expansion.
      ]). Many toxins cause liver damage and subsequently cell death in the parenchyma followed by liver regeneration (Table 1). Hepatotoxins can be used to induce selectively centrilobular (like acetaminophen) or periportal (like allyl alcohol) necrotic damages. Most of the hepatotoxins listed in Table 1 induce damage in the centrilobular parenchyma of the liver. Carbotetrachloride (CCl4) induces liver injury by its metabolites that arise from cytochrome P450-dependent breakdown. The highly reactive metabolite triggers lipid peroxidation in the hepatocytes which damages these (centrilobular) cells and induces necrosis. Under normal circumstances, acetaminophen (AAF) and paracetamol (APAP) undergo biotransformation by cytochrome P450 (glucuronidation and sulphation) and are excreted by the kidneys. After an overdose, the toxic metabolites accumulate and create adducts with DNA and protein leading to necrosis of the hepatocytes (for references see Table 1).
      Table 1
      • Akhurst B.
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      • Dumble M.L.
      • Knight B.
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      A modified choline-deficient, ethionine-supplemented diet protocol effectively induces oval cells in mouse liver.
      ,
      • Chiu C.C.
      • Huang G.T.
      • Chou S.H.
      • Chien C.T.
      • Chiou L.L.
      • Chang M.H.
      • et al.
      Characterization of cytokeratin 19-positive hepatocyte foci in the regenerating rat liver after 2-AAF/CCl4 injury.
      ,
      • Dabeva M.D.
      • Alpini G.
      • Hurston E.
      • Shafritz D.A.
      Models for hepatic progenitor cell activation.
      ,
      • Dahlke M.H.
      • Popp F.C.
      • Bahlmann F.H.
      • Aselmann H.
      • Jager M.D.
      • Neipp M.
      • et al.
      Liver regeneration in a retrorsine/CCl4-induced acute liver failure model: do bone marrow-derived cells contribute?.
      ,
      • Engelhardt N.V.
      • Baranov V.N.
      • Lazareva M.N.
      • Goussev A.I.
      Ultrastructural localisation of alpha-fetoprotin (AFP) in regenerating mouse liver poisoned with CCL4. 1. Reexpression of AFP in differentiated hepatocytes.
      ,
      • Engelhardt N.V.
      • Factor V.M.
      • Yasova A.K.
      • Poltoranina V.S.
      • Baranov V.N.
      • Lasareva M.N.
      Common antigens of mouse oval and biliary epithelial cells. Expression on newly formed hepatocytes.
      ,
      • Evarts R.P.
      • Nagy P.
      • Marsden E.
      • Thorgeirsson S.S.
      A precursor-product relationship exists between oval cells and hepatocytes in rat liver.
      ,
      • Factor V.M.
      • Radaeva S.A.
      • Thorgeirsson S.S.
      Origin and fate of oval cells in dipin-induced hepatocarcinogenesis in the mouse.
      ,
      • Fujio K.
      • Evarts R.P.
      • Hu Z.
      • Marsden E.R.
      • Thorgeirsson S.S.
      Expression of stem cell factor and its receptor, c-kit, during liver regeneration from putative stem cells in adult rat.
      ,
      • Gordon G.J.
      • Coleman W.B.
      • Hixson D.C.
      • Grisham J.W.
      Liver regeneration in rats with retrorsine-induced hepatocellular injury proceeds through a novel cellular response.
      ,
      • He X.Y.
      • Smith G.J.
      • Enno A.
      • Nicholson R.C.
      Short-term diethylnitrosamine-induced oval cell responses in three strains of mice.
      ,
      • Laconi E.
      • Oren R.
      • Mukhopadhyay D.K.
      • Hurston E.
      • Laconi S.
      • Pani P.
      • et al.
      Long-term, near-total liver replacement by transplantation of isolated hepatocytes in rats treated with retrorsine.
      ,
      • Laconi E.
      • Sarma D.S.
      • Pani P.
      Transplantation of normal hepatocytes modulates the development of chronic liver lesions induced by a pyrrolizidine alkaloid, lasiocarpine.
      ,
      • Lee J.H.
      • Ilic Z.
      • Sell S.
      Cell kinetics of repair after allyl alcohol-induced liver necrosis in mice.
      ,
      • Lemire J.M.
      • Shiojiri N.
      • Fausto N.
      Oval cell proliferation and the origin of small hepatocytes in liver injury induced by d-galactosamine.
      ,
      • Petersen B.E.
      • Zajac V.F.
      • Michalopoulos G.K.
      Hepatic oval cell activation in response to injury following chemically induced periportal or pericentral damage in rats.
      ,
      • Popp F.C.
      • Slowik P.
      • Eggenhofer E.
      • Renner P.
      • Lang S.A.
      • Stoeltzing O.
      • et al.
      No contribution of multipotent mesenchymal stromal cells to liver regeneration in a rat model of prolonged hepatic injury.
      ,
      • Preisegger K.H.
      • Factor V.M.
      • Fuchsbichler A.
      • Stumptner C.
      • Denk H.
      • Thorgeirsson S.S.
      Atypical ductular proliferation and its inhibition by transforming growth factor beta1 in the 3,5-diethoxycarbonyl-1,4-dihydrocollidine mouse model for chronic alcoholic liver disease.
      ,
      • Roskams T.
      • Yang S.Q.
      • Koteish A.
      • Durnez A.
      • DeVos R.
      • Huang X.
      • et al.
      Oxidative stress and oval cell accumulation in mice and humans with alcoholic and nonalcoholic fatty liver disease.
      ,
      • Schwarz M.
      • Buchmann A.
      • Wiesbeck G.
      • Kunz W.
      Effect of ethanol on early stages in nitrosamine carcinogenesis in rat liver.
      ,
      • Sell S.
      Comparison of oval cells induced in rat liver by feeding N-2-fluorenylacetamide in a choline-devoid diet and bile duct cells induced by feeding 4,4′-diaminodiphenylmethane.
      ,
      • Sells M.A.
      • Katyal S.L.
      • Shinozuka H.
      • Estes L.W.
      • Sell S.
      • Lombardi B.
      Isolation of oval cells and transitional cells from the livers of rats fed the carcinogen dl-ethionine.
      ,
      • Shinozuka H.
      • Lombardi B.
      • Sell S.
      • Iammarino R.M.
      Enhancement of dl-ethionine-induced liver carcinogenesis in rats fed a choline-devoid diet.
      ,
      • Smith P.G.
      • Tee L.B.
      • Yeoh G.C.
      Appearance of oval cells in the liver of rats after long-term exposure to ethanol.
      ,
      • Zhou X.F.
      • Wang Q.
      • Chu J.X.
      • Liu A.L.
      Effects of retrorsine on mouse hepatocyte proliferation after liver injury.
      used experimental models for LPC-mediated regeneration.
      Only representative publications are listed. The most frequently used hepatotoxins in rodents are highlighted in bold. AAF, 2-Acetylamino-fluorene; APAP, N-acetyl-p-aminophenol; AA, allyl alcohol; PH, partial hepatectomy; CCl4, carbone tetra-chloride; CDE, choline-deficient, ethionine-supplemented diet; DDC, 3-diethoxycarbonyl-1,4-dihydrocollidine; DIPIN, 1,4-bis[N,N′-di(ethylene)phosphamide]piperazine; DEN.V, diethylnitrosamine.
      Unlike hepatectomy, the hepatotoxic models of liver regeneration are rather easy to perform but difficult to standardize and one often observes a low reproducibility. The regenerative response largely depends on the dose and mode of administration of the hepatotoxins [
      • Diehl A.M.
      Nutrition, hormones, metabolism, and liver regeneration.
      ]. The toxins can also interfere with the cellular and molecular mechanisms of liver regeneration by creating membrane damage, inducing inflammatory reactions or even activate the non-parenchymal cells (in particular Kupffer cells and hepatic stellate cells) [
      • Czaja A.J.
      • dos Santos R.M.
      • Porto A.
      • Santrach P.J.
      • Moore S.B.
      Immune phenotype of chronic liver disease.
      ]. Finally, in these experimental models the process of liver damage and repair are interwoven, making the interpretation of the results more complex [
      • Nostrant T.T.
      • Miller D.L.
      • Appelman H.D.
      • Gumucio J.J.
      Acinar distribution of liver cell regeneration after selective zonal injury in the rat.
      ].

      Is there more than one liver progenitor cell?

      Notwithstanding the disagreements on the molecular signature of the LPCs and the isolation- and activation-strategy used, the investigators do agree on at least five similar parameters that one can apply to LPC-mediated liver regeneration in rodents.
      First of all, different experimental protocols of LPC-activation lead to a detection of a similar population of small cells in the periportal zone that proliferates extensively and, upon migration into the lobule, differentiate into hepatobiliary lineages. Mechanisms by which these cells are activated during liver regeneration have recently been addressed [
      • Bird T.G.
      • Lorenzini S.
      • Forbes S.J.
      Activation of stem cells in hepatic diseases.
      ,
      • Duncan A.W.
      • Dorrell C.
      • Grompe M.
      Stem cells and liver regeneration.
      ,
      • Erker L.
      • Grompe M.
      Signaling networks in hepatic oval cell activation.
      ,
      • Gaudio E.
      • Carpino G.
      • Cardinale V.
      • Franchitto A.
      • Onori P.
      • Alvaro D.
      New insights into liver stem cells.
      ,
      • Oben J.A.
      • Roskams T.
      • Yang S.
      • Lin H.
      • Sinelli N.
      • Li Z.
      • et al.
      Sympathetic nervous system inhibition increases hepatic progenitors and reduces liver injury.
      ]. It is believed that 3 important cell signalling axes are involved in the activation of LPCs: SCF/c-Kit [
      • Hu B.
      • Colletti L.M.
      Stem cell factor and c-kit are involved in hepatic recovery after acetaminophen-induced liver injury in mice.
      ] SDF1/CXCR4 important for the migration [
      • Hatch H.M.
      • Zheng D.
      • Jorgensen M.L.
      • Petersen B.E.
      SDF-1alpha/CXCR4: a mechanism for hepatic oval cell activation and bone marrow stem cell recruitment to the injured liver of rats.
      ,
      • Swenson E.S.
      • Kuwahara R.
      • Krause D.S.
      • Theise N.D.
      Physiological variations of stem cell factor and stromal-derived factor-1 in murine models of liver injury and regeneration.
      ] and TWEAK/Fn14 [
      • Jakubowski A.
      • Ambrose C.
      • Parr M.
      • Lincecum J.M.
      • Wang M.Z.
      • Zheng T.S.
      • et al.
      TWEAK induces liver progenitor cell proliferation.
      ] for the activation of the LPCs. This part has gained much interest since research that elucidates the factors that govern proliferation and differentiation of LPCs in response to liver injury could eventually be administered in vivo or used for expansion and differentiation of isolated adult LPCs in large numbers in vitro.
      Second, the presence of several markers expressed by the LPCs, following the various liver injury models, suggests common characteristics in terms of their molecular footprint (Table 2). They have a phenotype that is transitional between hepatocytes and biliary cells (KT-7, -8, -18, -19; ALB; AAT; CD24; c-Met), are associated with immature foetal hepatoblasts (AFP) and neuroepithelial cells (N-CAM; Chromogranin A) and are strongly related to extrahepatic cell types by sharing some haematopoietic markers such as Thy-1 (CD90), Sca-1, CD34 and CD133.
      Table 2Commonly used markers for the identification of LPCs in rodents.
      For an extended overview of the potential markers expressed on LPCs we refer to a multitude of articles and reviews and their related Refs.
      • Alison M.R.
      • Islam S.
      • Lim S.
      Stem cells in liver regeneration, fibrosis and cancer: the good, the bad and the ugly.
      ,
      • Bird T.G.
      • Lorenzini S.
      • Forbes S.J.
      Activation of stem cells in hepatic diseases.
      ,
      • Gaudio E.
      • Carpino G.
      • Cardinale V.
      • Franchitto A.
      • Onori P.
      • Alvaro D.
      New insights into liver stem cells.
      ,
      • Santoni-Rugiu E.
      • Jelnes P.
      • Thorgeirsson S.S.
      • Bisgaard H.C.
      Progenitor cells in liver regeneration: molecular responses controlling their activation and expansion.
      .
      The third common trait of LPCs induced by different injuries is their heterogeneity. Immunophenotypic characterizations on injured tissues reveal that at least two subtypes of cells are emerging from the portal field. One is a population of cells that forms duct-like structures and expresses bile duct as well as hepatocytic markers (i.e. the oval cells) [
      • Alison M.R.
      • Vig P.
      • Russo F.
      • Bigger B.W.
      • Amofah E.
      • Themis M.
      • et al.
      Hepatic stem cells: from inside and outside the liver?.
      ,
      • Newsome P.N.
      • Hussain M.A.
      • Theise N.D.
      Hepatic oval cells: helping redefine a paradigm in stem cell biology.
      ]. The other population consists of non-ductular cells that can be detected between and distally from the ductules with fibroblastic characteristics (the accompanying cells) [
      • Dezso K.
      • Jelnes P.
      • Laszlo V.
      • Baghy K.
      • Bodor C.
      • Paku S.
      • et al.
      Thy-1 is expressed in hepatic myofibroblasts and not oval cells in stem cell-mediated liver regeneration.
      ,
      • Dudas J.
      • Mansuroglu T.
      • Batusic D.
      • Ramadori G.
      Thy-1 is expressed in myofibroblasts but not found in hepatic stellate cells following liver injury.
      ,
      • Dudas J.
      • Mansuroglu T.
      • Batusic D.
      • Saile B.
      • Ramadori G.
      Thy-1 is an in vivo and in vitro marker of liver myofibroblasts.
      ,
      • Koenig S.
      • Probst I.
      • Becker H.
      • Krause P.
      Zonal hierarchy of differentiation markers and nestin expression during oval cell mediated rat liver regeneration.
      ,
      • Van Hul N.K.
      • Abarca-Quinones J.
      • Sempoux C.
      • Horsmans Y.
      • Leclercq I.A.
      Relation between liver progenitor cell expansion and extracellular matrix deposition in a CDE-induced murine model of chronic liver injury.
      ]. This explains the expression of Thy-1 (CD90) in a portion of LPC enriched cell populations.
      Although the identity of LPCs is far from clear, a large set of data favours the location of the LPC niche in the periportal regions [
      • Forbes S.
      • Vig P.
      • Poulsom R.
      • Thomas H.
      • Alison M.
      Hepatic stem cells.
      ,
      • Paku S.
      • Schnur J.
      • Nagy P.
      • Thorgeirsson S.S.
      Origin and structural evolution of the early proliferating oval cells in rat liver.
      ,
      • Petersen B.
      • Shupe T.
      Location is everything: the liver stem cell niche.
      ,
      • Roskams T.
      Different types of liver progenitor cells and their niches.
      ,
      • Roskams T.
      • Cassiman D.
      • De Vos R.
      • Libbrecht L.
      Neuroregulation of the neuroendocrine compartment of the liver.
      ,
      • Theise N.D.
      • Saxena R.
      • Portmann B.C.
      • Thung S.N.
      • Yee H.
      • Chiriboga L.
      • et al.
      The canals of Hering and hepatic stem cells in humans.
      ]. Indeed, the fourth common trait is that the canals of Hering are the most likely origin of the LPCs in adult tissue. Nonetheless, Kuwahara has demonstrated that the liver has a multi-tiered, flexible system of regeneration rather than a single LPC location [
      • Kuwahara 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.
      ]. He enumerated four distinct niches: the canal of Hering, the intralobular bile ducts, the peri-ductal cells, and the peri-biliary hepatocytes. These results not only confirm several different, and often contradictory, lines of investigation regarding the intra-hepatic location of the LPCs, they also summarize the different niches that have been observed under different injury models so far [
      • Baumann U.
      • Crosby H.A.
      • Ramani P.
      • Kelly D.A.
      • Strain A.J.
      Expression of the stem cell factor receptor c-kit in normal and diseased pediatric liver: identification of a human hepatic progenitor cell?.
      ,
      • Saxena R.
      • Theise N.D.
      • Crawford J.M.
      Microanatomy of the human liver-exploring the hidden interfaces.
      ,
      • Theise N.D.
      • Saxena R.
      • Portmann B.C.
      • Thung S.N.
      • Yee H.
      • Chiriboga L.
      • et al.
      The canals of Hering and hepatic stem cells in humans.
      ,
      • Yavorkovsky L.
      • Lai E.
      • Ilic Z.
      • Sell S.
      Participation of small intraportal stem cells in the restitutive response of the liver to periportal necrosis induced by allyl alcohol.
      ,
      • Zajicek G.
      • Oren R.
      • Weinreb Jr., M.
      The streaming liver.
      ]. The different niches are thought to act as microenvironments, made up of cells, basal membrane (BM) and extracellular matrix (ECM) that can have an effect on LPC-activation and proliferation. The LPC niches are most likely surrounded by hepatic stellate cells [
      • Paku S.
      • Schnur J.
      • Nagy P.
      • Thorgeirsson S.S.
      Origin and structural evolution of the early proliferating oval cells in rat liver.
      ,
      • Roskams T.
      Relationships among stellate cell activation, progenitor cells, and hepatic regeneration.
      ,
      • Roskams T.
      • Cassiman D.
      • De Vos R.
      • Libbrecht L.
      Neuroregulation of the neuroendocrine compartment of the liver.
      ] and Kupffer cells [
      • Holt M.P.
      • Cheng L.
      • Ju C.
      Identification and characterization of infiltrating macrophages in acetaminophen-induced liver injury.
      ], which play a crucial role in fibrogenesis. Depending on their location within the hepatic lobule, their activation status, the nature and severity of the injury, hepatic stellate cells and Kupffer cells will not have the same impact on the LPC compartment. As a result, the immunophenotype of LPCs isolated from differentially injured rodents will be different [
      • Erker L.
      • Grompe M.
      Signaling networks in hepatic oval cell activation.
      ,
      • Santoni-Rugiu E.
      • Jelnes P.
      • Thorgeirsson S.S.
      • Bisgaard H.C.
      Progenitor cells in liver regeneration: molecular responses controlling their activation and expansion.
      ]. Nerves [
      • Roskams T.
      • Cassiman D.
      • De Vos R.
      • Libbrecht L.
      Neuroregulation of the neuroendocrine compartment of the liver.
      ] basement membrane [
      • Paku S.
      • Nagy P.
      • Kopper L.
      • Thorgeirsson S.S.
      2-Acetylaminofluorene dose-dependent differentiation of rat oval cells into hepatocytes: confocal and electron microscopic studies.
      ] and ECM [
      • Van Hul N.K.
      • Abarca-Quinones J.
      • Sempoux C.
      • Horsmans Y.
      • Leclercq I.A.
      Relation between liver progenitor cell expansion and extracellular matrix deposition in a CDE-induced murine model of chronic liver injury.
      ,
      • Zhang W.
      • Chen X.P.
      • Zhang W.G.
      • Zhang F.
      • Xiang S.
      • Dong H.H.
      • et al.
      Hepatic non-parenchymal cells and extracellular matrix participate in oval cell-mediated liver regeneration.
      ] are also involved in the regenerative process increasing the influence of the microenvironment on the activation of LPCs.
      Finally, when using experimental rodent models of liver injury, investigators collectively observed that a strong inflammatory response occurs with the infiltration of immune cells into the liver; this results in a surge of cytokine expression, and in particular IL-6 and TNF-α and -β [
      • Duncan A.W.
      • Dorrell C.
      • Grompe M.
      Stem cells and liver regeneration.
      ,
      • Erker L.
      • Grompe M.
      Signaling networks in hepatic oval cell activation.
      ,
      • Knight B.
      • Matthews V.B.
      • Olynyk J.K.
      • Yeoh G.C.
      Jekyll and Hyde: evolving perspectives on the function and potential of the adult liver progenitor (oval) cell.
      ]. Knight and co-workers documented a close correlation between inflammation, cytokine production and the expansion of oval cells in the liver during experimental chronic injury (CDE treatment) in C57BL/6 mice [
      • Knight B.
      • Akhurst B.
      • Matthews V.B.
      • Ruddell R.G.
      • Ramm G.A.
      • Abraham L.J.
      • et al.
      Attenuated liver progenitor (oval) cell and fibrogenic responses to the choline deficient, ethionine supplemented diet in the BALB/C inbred strain of mice.
      ]. They showed that the oval cell response to a CDE treatment was inhibited in mice lacking Th1 immune signalling (BALB/C mice) compared to the C57BL/6 mice that were not deficient in Th1 response. Since then, other investigators proved that the immune system is a key component in the activation of the oval cell compartment [
      • Erker L.
      • Grompe M.
      Signaling networks in hepatic oval cell activation.
      ].

      Are cholangiocytes progenitor cells?

      A question that is frequently asked is whether cholangiocytes are in fact LPCs, since LPCs are believed to originate from the canals of Hering. This channel is lined partially by cholangiocytes and partly by hepatocytes, and it serves to conduct bile from bile canaliculi to terminal bile ducts in portal tracts [
      • Saxena R.
      • Theise N.
      Canals of Hering: recent insights and current knowledge.
      ]. Because the canal of Hering forms the biliary-hepatocytic interface, it makes biological sense that any LPC with the potential for biphenotypic differentiation is located at this interface. Sharing a close anatomical location, it would not be unreasonable to assume that cholangiocytes from the canal of Hering are progenitor cells. At present, no experiments have been reported that can fully underline this assumption. Cholangiocytes proliferate under various pathological conditions, and for instance, after PH or bile duct ligation in rats, they proliferate from pre-existing ducts in the portal field [
      • Alpini G.
      • Lenzi R.
      • Sarkozi L.
      • Tavoloni N.
      Biliary physiology in rats with bile ductular cell hyperplasia. Evidence for a secretory function of proliferated bile ductules.
      ]. Both oval cells and cholangiocytes are known to express some intracellular and membrane proteins including EpCAM, ABCG2, prominin-1, KT-7 and KT-19. In addition, Okabe and co-workers demonstrated that EpCAM is expressed in both mouse cholangiocytes and oval cells, whereas its related protein, TROP2, is expressed exclusively in oval cells [
      • Okabe M.
      • Tsukahara Y.
      • Tanaka M.
      • Suzuki K.
      • Saito S.
      • Kamiya Y.
      • et al.
      Potential hepatic stem cells reside in EpCAM+ cells of normal and injured mouse liver.
      ]. This establishes TROP2 as a marker to distinguish oval cells from cholangiocytes and might help to determine whether cholangiocytes are part of the LPC/oval cell response.

      Location is everything

      As Petersen and Shupe stated last year [
      • Petersen B.
      • Shupe T.
      Location is everything: the liver stem cell niche.
      ], “location is everything” (Fig. 2). LPCs are found in the canals of Hering, which represent a fertile environment and confers distinct advantages for these cells. The particular zonation of hepatocytes purges the vicinity of LPCs from high excess of exogenous but also endogenous molecules. The organization of the sinusoidal framework displays heterogeneity throughout the length of the sinusoid, simultaneously on the size, the number and the distribution of the fenestrae, but also on the infrastructure of the sinusoids network [
      • Horn T.
      • Henriksen J.H.
      • Christoffersen P.
      The sinusoidal lining cells in “normal” human liver. A scanning electron microscopic investigation.
      ,
      • McCuskey R.S.
      • Ekataksin W.
      • LeBouton A.V.
      • Nishida J.
      • McCuskey M.K.
      • McDonnell D.
      • et al.
      Hepatic microvascular development in relation to the morphogenesis of hepatocellular plates in neonatal rats.
      ,
      • Vidal-Vanaclocha F.
      • Barbera-Guillem E.
      Fenestration patterns in endothelial cells of rat liver sinusoids.
      ,
      • Wisse E.
      • Braet F.
      • Luo D.
      • De Zanger R.
      • Jans D.
      • Crabbe E.
      • et al.
      Structure and function of sinusoidal lining cells in the liver.
      ,
      • Wisse E.
      • De Zanger R.B.
      • Charels K.
      • Van Der Smissen P.
      • McCuskey R.S.
      The liver sieve: considerations concerning the structure and function of endothelial fenestrae, the sinusoidal wall and the space of Disse.
      ].
      Figure thumbnail gr2
      Fig. 2Schematic representation of the cellular complexity of the liver. Two major epithelial cell types constitute this organ: hepatocytes and cholangiocytes (or bile duct cells). Kupffer cells, sinusoidal cells, stellate cells, myofibroblasts and fibroblasts are resident liver cells. The bile produced by hepatocytes is initially secreted into the bile canaliculi, which are located between the cytoplasmic membranes of two adjacent hepatocytes. Bile canaliculi are connected with bile ducts (BD) through the interposition of the canal of Hering (CoH) (the niche of the liver progenitor cells). Terminal branches of the portal vein (PV) and hepatic artery (HA) converge and mix as they enter sinusoids in the liver. The blood flows through the sinusoids and empties into the central vein (CV) of each lobule. The locations of hepatocytes, liver sinusoidal cells, extracellular matrix, basal membrane and hepatic stellate cells are well defined. All these cells could interact and cross-talk with the liver progenitor cells.
      The sinusoids surrounding the portal tracts act as a selective barrier and ensure a blood flow rich in nutrients and oxygen by comparison to its counterpart, the sinusoids around the central vein. If necessary, the diameter of the sinusoids can change by varying the contractile properties of stellate and endothelial cells. Specific elements of the ECM in portal tracts are dissimilar to those found in the central vein and throughout the sinusoids [
      • Martinez-Hernandez A.
      • Amenta P.S.
      The extracellular matrix in hepatic regeneration.
      ,
      • Martinez-Hernandez A.
      • Amenta P.S.
      The hepatic extracellular matrix. II. Ontogenesis, regeneration and cirrhosis.
      ,
      • Martinez-Hernandez A.
      • Delgado F.M.
      • Amenta P.S.
      The extracellular matrix in hepatic regeneration. Localization of collagen types I, III, IV, laminin, and fibronectin.
      ]. These discrepancies eventually can lead to different attachment efficiencies, growth, and morphology of LPCs thus explaining their location in the canals of Hering and not elsewhere. Recently, McClelland and colleagues showed compelling evidence of such a scenario [
      • McClelland R.
      • Wauthier E.
      • Uronis J.
      • Reid L.
      Gradients in the liver’s extracellular matrix chemistry from periportal to pericentral zones: influence on human hepatic progenitors.
      ]. They reported the influence of the ECM chemistry on human cultured LPCs by showing that with a composition similar to the matrix found in the portal tract, the LPCs had a better attachment efficiency and a higher growth rate. In contrast, the mimicked conditions found in the central vein elicited growth arrest, differentiation and even inhibited attachment. Another approach is the development of miniature bio-artificial livers that mimic the niche of LPCs by combining multiple cell types and ECM into one device [
      • Streetz K.L.
      Bio-artificial liver devices –tentative, but promising progress.
      ]. These efforts aim to determine the microenvironment necessary for in vitro LPC expansion and/or differentiation of progenitor cells.
      One of the major drawbacks for LPCs identification/characterization is due to the difficulties of their extraction; largely explained by the cellular complexity of the organ in which they reside. The impressive heterogeneity of cells, the nature of physical links between all of them, and the complex macromolecular structural network represented by the ECM and the BM constitutes an environment that protects the LPCs during their lifespan (Fig. 2). This complexity also hampers their extraction. One of the issues is linked to the various functions carried out by hepatocytes and their zonation i.e. depending on their specific location within the liver lobule, hepatocytes’ function differs. Similarly, such zonal heterogeneity has been shown to be present in the non-parenchymal cell compartment, including Kupffer cells [
      • Sleyster E.C.
      • Knook D.L.
      Relation between localization and function of rat liver Kupffer cells.
      ] endothelial cells [
      • Wisse E.
      • De Zanger R.B.
      • Charels K.
      • Van Der Smissen P.
      • McCuskey R.S.
      The liver sieve: considerations concerning the structure and function of endothelial fenestrae, the sinusoidal wall and the space of Disse.
      ] and stellate cells [
      • Wake K.
      “Sternzellen” in the liver: perisinusoidal cells with special reference to storage of vitamin A.
      ], as well as, in the ECM compartment [
      • Reid L.M.
      • Fiorino A.S.
      • Sigal S.H.
      • Brill S.
      • Holst P.A.
      Extracellular matrix gradients in the space of Disse: relevance to liver biology.
      ].
      The location of the LPCs brings them in close anatomic relationship with non-parenchymal cells, in particular with hepatic stellate cells, both in normal and injured liver [
      • Alpini G.
      • Aragona E.
      • Dabeva M.
      • Salvi R.
      • Shafritz D.A.
      • Tavoloni N.
      Distribution of albumin and alpha-fetoprotein mRNAs in normal, hyperplastic, and preneoplastic rat liver.
      ,
      • Yin L.
      • Lynch D.
      • Ilic Z.
      • Sell S.
      Proliferation and differentiation of ductular progenitor cells and littoral cells during the regeneration of the rat liver to CCl4/2-AAF injury.
      ,
      • Yin L.
      • Lynch D.
      • Sell S.
      Participation of different cell types in the restitutive response of the rat liver to periportal injury induced by allyl alcohol.
      ]. Both cells have neuroendocrine features (see Table 2 for LPCs and for stellate cells see [
      • Hautekeete M.L.
      • Geerts A.
      The hepatic stellate (Ito) cell: its role in human liver disease.
      ,
      • Knittel T.
      • Aurisch S.
      • Neubauer K.
      • Eichhorst S.
      • Ramadori G.
      Cell-type-specific expression of neural cell adhesion molecule (N-CAM) in Ito cells of rat liver. Up-regulation during in vitro activation and in hepatic tissue repair.
      ,
      • Levy M.T.
      • McCaughan G.W.
      • Abbott C.A.
      • Park J.E.
      • Cunningham A.M.
      • Muller E.
      • et al.
      Fibroblast activation protein: a cell surface dipeptidyl peptidase and gelatinase expressed by stellate cells at the tissue remodelling interface in human cirrhosis.
      ]) suggesting that the cell types form a neuroendocrine compartment of the liver, which could be under the control of the central nervous system. Interactions between diverse systems create a regulatory “brain-stellate cells-LPCs triad,” adding yet another dimension to the concept of the LPC niche [
      • Roskams T.
      • Cassiman D.
      • De Vos R.
      • Libbrecht L.
      Neuroregulation of the neuroendocrine compartment of the liver.
      ]. Unfortunately, the mechanistics and the physical interactions between the three components of the triad have not been elucidated yet. Indeed, this remains a big challenge because understanding the control mechanisms of the triad could eventually be used in liver transplantation (the nerves being cut in the recipient) to create the right environment for re-innervation of diseased tissue after surgery, and thereby the survival of the graft.

      Isolation and characterization of LPCs from rodents: practical issues

      Destruction of the tissue

      In order to guarantee LPC extraction from the liver organ, cell–cell and cell–matrix interaction networks (Fig. 2) have to be destroyed enzymatically to get a suspension of single cells. The goal is to collect as many viable cells as possible and obtain a good dissociation efficiency while considering the best possible antigen retention. These parameters are of importance for the liver digestion and they are related to the choice of digestive enzymes. It has already been demonstrated that parameters, such as digestion time and enzyme activity, which constitute the dissociation efficiency, have a significant effect on cell yield and viability [
      • Panchision D.M.
      • Chen H.L.
      • Pistollato F.
      • Papini D.
      • Ni H.T.
      • Hawley T.S.
      Optimized flow cytometric analysis of central nervous system tissue reveals novel functional relationships among cells expressing CD133, CD15, and CD24.
      ,
      • Pilgaard L.
      • Lund P.
      • Rasmussen J.G.
      • Fink T.
      • Zachar V.
      Comparative analysis of highly defined proteases for the isolation of adipose tissue-derived stem cells.
      ]. However, the right balance in these digestion parameters is not necessarily linked to the highest cell viability or the most efficient tissue digestion. Consistent with studies in various tissues, differences in the aggressiveness of digestive enzymes are obvious on some cell surface markers. For instance, Panchision and co-workers showed that the flow cytometric analysis of the neural antigens (CD133, CD15 and CD24) on neuronal progenitor cells is affected by the manner of dissociation [
      • Panchision D.M.
      • Chen H.L.
      • Pistollato F.
      • Papini D.
      • Ni H.T.
      • Hawley T.S.
      Optimized flow cytometric analysis of central nervous system tissue reveals novel functional relationships among cells expressing CD133, CD15, and CD24.
      ]. CD24 antigenicity is lost by papain treatment whereas it is retained during Liberase-1, Tryp-LE and Accutase treatment. In contrast, while well preserved in presence of Liberase-1 and Accutase, CD133 antigenicity is lost by a cruder preparation of trypsin. The most popular couple used is collagenase/pronase using a multiple-step digestion protocol [
      • Yasui O.
      • Miura N.
      • Terada K.
      • Kawarada Y.
      • Koyama K.
      • Sugiyama T.
      Isolation of oval cells from Long–Evans Cinnamon rats and their transformation into hepatocytes in vivo in the rat liver.
      ]. Usually, the vascular system of the liver is used as the best route to deliver efficiently in situ the enzymatic solution. The pre-digested liver is then removed out of the animal, minced and exposed to a new digestion step. Typically, either a purified type of collagenase or a crude collagenase mixture is applied often leading to a lot-to-lot variability in collagenase activity and enzyme composition. Besides the earlier discussed data on non-liver tissues like the central nervous system [
      • Panchision D.M.
      • Chen H.L.
      • Pistollato F.
      • Papini D.
      • Ni H.T.
      • Hawley T.S.
      Optimized flow cytometric analysis of central nervous system tissue reveals novel functional relationships among cells expressing CD133, CD15, and CD24.
      ] and adipose tissue [
      • Pilgaard L.
      • Lund P.
      • Rasmussen J.G.
      • Fink T.
      • Zachar V.
      Comparative analysis of highly defined proteases for the isolation of adipose tissue-derived stem cells.
      ], effects of different dissociation methods on the analysis of important cell surface markers on the LPCs is essentially not described in the literature.
      While one can choose the type of enzyme, unfortunately, some parameters cannot always be controlled for e.g. the perfusion efficiency and intra-species variability. It is noteworthy that the optimized parameters of digestion validated for a healthy liver may be different for injured livers wherein ECM molecules are overexpressed and could eventually modify the established digestion efficiency. Therefore, concentration and digest times have to be continuously evaluated for an efficient digestion.

      Isolation and enrichment of LPCs

      LPCs represent only a small portion of the entire liver cell population but they can be isolated by using some of their specific properties, such as their size, density, antigenicity and functions (Fig. 3). The first attempts to isolate LPC from whole liver were performed on carcinogen-treated rats followed by centrifugal elutriation [
      • Overturf K.
      • Al-Dhalimy M.
      • Finegold M.
      • Grompe M.
      The repopulation potential of hepatocyte populations differing in size and prior mitotic expansion.
      ]. Largely due to low yield and high cost, this approach had been discontinued and investigators developed more attractive methods which involved the use of isopycnic centrifugation based on sorting cells according to their size and density. Fig. 3 illustrates the experimental procedures that are currently used for enrichment of adult LPCs. After digestion of the liver, hepatocytes (PF or parenchymal fraction) are excluded from the non-parenchymal cell fractions (NPF) by repeated low-speed centrifugations. This step is already limiting due to possible cell–cell adhesions between LPCs and hepatocytes which will be then pelleted together during this centrifugation. As a result, the number of progenitor cells is probably underestimated. LPCs may be purified by centrifugation through a discontinuous gradient. The fraction of interest (NFP), containing enriched LPCs, is subsequently taken out, washed and centrifuged to collect the cells for immediate seeding or use in subsequent enrichment steps [
      • Grozdanov P.N.
      • Yovchev M.I.
      • Dabeva M.D.
      The oncofetal protein glypican-3 is a novel marker of hepatic progenitor/oval cells.
      ,
      • Koenig S.
      • Probst I.
      • Becker H.
      • Krause P.
      Zonal hierarchy of differentiation markers and nestin expression during oval cell mediated rat liver regeneration.
      ,
      • Shupe T.D.
      • Piscaglia A.C.
      • Oh S.H.
      • Gasbarrini A.
      • Petersen B.E.
      Isolation and characterization of hepatic stem cells, or “oval cells”, from rat livers.
      ].
      Figure thumbnail gr3
      Fig. 3Different approaches used to isolate and characterize LPCs from rodents. Different solutions can be use to enrich the LPCs obtained by digestion/perfusion of the liver: Percoll, OptiPrep, Nycodenz or Ficoll-gradient. Characterization of the obtained cells can be done at different levels of purity/complexity or on total liver.
      Usually, this sort of isolation is based on the recognition (or activity) of molecules that are over expressed in these stem/progenitor cells. These molecules could be surface markers recognizable by antibodies and the function could be the overexpression of pumps, which are involved in expulsion of dangerous molecules from the LPCs. Two commonly used methodologies, magnetic activated cell sorting (MACS), and high-speed fluorescence-activated cell sorting (FACS), have been employed to obtain enriched populations of various stem/progenitor cells based on the cellular surface markers. In the liver, however, few specific markers are available until now, and only recently LPCs have been identified and isolated based on (a combination of) some non-specific cell surface markers i.e. c-Kit, CD45, TER119, c-Met, EpCAM, Sca-1, prominin-1 [
      • Rountree C.B.
      • Barsky L.
      • Ge S.
      • Zhu J.
      • Senadheera S.
      • Crooks G.M.
      A CD133-expressing murine liver oval cell population with bilineage potential.
      ,
      • Shafritz D.A.
      • Oertel M.
      • Menthena A.
      • Nierhoff D.
      • Dabeva M.D.
      Liver stem cells and prospects for liver reconstitution by transplanted cells.
      ,
      • Yovchev M.I.
      • Grozdanov P.N.
      • Joseph B.
      • Gupta S.
      • Dabeva M.D.
      Novel hepatic progenitor cell surface markers in the adult rat liver.
      ,
      • Yovchev M.I.
      • Grozdanov P.N.
      • Zhou H.
      • Racherla H.
      • Guha C.
      • Dabeva M.D.
      Identification of adult hepatic progenitor cells capable of repopulating injured rat liver.
      ].
      Another widely used method for the isolation of stem/progenitor cells is based on the efflux of the fluorescent dye Hoechst33343 determining a so-called side population (SP) [
      • Challen G.A.
      • Little M.H.
      A side order of stem cells: the SP phenotype.
      ]. This property to expel the dye is in large part due to the high expression of the ABC-transporter ABCG2 [
      • Zhou S.
      • Schuetz J.D.
      • Bunting K.D.
      • Colapietro A.M.
      • Sampath J.
      • Morris J.J.
      • et al.
      The ABC transporter Bcrp1/ABCG2 is expressed in a wide variety of stem cells and is a molecular determinant of the side-population phenotype.
      ]. Recently, an SP was also detected in murine liver that represented a small population of cells with progenitor-like characteristics [
      • Challen G.A.
      • Little M.H.
      A side order of stem cells: the SP phenotype.
      ,
      • Wulf G.G.
      • Luo K.L.
      • Jackson K.A.
      • Brenner M.K.
      • Goodell M.A.
      Cells of the hepatic side population contribute to liver regeneration and can be replenished with bone marrow stem cells.
      ]. These studies add to the growing belief that the SP fraction more or less equates with the LPC population in normal tissue [
      • Alison M.R.
      • Poulsom R.
      • Otto W.R.
      • Vig P.
      • Brittan M.
      • Direkze N.C.
      • et al.
      Plastic adult stem cells: will they graduate from the school of hard knocks?.
      ].
      Simple isolation techniques (without any fractionation step) of cells from the non-injured liver have also been carried out successfully, based on their similarities to oval cells and haematopoietic stem cells [
      • Sahin M.B.
      • Schwartz R.E.
      • Buckley S.M.
      • Heremans Y.
      • Chase L.
      • Hu W.S.
      • et al.
      Isolation and characterization of a novel population of progenitor cells from unmanipulated rat liver.
      ]. Azuma and co-workers developed a new enrichment system for LPCs from normal adult liver using their cell aggregate formation properties [
      • Azuma H.
      • Hirose T.
      • Fujii H.
      • Oe S.
      • Yasuchika K.
      • Fujikawa T.
      • et al.
      Enrichment of hepatic progenitor cells from adult mouse liver.
      ]. These cells are capable of growth and maturation along the hepatocyte lineage, indicating that they are LPCs.

      Characterization of LPCs

      Two strategies are commonly used to characterize LPCs (Fig. 3). The first involves the use of tissues from a normal or damaged liver that are collected directly after treatment and subsequently subjected to further analysis. The second strategy involves using cells obtained after the enrichment step; these are collected and ready for testing by different approaches.
      Studies based on tissue involves the use of antibodies that recognize specific targets expected to be only present on LPCs. Table 2 gives an overview of the many antigens used to identify LPCs in both normal and damaged liver. It is clear that amongst those markers there are very few that are not shared with other (liver) cell populations and even less that can be used for isolation by MACS or FACS. One can imagine that due to the complexity of the liver, it is difficult to characterize LPCs by techniques like Western blotting or real time PCR of total liver tissue. Some investigators have developed methods of labelling and micro-dissecting rodent cells within an extraordinarily short period of time using laser capture micro-dissection (LCM) [
      • Blatt R.
      • Srinivasan S.
      Defining disease with laser precision: laser capture microdissection in gastroenterology.
      ,
      • Gehring S.
      • Sabo E.
      • San Martin M.E.
      • Dickson E.M.
      • Cheng C.W.
      • Gregory S.H.
      Laser capture microdissection and genetic analysis of carbon-labeled Kupffer cells.
      ]. Using this technique in combination with LPC markers like K19 on normal and injured livers will probably gain more insight in the signalling pathways that regulate the LPCs but will also yield LPC-specific cell surface markers.
      Concerning the characterization of the LPCs after they have been isolated and enriched, three methods have been developed. The first method is aimed to prevent the ‘tissue culture-induced results’ and uses the freshly isolated cells as soon as possible without any culturing step. The second characterization approach is to analyze the differentiation capacities of the enriched LPCs, mainly by analyzing growth factor, cytokine or matrix-induced differentiation toward hepatocyte and cholangiocyte lineages [
      • Alison M.R.
      • Islam S.
      • Lim S.
      Stem cells in liver regeneration, fibrosis and cancer: the good, the bad and the ugly.
      ,
      • Oertel M.
      • Menthena A.
      • Chen Y.Q.
      • Teisner B.
      • Jensen C.H.
      • Shafritz D.A.
      Purification of fetal liver stem/progenitor cells containing all the repopulation potential for normal adult rat liver.
      ,
      • Snykers S.
      • Henkens T.
      • De Rop E.
      • Vinken M.
      • Fraczek J.
      • De Kock J.
      • et al.
      Role of epigenetics in liver-specific gene transcription, hepatocyte differentiation and stem cell reprogrammation.
      ]. The third approach is based on the functional characterization of LPCs i.e. whether isolated/enriched LPCs can rescue an injured liver. For instance, LPCs isolated from the liver of d-galactosamine treated rats engraft and undergo 5–7 rounds of cell division, as opposed to adult hepatocytes that undergo no more than 2–3 cell divisions under the same conditions [
      • Dabeva M.D.
      • Hwang S.G.
      • Vasa S.R.
      • Hurston E.
      • Novikoff P.M.
      • Hixson D.C.
      • et al.
      Differentiation of pancreatic epithelial progenitor cells into hepatocytes following transplantation into rat liver.
      ]. LPCs isolated from the livers of DDC-fed mice and transplanted into FAH null mice, repopulate the compromised liver with higher efficiency compared to hepatocytes [
      • Wang X.
      • Foster M.
      • Al-Dhalimy M.
      • Lagasse E.
      • Finegold M.
      • Grompe M.
      The origin and liver repopulating capacity of murine oval cells.
      ]. Sca-1+ LPCs from GFP transgenic mice induced by a DDC diet were able to repopulate approximately 50% of a liver when transplanted into monocrotaline-treated mice in conjunction with PH [
      • Song S.
      • Witek R.P.
      • Lu Y.
      • Choi Y.K.
      • Zheng D.
      • Jorgensen M.
      • et al.
      Ex vivo transduced liver progenitor cells as a platform for gene therapy in mice.
      ]. It is needless to say that these kinds of assays are indispensable for making claims with respect to the identity of the isolated LPCs.
      The majority of studies on LPCs depict gene expressions, probably reflecting the difficulty to obtain relatively large amounts of samples to perform protein studies. So far only one paper describes the use of proteomics showing a proteomic analysis of the c-Kit(CD45/Ter119)-LPC population in foetal mice (BALB/C strain) [
      • He Y.F.
      • Liu Y.K.
      • Lu H.J.
      • Chen J.
      • Yang P.Y.
      Comparative proteomic analysis of primary mouse liver c-Kit-(CD45/TER119)-stem/progenitor cells.
      ]. This 2-dimensional proteome map was possible by enrichment of the c-Kit(CD45/Ter119)-LPCs using successively two MACS procedures to deplete the red blood cells and the fibroblast-related cells.
      Finally, during the characterization of LPCs, either on tissue sections or cells, another limiting step is related to the use of antibodies. An antibody can recognize different parts of the protein, either glycosylated or phosphorylated, thereby determining antibody specificity. For instance, antibodies recognizing differently glycosylated forms of Prom1/CD133 are used to isolate progenitor cells from various tissues. Unfortunately, only few antibodies are able to recognize a specific glycosylated form of Prom1/CD133 that is strongly associated with “stemness” [
      • Karbanova J.
      • Missol-Kolka E.
      • Fonseca A.V.
      • Lorra C.
      • Janich P.
      • Hollerova H.
      • et al.
      The stem cell marker CD133 (Prominin-1) is expressed in various human glandular epithelia.
      ].

      Is there room for improvement?

      While the biological features of stem/progenitor cells justify the hope for future clinical applications, LPC therapy is still a bench issue that is far from the bedside [
      • Sancho-Bru P.
      • Najimi M.
      • Caruso M.
      • Pauwelyn K.
      • Cantz T.
      • Forbes S.
      • et al.
      Stem and progenitor cells for liver repopulation: can we standardise the process from bench to bedside?.
      ]. The problems are largely due to the ‘artificial’ strategy that researchers have to use to get sufficient amounts of LPCs, i.e. the application of different rodent injury models. Only some studies demonstrated a population of progenitor cells exhibiting similarities to LPCs that could be isolated from non-injured adult rodent livers [
      • Conigliaro A.
      • Colletti M.
      • Cicchini C.
      • Guerra M.T.
      • Manfredini R.
      • Zini R.
      • et al.
      Isolation and characterization of a murine resident liver stem cell.
      ,
      • Fougere-Deschatrette C.
      • Imaizumi-Scherrer T.
      • Strick-Marchand H.
      • Morosan S.
      • Charneau P.
      • Kremsdorf D.
      • et al.
      Plasticity of hepatic cell differentiation: bipotential adult mouse liver clonal cell lines competent to differentiate in vitro and in vivo.
      ,
      • Sahin M.B.
      • Schwartz R.E.
      • Buckley S.M.
      • Heremans Y.
      • Chase L.
      • Hu W.S.
      • et al.
      Isolation and characterization of a novel population of progenitor cells from unmanipulated rat liver.
      ]. This raises the question whether the choice of liver disease animal model influences the type of LPCs isolated?

      Animal subjects

      The strain, age and gender differences of animal subjects represent a variable in the identification/characterization of LPCs. For mice, most of the strains being used are BALB/C and C57BL/6, whereas for rat, Fisher 344 and Sprague–Dawley are generally used. The age of the animals commonly used varies between 3 and 16 weeks old for mice, and 120–230 g for rats. As the self-renewal and differentiation capacity of young and aged stem/progenitor cells are interconnected [
      • Sharpless N.E.
      • DePinho R.A.
      How stem cells age and why this makes us grow old.
      ] it is difficult to compare the amount and quality of the LPCs isolated from animals with different ages.

      Different types of liver injury lead to activation of LPCs

      Mainly because of the great variability in the methods used to activate the LPC compartment, it is difficult to compare the different studies that have already been performed (Table 1). Differing treatments have a completely different impact on the liver. For instance, phenobarbital/cocaine and allyl alcohol injury models induce periportal injury [
      • Rosenberg D.
      • Ilic Z.
      • Yin L.
      • Sell S.
      Proliferation of hepatic lineage cells of normal C57BL and interleukin-6 knockout mice after cocaine-induced periportal injury.
      ,
      • Yavorkovsky L.
      • Lai E.
      • Ilic Z.
      • Sell S.
      Participation of small intraportal stem cells in the restitutive response of the liver to periportal necrosis induced by allyl alcohol.
      ]. Hepatocytes in non-injured zones start to proliferate, followed by proliferation of cholangiocytes and LPCs. By 10 days the injured zone is completely repaired and no dividing cells remain. Interestingly, the appearance of LPCs is only detected after several days in the phenobarbital/cocaine model [
      • Rosenberg D.
      • Ilic Z.
      • Yin L.
      • Sell S.
      Proliferation of hepatic lineage cells of normal C57BL and interleukin-6 knockout mice after cocaine-induced periportal injury.
      ] while they can already be recognized within hours in the APAP models [
      • Kofman A.V.
      • Morgan G.
      • Kirschenbaum A.
      • Osbeck J.
      • Hussain M.
      • Swenson S.
      • et al.
      Dose- and time-dependent oval cell reaction in acetaminophen-induced murine liver injury.
      ,
      • Paku S.
      • Nagy P.
      • Kopper L.
      • Thorgeirsson S.S.
      2-Acetylaminofluorene dose-dependent differentiation of rat oval cells into hepatocytes: confocal and electron microscopic studies.
      ]. This discrepancy can be explained by the fact that in the case of the APAP injury, the lesion is central and anatomically preserves the zone where LPC reactions take place. In contrast, the phenobarbital/cocaine treatment leads to damage in the periportal area. These findings seem to indicate that an injury close to the LPC compartment will take more time to generate an LPC reaction than an injury that affects a remote area.
      Van Hul and co-workers showed that, in the CDE model, ECM deposition and activation of matrix producing cells occurred in an initial phase, prior to LPCs expansion, and in front of LPCs along the porto-veinous gradient of lobular invasion [
      • Van Hul N.K.
      • Abarca-Quinones J.
      • Sempoux C.
      • Horsmans Y.
      • Leclercq I.A.
      Relation between liver progenitor cell expansion and extracellular matrix deposition in a CDE-induced murine model of chronic liver injury.
      ]. Those observations (in C57BL/6J mice) suggest a fundamental role for a hepatic microenvironment or niche during the process of activation and differentiation of LPCs. Studies in this and other injury models should reveal whether there is really a supportive role of ECM reconstruction in the LPC response, and whether it becomes one of the general characteristic for an LPC response.
      As a consequence of the above mentioned parameters, it is nearly impossible to give a systematic and comparative overview of the similarities and differences in the response of the LPC compartment in adult rats and mice subjected to various experimental models of liver injury. Fortunately, one recent study attempted to do this experimentally and the results speak for themselves. Jelnes and co-workers have used two of the most widely rodent strains: mouse (C57BL/6J) and rat (Fisher 344) and several commonly used protocols for LPC-mediated liver regeneration (AAF/PHx, CDE, DDC and APAP) [
      • Jelnes P.
      • Santoni-Rugiu E.
      • Rasmussen M.
      • Friis S.L.
      • Nielsen J.H.
      • Tygstrup N.
      • et al.
      Remarkable heterogeneity displayed by oval cells in rat and mouse models of stem cell-mediated liver regeneration.
      ]. They demonstrated that the reactions observed in rat and mouse protocols differ in several aspects when the regenerative response was evaluated by immuno positivity for the LPC markers (like, KT-19 ABCG2, AFP and Dlk/Pref1). The AAF/PHx protocol results in a reproducible activation of the LPC compartment in rat, whereas it is inadequate to induce the desired compartment in mice. The APAP model is more appropriate for oval cell activation in mouse. In contrast to rat, the DDC diet was found to induce very consistent and massive oval cell accumulation in mice. The CDE protocol induced the LPC compartment in both species, although there are differences in the phenotype of LPCs. A possible explanation for these differences is a different rate of metabolism of the diets in the two rodent models.
      LPCs constitute a heterogeneous population of proliferating progenitors found in rodent livers following carcinogenic treatments. However, during such treatments, not only do oval cells appear but a second population emerges from the periportal field as well [
      • Dudas J.
      • Mansuroglu T.
      • Batusic D.
      • Ramadori G.
      Thy-1 is expressed in myofibroblasts but not found in hepatic stellate cells following liver injury.
      ,
      • Koenig S.
      • Probst I.
      • Becker H.
      • Krause P.
      Zonal hierarchy of differentiation markers and nestin expression during oval cell mediated rat liver regeneration.
      ]. In general, the second population is positive for Thy-1 and nestin, probably reflecting the presence of fibroblasts, myofibroblasts and hepatic stellate cells. During the AAF/PHx injury model these two populations (LPCs and accompanying cells) are organized in a zonal hierarchy with a marker gradient form the inner to the outer zone of the proliferating progeny clusters [
      • Koenig S.
      • Probst I.
      • Becker H.
      • Krause P.
      Zonal hierarchy of differentiation markers and nestin expression during oval cell mediated rat liver regeneration.
      ]. Unfortunately such studies have not been carried out in other rodent injury models. In addition, whether there is a recruitment of pre-existing Thy-1+ and nestin+ cells in response to activation of the oval cell compartment, or whether potential differentiation of oval cells into Thy-1+ and nestin+ cells takes place is not defined yet.

      Working 9 to 12?

      A circadian rhythm of liver regeneration exists [
      • Barbason H.
      • Bouzahzah B.
      • Herens C.
      • Marchandise J.
      • Sulon J.
      • Van Cantfort J.
      Circadian synchronization of liver regeneration in adult rats: the role played by adrenal hormones.
      ,
      • LaBrecque D.R.
      • Feigenbaum A.
      • Bachur N.R.
      Diurnal rhythm: effects on hepatic regeneration and hepatic regenerative stimulator substance.
      ] e.g. the induction of liver regeneration by various hepatotoxins should preferentially be initiated at a standardized time of day (between 9:00 and 12:00 in the morning). While a link between circadian rhythm and the use of hepatotoxins has not been reported, the molecular components of the body’s circadian clock to adult stem cell physio-biology have been identified [
      • Gimble J.M.
      • Floyd Z.E.
      • Bunnell B.A.
      The 4th dimension and adult stem cells: can timing be everything?.
      ]. A highly conserved set of genes encoding the core circadian regulatory proteins (CCRP) has evolved across species [
      • Bozek K.
      • Relogio A.
      • Kielbasa S.M.
      • Heine M.
      • Dame C.
      • Kramer A.
      • et al.
      Regulation of clock-controlled genes in mammals.
      ]. The levels of these transcription factors and their activities oscillate rhythmically over a 24-h period [
      • Lowrey P.L.
      • Takahashi J.S.
      Mammalian circadian biology: elucidating genome-wide levels of temporal organization.
      ]. Interestingly, CCRP regulation has been found in some adult stem cell models, like haematopoietic [
      • Mendez-Ferrer S.
      • Lucas D.
      • Battista M.
      • Frenette P.S.
      Haematopoietic stem cell release is regulated by circadian oscillations.
      ], bone marrow-derived mesenchymal and adipose-derived stem cells [
      • Gimble J.M.
      • Floyd Z.E.
      • Bunnell B.A.
      The 4th dimension and adult stem cells: can timing be everything?.
      ]. Regarding the specific sequences consensus for such CCRP proteins in different tissues, it has been found that in the liver such CCRP sequences lie on hepatic genes like HFN-1, -3 and -4 [
      • Bozek K.
      • Relogio A.
      • Kielbasa S.M.
      • Heine M.
      • Dame C.
      • Kramer A.
      • et al.
      Regulation of clock-controlled genes in mammals.
      ]. We can hypothesize that such regulated mechanisms also exist in the oval cells, thereby maybe influencing the outcome of the hepatotoxic injury.

      Fine-tuning oval cells

      Numerous data support the concept of an intimate relationship between LPC expansion, ECM deposition and myofibroblastic cells chaperoning oval cells during their activation, emphasizing the importance of the established liver niche [
      • Dezso K.
      • Jelnes P.
      • Laszlo V.
      • Baghy K.
      • Bodor C.
      • Paku S.
      • et al.
      Thy-1 is expressed in hepatic myofibroblasts and not oval cells in stem cell-mediated liver regeneration.
      ,
      • Dudas J.
      • Mansuroglu T.
      • Batusic D.
      • Saile B.
      • Ramadori G.
      Thy-1 is an in vivo and in vitro marker of liver myofibroblasts.
      ,
      • Duncan A.W.
      • Dorrell C.
      • Grompe M.
      Stem cells and liver regeneration.
      ,
      • Kamo N.
      • Yasuchika K.
      • Fujii H.
      • Hoppo T.
      • Machimoto T.
      • Ishii T.
      • et al.
      Two populations of Thy1-positive mesenchymal cells regulate in vitro maturation of hepatic progenitor cells.
      ,
      • Koenig S.
      • Probst I.
      • Becker H.
      • Krause P.
      Zonal hierarchy of differentiation markers and nestin expression during oval cell mediated rat liver regeneration.
      ,
      • Van Hul N.K.
      • Abarca-Quinones J.
      • Sempoux C.
      • Horsmans Y.
      • Leclercq I.A.
      Relation between liver progenitor cell expansion and extracellular matrix deposition in a CDE-induced murine model of chronic liver injury.
      ,
      • Yang L.
      • Jung Y.
      • Omenetti A.
      • Witek R.P.
      • Choi S.
      • Vandongen H.M.
      • et al.
      Fate-mapping evidence that hepatic stellate cells are epithelial progenitors in adult mouse livers.
      ,
      • Yovchev M.I.
      • Grozdanov P.N.
      • Zhou H.
      • Racherla H.
      • Guha C.
      • Dabeva M.D.
      Identification of adult hepatic progenitor cells capable of repopulating injured rat liver.
      ]. Elegant evidence for the biological function of the TWEAK/Fn14 pathway identified TWEAK (TNF-like weak inducer of apoptosis) as a main component of the LPC niche; transgenic mice expressing TWEAK in hepatocytes displayed a spontaneous oval cell reaction and a reduction of oval cell response was observed when, in the DDC mouse model, TWEAK blocking antibodies or Fn14 null mice were used. [
      • Jakubowski A.
      • Ambrose C.
      • Parr M.
      • Lincecum J.M.
      • Wang M.Z.
      • Zheng T.S.
      • et al.
      TWEAK induces liver progenitor cell proliferation.
      ]. The potential use of recombinant TWEAK, or agonists to Fn14, to enable LPC expansion in vitro and in vivo is an exciting prospect [
      • Burkly L.C.
      • Michaelson J.S.
      • Hahm K.
      • Jakubowski A.
      • Zheng T.S.
      TWEAKing tissue remodeling by a multifunctional cytokine: role of TWEAK/Fn14 pathway in health and disease.
      ].

      Liver regeneration: will LPCs be better than hepatocytes?

      Low liver-engraftment rates and poor survival of transplanted cells hamper the efficiency of clinical and experimental hepatocyte transplantation [
      • Fox I.J.
      • Chowdhury J.R.
      • Kaufman S.S.
      • Goertzen T.C.
      • Chowdhury N.R.
      • Warkentin P.I.
      • et al.
      Treatment of the Crigler-Najjar syndrome type I with hepatocyte transplantation.
      ]. Due to their large diameter (20–40 μm), up to 70% of transplanted hepatocytes get trapped in the hepatic sinusoids, which leads to temporary obstruction with subsequent portal hypertension [
      • Weber A.
      • Groyer-Picard M.T.
      • Franco D.
      • Dagher I.
      Hepatocyte transplantation in animal models.
      ], poor engraftment rate and finally the demand for a high amount of transplantable cells (up to 2 × 107 hepatocytes in rodent models) [
      • Allen K.J.
      • Soriano H.E.
      Liver cell transplantation: the road to clinical application.
      ]. For this reason, alternative administration of stem/progenitor cells is considered to be a promising future treatment option for numerous acute or chronic liver diseases [
      • Sandhu J.S.
      • Petkov P.M.
      • Dabeva M.D.
      • Shafritz D.A.
      Stem cell properties and repopulation of the rat liver by fetal liver epithelial progenitor cells.
      ]. In contrast to hepatocytes, their high accessibility from various tissues and their small overall size predisposes stem cells to be a feasible and efficient alternative therapy. At present, research has succeeded in obtaining transplantable progenitor/stem cells from liver, bone marrow, umbilical cord blood, Whartons’s jelly stem cells, skin and adipose tissue. Few approaches have been developed to reduce the rejection of transplanted cells and to improve the poor cell engraftment rate in order to reduce the overall required number of cells to administer. One proposed method to decrease rejection rate and to increase engraftment rate of transplanted cells is the (co)administration of mesenchymal stem cells; not only due to their proven immunomodulatory and immunosuppressive properties, but also because they may provide an appropriate peri-cellular and extracellular environment. Although the expected transplantation efficiencies of stem cells is much higher [
      • Sandhu J.S.
      • Petkov P.M.
      • Dabeva M.D.
      • Shafritz D.A.
      Stem cell properties and repopulation of the rat liver by fetal liver epithelial progenitor cells.
      ] due to their small size, it should nevertheless be kept in mind that these cells have to differentiate into functional hepatic cells, this process takes time and does not consistently occur in a diseased liver which could influence the final outcome.

      Conclusion

      It remains unclear whether LPCs that have been generated through different protocols in different species have the same characteristics. Moreover, before LPCs can be safely and effectively used in patients many hurdles remain to be overcome. In contrast to other stem cell systems, the molecular characterization of the LPCs still suffers from the lack of specific markers that can unambiguously and specifically label LPCs, thus enabling their identification. The lack of such markers also hinders the optimization of conditions that would keep an LPC culture in a stem cell state, thereby limiting the amount of cells available for any further characterization or transplantation. Recent techniques like LCM or the development of miniature artificial liver devices may thus help to accelerate the identification and characterization of the LPCs and pave the way for future applications.

      Acknowledgments

      The authors who have taken part in this study declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript. We remember Professor Albert Geerts who passed away during the preparations of this review. We are grateful to him for all his enthusiasm and support. This paper is in his honour. The literature on liver progenitor cells is extensive, and numerous important studies from many colleagues were not mentioned here owing to space limitations. We apologize for not citing their work. The work in the CYTO Lab is funded by the Vrije Universiteit Brussel (VUB) through different OZR grants, by the Fund for Scientific Research-Flanders (FWO-V) ( G.0229.08 and G.0651.06 ), BELSPO ( IUAP-VI, P6/36 ) and the Brussels region (ISRIB/“BRUSTEM”).

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