The quest for liver progenitor cells: A practical point of view Academic research paper on "Biological sciences"

Review

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EUROPEAN ASSOCIATION FOR THE STUDY OF THE LIVER I

JOURNAL OF HEPATOLOGY

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

Laurent Dolle1, Jan Best1, Jie Mei1, Feras Al Battah1, Hendrik Reynaert1,2, Leo A. van Grunsven1'*,

Albert Geerts1,f

1 Department of Cell Biology, Vrije Universiteit Brüssel (V.U.B.), Belgium; 2Department of Physiology, Vrije Universiteit Brüssel (V.U.B.), Belgium

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. © 2009 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Keywords: Liver progenitor cells; Oval cells; Liver regeneration; Liver injury; Stem cells.

"Corresponding author. Address: Department of Cell Biology, Vrije Universiteit Brüssel (V.U.B.), Faculty of Medicine and Pharmacy, Laarbeeklaan 103, 1090 Brüssel, Belgium. Tel.: +32 2 477 4419; fax: +32 2 477 4412. E-mail address: Leo.van.grunsven@vub.ac.be (L.A. van Grunsven). f Prof. Geerts passed away during the completion of the study. Abbreviations: DNA, deoxyribonucleic acid; LPC, liver progenitor cell; CCl4, carb-otetrachloride; AAF, 2-acetylaminofluorene; APAP, N-acetyl-p-aminophenol; SCF, stem cell factor; SDF1, stromalcell-derived factor-1; CXCR4, CXC chemokine re-ceptor4; TWEAK, tumor necrosis factor-like weak inducer of apoptosis; KT, cyt-okeratin; 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, ethi-onine-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; CC-RP, core circadian regulatory protein; HNF, hepatocyte nuclear factor; Cx, Conn-exin; 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 [37] (for instance, what criteria make a patient eligible to receive a donor organ?). Additionally, the technically demanding "state-of-the-art" surgery [16], and especially the cost and risks of a life long immunosuppression [99] 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 [149]. However, the low liver-engraftment rate and survival of transplanted hepatocytes hamper this procedure [54]. 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 [13]. 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 [24]. 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 [3,47,135,156]. 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 cholangio-cytes to restore liver function and cell mass [7,43,48,140,152]. 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 [4,61,104,148].

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 [50,51,173] and opponents [20,144]. 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. cholan-giocytes and other non-parenchymal cells. For instance, cholan-giocytes also have low basal DNA synthesis but they proliferate in a number of experimental models of cholestasis [10,11].

It is only upon extensive and chronic liver injury that another cell type is activated: the liver progenitor cells (LPCs) [17,36,40]. 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 [109,119,160].

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) [60,97,98,154,156]. 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 [4,17,36,86] (Fig. 1). Following 70% partial hepatectomy, rat liver completely recovers its initial volume at day 20 [67], while after right lobe transplantation in humans, donor and recipient livers reached their original weight by 60 days after surgery [90]. 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 [49,98,134] (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 [107,118]. Grompe's group has demonstrated that adult hepatocytes expand clonally and may be serially trans-

planted [106,107]. These studies raise the possibility that hepato-cytes may display multipotentiality, one of the defining characteristics of stem cells.

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 [21,45,46]. 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 [23,44,154]. 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 hepa-tocytes to replenish the organ, thus promoting the activation of a secondary intra-hepatic regenerative compartment [2,46,48,137, 139,142] (Fig. 1). This so-called ''oval cells compartment" consists of 'small ovoid cells with scant lightly basophilic cytoplasm and pale blue-staining nuclei' [43]. 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 [109,124,131,155]. The oval cell compartment can probably not be attributed to a single cell type [175]. 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 [140]. Proliferating oval cells constitute a heterogeneous population justifying the different names used to describe them: ductular progenitor cells [108,109], atypical ductular cells [73], peri-ductular liver progenitor cells [136,137] or individual progenies [170].

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 [2,36,110,127,129]). 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. Car-botetrachloride (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

Fig. 1. Schematic 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.

(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).

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 [33]. 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) [28]. Finally, in these experimental models the process of liver damage and repair are interwoven, making the interpretation of the results more complex [101].

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 [17,36,40,56,102]. It is believed that 3 important cell signalling axes are involved in the activation of LPCs: SCF/c-Kit [70] SDF1/CXCR4 important for the migration [63,153] and TWEAK/Fn14 [71] for the activation of the LPCs. This

Table 1. Commonly used experimental models for LPC-mediated regeneration.

Nature of the regime Rodent model Reference

Phénobarbital Mouse [119]

AAF/PH Mouse and rat [41,55,116,142]

AAF/CCL4 Rat [26,113,168]

AAF/AA Rat [113]

CD/AAF Rat [133,138]

CDE Rat and mouse [1143]

DDC Mouse and rat [116,160]

DIPIN + PH Mouse and rat [39,42]

D-Calactosamine + PH Rat and mouse [87,29,85]

Lasiocarpine + PH Rat [84]

Retrorsine + PH Rat [59,83]

Retrorsine + CC14 Rat [31,177]

Retrorsine + allyl alcohol [115]

APAP Mouse [80]

AA Rat and mouse [85,167]

CCI4 Mouse [38]

DEN Rat and mouse [65,132]

Long-term ethanol Rat and mouse [123,147]

Only representative publications are listed. The most frequently used hepato-toxins 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)phos-phamide]piperazine; DEN.V, diethylnitrosamine.

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 neu-

roepithelial 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.

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) [7,100]. The other population consists of non-ductular cells that can be detected between and distally from the ductules with fibroblastic characteristics (the accompanying cells) [32,34,35,79,157]. 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 [52,109,112,120,122,155]. 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 [81]. 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 [15,131,155,167,173]. 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 [109,121,122] and Kupffer cells [68], 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

Table 2. Commonly used markers for the identification of LPCs in rodents.

Marker Location

Adult hepatocyte related Albumin (ALB), alpha anti-trypsine (AAT), hepatocyte nuclear factor 4 (HNF4), cytokeratins (KT-8, KT-18) ATP binding cassette G2 (ABCG2), E-cadherin, c-Met, c-Kit (or CD117) Intracellular Cell membrane

Foetal hepatocyte related Alpha fetoprotein (AFP), muscle pyruvate kinase (MPK), glutathione S transferase (GST), gamma glutamyl transpeptidase (GGT), delta-like protein (Dlk), aldolase A and C Intracellular

Fnl4 (or TWEAK receptor), CD13, CD24, CD29 (or integrin-bl), CD26 (or dipeptidylpeptidase IV), CD49f (or integrin-a6), ATP binding cassette G2 (ABCG2) Cell membrane

Adult biliary related Cytokeratins (KT-7, KT-14, KT-19), muscle pyruvate kinase (MPK), Glutathione S transferase (GST), gamma glutamyl transpeptidase (GGT), OV-6 (KT-14 and KT-19), OC (2,3, 4, 5), A6 antigen OV-1, epithelial cell adhesion molecule (EpCAM), c-Met, CD133 (prominin-1), c-Kit (or CD117), Connexin 43 (Cx43), CD24, MUC1 (or Mucin-1), cadherin 22, CD44, CD49f (or integrin-a6) Intracellular Cell membrane

Adult haematopoietic related Thymus antigen 1 (or Thy-1 or CD90), c-Kit (or CD117), stem cell antigen 1 (Sca-1), CXCR4, CD34, Flt-3 (or CD135) Cell membrane

Neuroepithelial related Chromogranin A (Chrom-A), S-100, parathyroid hormone-related peptide (PTHrP) CD56 (or N-CAM), neurotrophin receptors Intracellular Cell membrane

(Myo)fribroblastic related Nestin CD44, Fnl4 (or TWEAK receptor), thymus antigen 1 (or Thy-1 or CD90) Intracellular Cell membrane

Progenitor related TROP2, Connexin 32 (Cx32) Cell membrane

For an extended overview of the potential markers expressed on LPCs we refer to a multitude of articles and reviews and their related Refs. [5,17,56,129].

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 [40,129]. Nerves [122] basement membrane [108] and ECM [157,174] 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-a and -p [36,40,77]. Knight and co-workers documented a close correlation between inflammation, cyto-kine production and the expansion of oval cells in the liver during experimental chronic injury (CDE treatment) in C57BL/6 mice [76]. 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 [40].

partly by hepatocytes, and it serves to conduct bile from bile can-aliculi to terminal bile ducts in portal tracts [130]. 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 cholan-giocytes 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 [10]. 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 [105]. 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.

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

Location is everything

As Petersen and Shupe stated last year [112], "location is everything" (Fig. 2). LPCs are found in the canals of Hering, which

Fig. 2. Schematic 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.

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 [69,95,158,162,163].

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 [91-93]. 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 [94]. 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 [151]. 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 hepato-cytes 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 [146] endothelial cells [163] and stellate cells [159], as well as, in the ECM compartment [117].

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 [9,168,169]. Both cells have neuroendocrine features (see Table 2 for LPCs and for stellate cells see [64,78,88]) 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 [122]. 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 [111,114]. 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 [111]. 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 [166]. 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 [111] and adipose tissue [114], 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 [106]. 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 cen-trifugation 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

Fig. 3. Different 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.

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 [62,79,145].

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 overexpres-

sion 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 [125,140,171,172].

Another widely used method for the isolation of stem/progenitor cells is based on the efflux of the fluorescent dye Hoe-chst33343 determining a so-called side population (SP) [25]. This property to expel the dye is in large part due to the high expression of the ABC-transporter ABCG2 [176]. Recently, an SP

was also detected in murine liver that represented a small population of cells with progenitor-like characteristics [25,164]. These studies add to the growing belief that the SP fraction more or less equates with the LPC population in normal tissue [6].

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 [126]. Azuma and co-workers developed a new enrichment system for LPCs from normal adult liver using their cell aggregate formation properties [12]. 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) [18,57]. 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 cholan-giocyte lineages [5,103,148]. 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 [30]. LPCs isolated from the livers of DDC-fed mice and transplanted into FAH null mice, repopulate the compromised liver with higher efficiency compared to hepa-tocytes [160]. 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 [150]. 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) [66]. 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 "sternness" [75].

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 [127]. 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 [27,53,126]. 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 [141] 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 [119,167]. Hepatocytes in non-injured zones start to proliferate, followed by proliferation of cholangio-cytes 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 phenobar-bital/cocaine model [119] while they can already be recognized within hours in the APAP models [80,108]. 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 [157]. 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) [72]. 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 [34,79]. 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 [79]. 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 [14,82] 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 [58]. A highly conserved set of genes encoding the core circadian regulatory proteins (CCRP) has evolved across species [19]. The levels of these transcription factors and their activities oscillate rhythmically over a 24-h period [89]. Interestingly, CCRP regulation has been found in some adult stem cell models, like haematopoietic [96], bone marrow-derived mesenchymal and adipose-derived stem cells [58]. 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 [19]. 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 [32,35,36,74,79, 157,165,172]. 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. [71]. The potential use of recombinant TWEAK, or agonists to Fn14, to enable LPC expansion in vitro and in vivo is an exciting prospect [22].

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 hepato-cyte transplantation [54]. Due to their large diameter (2040 im), up to 70% of transplanted hepatocytes get trapped in the hepatic sinusoids, which leads to temporary obstruction with subsequent portal hypertension [161], poor engraftment rate and finally the demand for a high amount of transplantable cells (up to 2 x 107 hepatocytes in rodent models) [8]. 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 [128]. 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 trans-plantable 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 engraft-ment 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 [128] 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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