Journal of Pathology J Pathol 2002; 197: 510–518. Published online 30 May 2002 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002 / path.1163
Review Article
Hepatic stem cells Stuart Forbes1, Pamela Vig2, Richard Poulsom2, Howard Thomas1 and Malcolm Alison3* 1
Department of Hepatology, Imperial College, London, UK Histopathology Unit, Cancer Research UK, 44 Lincoln’s Inn Fields, London, UK 3 Department of Histopathology, Imperial College, London, UK 2
* Correspondence to: Malcolm Alison, Department of Histopathology, Faculty of Medicine, Imperial College School of Science, Technology and Medicine, Hammersmith Hospital, Du Cane Road, London W12 0NN, UK. E-mail:
[email protected]
Abstract The liver in an adult healthy body maintains a balance between cell gain and cell loss. Though normally proliferatively quiescent, hepatocyte loss such as that caused by partial hepatectomy, uncomplicated by virus infection or inflammation, invokes a rapid regenerative response to restore liver mass. This restoration of moderate cell loss and ‘wear and tear’ renewal is largely achieved by hepatocyte self-replication. Furthermore, hepatocyte transplants in animals have shown that a certain proportion of hepatocytes can undergo significant clonal expansion, suggesting that hepatocytes themselves are the functional stem cells of the liver. More severe liver injury can activate a potential stem cell compartment located within the intrahepatic biliary tree, giving rise to cords of bipotential so-called oval cells within the lobules that can differentiate into hepatocytes and biliary epithelial cells. A third population of stem cells with hepatic potential resides in the bone marrow; these haematopoietic stem cells can contribute to the albeit low renewal rate of hepatocytes, make a more significant contribution to regeneration, and even completely restore normal function in a murine model of hereditary tyrosinaemia. How these three stem cell populations integrate together to achieve a homeostatic balance is not known. This review focuses on two major aspects of liver stem cell biology: firstly, the identity of the liver stem cells, and secondly, their potential value in the treatment of major liver disease. Copyright # 2002 John Wiley & Sons, Ltd. Keywords: stem cells; hepatocytes; cholangiocytes; haematopoietic stem cells; bone marrow; transplantation; gene therapy
The nature of liver stem cells Perhaps born out of necessity from the plethora of potentially cell-damaging xenobiotics that assail the liver, plus a myriad of other cellular insults, e.g. hepatotropic viruses, the liver can invoke not just one, but three apparently phenotypically distinct cell lineages to contribute to regenerative growth after damage.
Hepatocytes In response to parenchymal cell loss, the hepatocytes are the cells that normally restore the liver mass, rapidly re-entering the cell cycle from the G0 phase. However, even after a two-thirds partial hepatectomy (Figure 1), the remaining cells only have to cycle at the very most two to three times to restore pre-operative cell number, since we know that all remaining hepatocytes traverse the cell cycle at least once. This seemingly modest response led to the incorrect assumption that hepatocytes had only limited division potential and thus were not true stem cells. A crucial property that defines a stem cell is its ability to give rise to a large family of descendants and, importantly, at least some hepatocytes can do this. Hepatocyte transplantation protocols, developed because of the shortage of livers for whole-organ transplantation, Copyright # 2002 John Wiley & Sons, Ltd.
have shown that the transplanted cells are capable of significant clonal expansion within the diseased liver of a recipient (see below).
Cholangiocytes When either massive damage is inflicted upon the liver or regeneration after damage is compromised, a potential stem cell compartment located within the smallest branches of the intrahepatic biliary tree is activated. This so-called ‘oval cell’ or ‘ductular reaction’ amplifies the biliary population before these cells differentiate into hepatocytes [1–5]. In rats and mice, this response emanates from the smaller interlobular bile ducts and canals of Hering that barely extend beyond the limiting plate (Figure 2A), and the resultant oval cells (Figure 2B) form arborizing ducts between the liver cell plates (Figure 2C) before these cells differentiate into hepatocytes (Figure 2D). Elegant three-dimensional reconstructions of serial sections of human liver immunostained for cytokeratin-19 (CK19) have shown that the smallest biliary ducts, the canals of Hering, unlike those in rodents actually normally extend into the proximate third of the lobule [6] and it is envisaged that these canals react to massive liver damage (akin to a trip-wire), proliferating and then differentiating into hepatocytes (Figure 3). Oval cell numbers in human liver rise with increasing severity of
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Figure 1. (A) Partial hepatectomy (PH) in a rat involves resection of the left lateral (LL) and median (M) lobes comprising two-thirds of the liver mass and the pre-operative mass is restored within 10 days. (B) Twenty-four hours after PH, many hepatocytes are in DNA synthesis, as indicated by bromodeoxyuridine labelling, but note the relative absence of labelling around the hepatic vein (HV) at this time, though within 48 h after PH almost all hepatocytes will have traversed the cell cycle at least once
liver disease [7] and this ductular reaction is widely accepted to be a stem cell response rather than a ductular metaplasia of ‘damaged’ hepatocytes. Nonparenchymal epithelial cells from adult porcine liver,
essentially ductal epithelia, are also capable of differentiating into both biliary epithelia and hepatocytes in vitro [8]. Moreover, antigens traditionally associated with haematopoietic cells can also be expressed by oval
Figure 2. Hepatic stem cell activation in the rat liver. (A) Normal rat liver immunostained for CK19 to highlight biliary epithelia. Note that all positive cells are within or very close to the portal tract. (B) After hepatocyte loss in a liver where hepatocyte regeneration is compromised, oval cells increase in number – an electron micrograph of oval cells with one in mitosis (M). (C) Oval cell reaction in which the biliary cells form branching ducts radiating from the portal tract (CK19 immunostain). (D) The biliary cells observed in C eventually differentiate into hepatocytes that have not yet acquired the cytochrome P450 immunoreactivity of the older and larger hepatocytes; residual ductular structures are still apparent (arrows) Copyright # 2002 John Wiley & Sons, Ltd.
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Figure 3. (A) The canals of Hering extend into the proximate third of the hepatic lobule in the human liver (see [ref. 6]) and major damage (*) activates the lining cells to divide and probably differentiate into hepatocytes. (B) Human liver with a ductular reaction in response to major parenchymal damage (CK8 immunostain)
cells, including c-kit, flt-3, Thy-1, and CD34 [9–12]. This may be no more than coincidental, but it has given support to the notion that at least some hepatic oval cells are directly derived from a precursor of bone marrow origin, particularly when the biliary tree is damaged [13], though we firmly believe that many/ most oval cells are derived from the direct intrahepatic proliferation of cells already located within the biliary tree [1–5].
Bone marrow Within an adult tissue, the locally resident stem cells were formerly considered to be capable of only giving rise to the cell lineage(s) normally present. However, adult haematopoietic stem cells (HSCs) in particular appear to be much more flexible: removed from their usual niche, they are capable of differentiating into all manner of tissues including skeletal and cardiac muscle, endothelia, and a variety of epithelia including neuronal cells, pneumocytes, and hepatocytes. Some oval cells/hepatocytes were first revealed to be derived from circulating bone marrow cells in the rat: Petersen et al. [14] followed the fate of syngeneic male bone marrow cells transplanted into lethally irradiated female recipient animals whose livers were subsequently injured by a regime of 2-acetylaminofluorene (which blocks hepatocyte regeneration) and carbon tetrachloride (which causes hepatocyte necrosis) designed to cause oval cell activation. Y-chromosomepositive oval cells were found at 9 days after liver injury and some Y-chromosome-positive hepatocytes were seen at 13 days when oval cells were differentiating into hepatocytes. Additional evidence for hepatic engraftment of bone marrow cells was forthcoming from a rat whole-liver transplant model. Lewis rats expressing the MHC class II antigen L21-6 were recipients of livers from Brown Norway rats that were negative for L21-6. Subsequently, ductular structures in the transplants contained both L21-6-negative and L21-6-positive cells, indicating that some biliary epithelium was of in situ derivation and some was of Copyright # 2002 John Wiley & Sons, Ltd.
recipient origin, presumably from circulating bone marrow cells. Using a similar gender mismatch bone marrow transplantation approach in mice to track the fate of bone marrow cells, Theise et al. [15] reported that over a 6-month period 1–2% of hepatocytes in the murine liver may be derived from bone marrow in the absence of any obvious liver damage, suggesting that bone marrow contributes to normal ‘wear and tear’ renewal. It was thought unlikely that the bone marrow transplant contained a liver progenitor cell that was not of bone marrow origin, since 200 CD34+linx marrow cells produced the same degree of hepatic engraftment as 20 000 unfractionated bone marrow cells. In two contemporaneous papers, Alison et al. [16] and Theise et al. [17] have demonstrated that hepatocytes can also be derived from bone marrow cell populations in humans. Two approaches were adopted – first the livers of female patients who had previously received a bone marrow transplant from a male donor were examined for cells of donor origin using a DNA
Figure 4. (A) Repopulating nodules detected by X-gal histochemistry in the FAH–/– mouse 7 months after transplantation with 1r106 bone marrow cells from ROSA26 mice. (B) FAH staining of a nodule; the dark red area is FAH-positive hepatocytes adjacent to an FAH-negative area. From Lagasse et al. [24], with permission J Pathol 2002; 197: 510–518.
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probe specific for the Y-chromosome, localized using in situ hybridization. Secondly, Y-positive cells were sought in female livers engrafted into male patients but which were later removed for recurrent disease. In both sets of patients, Y-chromosome-positive hepatocytes were readily identified. The degree of hepatic engraftment of HSCs into human liver is highly variable, but is most likely related to the severity of parenchymal damage, with up to 40% of hepatocytes and cholangiocytes derived from bone marrow in a liver transplant recipient with recurrent hepatitis C [17]. Subsequent human investigations with G-CSF mobilized CD34+ stem cells have shown that these cells are also able to transdifferentiate into hepatocytes, with 4–7% of hepatocytes in female livers being Ychromosome-positive after a bone marrow transplant from a male donor [18]. Many claims for stem cell plasticity, such as those cited above, rely on the recognition of a Y-chromosome in a female recipient of a transplant from a male donor. However, some recent publications have called into question the validity of these observations, suggesting that some claims for transdifferentiation could be merely due to the fusion of bone marrow cells with the differentiated cells in the new organ, e.g. liver. Two studies suggest that cells from one source can fuse with cells from another (albeit embryonic stem cells) and the resultant tetraploid hybrids adopt the phenotype of the recipient cells [19,20]. When bone marrow from GFP transgenic mice was mixed with embryonic stem (ES) cells, a very small proportion (2–11 hybrid clones per 106 marrow cells) of bone marrow cells fused with ES cells; these cells could subsequently adopt many of the phenotypes typical of ES cell differentiation [19]. However, it should be noted that the frequency of hybrid cell formation was not increased by using the Sca1+Linx fraction; thus, the haematopoietic stem cells are not likely to be involved in these fusions and these are the bone marrow fraction thought to be largely responsible for liver engraftment (see below). Such a low level of fusion also makes it unlikely that such hybrids could be responsible for the apparent widespread liver colonization of marrowderived cells seen in some models of metabolic liver disease (see below). A very low frequency of fusion (one event per 100 000 CNS cells) has also been reported when mouse CNS cells are mixed with ES cells and here the derived hybrid cells were able to show multi-lineage potential when injected into blastocysts, most prominently into liver [20]. These reports do suggest that we should look at the genotype of cells claimed to have been generated from tissue of another type; however, if fusion does occur, then indeed most organs will naturally have many polyploid epithelial cells and this does not seem to be the case. With this in mind, it is interesting to look at epithelial tissue from mothers of male offspring. Postpartum exacerbation of thyroiditis is sometimes observed and could be due to transplacentally acquired fetal cells that cause an alloimmune disease previously Copyright # 2002 John Wiley & Sons, Ltd.
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regarded as an autoimmune disease [21]. Particularly noteworthy was one female patient with clusters of fully differentiated thyroid follicular cells bearing one X- and one Y-chromosome; of course, the source of the transdifferentiated cells was the fetus rather than a deliberate transplant, but nevertheless no follicular cells were XXXY, suggesting that cell fusion was not responsible for the phenomenon. In a similar vein, an investigation by FISH of the karyotype of the male donor peripheral blood stem cells that had apparently differentiated into epidermal, hepatic, and gastric mucosal cells in human female recipients clearly demonstrated the presence of only one X- and one Y-chromosome [18]. Since bone marrow could potentially be used either to increase the functional capability of an ailing liver or to deliver therapeutic genes (e.g. for single gene defects, anti-inflammatory cytokines), it becomes important to explore the functional capabilities of these cells. The technique of Y-chromosome detection also allows one to examine the ploidy status of these hepatocytes, a factor of considerable relevance, since polyploidization is an integral feature of hepatocyte differentiation and replication [22]. We have identified both diploid and polyploid hepatocytes of haematopoietic origin in female mice that have been given a male bone marrow transplant after whole-body lethal irradiation [23]. We have also identified Ychromosome-positive hepatocytes of both diploid and polyploid class within liver biopsies both from a female who has received a bone marrow transplant from a male donor and from a male patient who had received a female orthotopic liver transplant. Moreover, the Y-positive hepatocytes were often present in fractal clones, further suggestive of intrahepatic division after engraftment. These observations suggest that hepatocytes derived from bone marrow cells have the ability to undergo polyploidization in mice and man, further indicating that they have the potential to function as normal hepatocytes and contribute towards liver regeneration. Furthermore, in mice, the ability of bone marrow cells to cure a metabolic liver disease has been established [24]. Female mice deficient in the enzyme fumarylacetoacetate hydrolase (FAH–/–, a model of fatal hereditary tyrosinaemia type 1), a key component of the tyrosine catabolic pathway, can be biochemically rescued by 106 unfractionated bone marrow cells that are wild type for FAH (Figure 4). Furthermore, only purified HSCs (c-kithighThylowLinxSca-1+) were capable of this functional repopulation, with as few as 50 of these cells being capable of hepatic engraftment when haematopoiesis was supported by 2r105 FAH–/– congenic adult female bone marrow cells. Functionality of marrow-derived cells has also been established in a different model, the irradiated dipeptidyl peptidase IV-negative (DPPIVx) female rat transplanted with male DPPIV+ bone marrow, and clusters of hepatocytes expressing DPPIV on their bile canalicular surface were seen to emerge [13]. In another model of rat liver parenchymal damage, J Pathol 2002; 197: 510–518.
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it is highly likely that the periductular cells that proliferate in response to allyl alcohol-induced periportal damage are haematopoietic in origin [25]. Also in the rat, a population of bone marrow-derived hepatocyte stem cells (BDHSCs) has been identified on the basis of being beta 2 microglobulin-negative and Thy-1-positive (b2mx/Thy-1+) [26]. These cells were more numerous in damaged liver and expressed albumin, even in the liver. After these BDHSCs were co-cultured with cholestatic hepatocytes (separated by a semi-permeable membrane), they differentiated into hepatocytes and were able to metabolize ammonia into urea as efficiently as existing hepatocytes; prior coculture with healthy hepatocytes was not sufficient to achieve this. Thus, hepatocyte damage (? functional demand) seems a prerequisite not only for engraftment, but also for hepatic differentiation of bone marrowderived cells. In a more widespread study, it has been claimed that even a single cell from a male mouse bone marrow population (lineage-depleted and enriched for CD34+ and Sca-1+ by in vivo homing to the bone marrow) can, when injected into female recipients along with 2r104 female supportive haematopoietic progenitor cells, give rise to a variable proportion of epithelial cells in some organs: at 11 months, a surprisingly high proportion of pneumocytes were Y-chromosomepositive, but less than 1% of biliary cells and no hepatocytes were Y-chromosome-positive [27]. The high level of lung engraftment was attributed to lung damage caused by either the lethal irradiation to facilitate bone marrow transplantation or viral infection in the temporarily immunosuppressed animals. While it seems logical to believe that parenchymal damage is a stimulus to hepatic engraftment by HSCs, the molecules that mediate this homing reaction to the liver are unknown. Petrenko et al. [28] speculated that in mice the molecule AA4 (murine homologue of the C1q receptor protein) may be involved in the homing of haematopoietic progenitors to the fetal liver – maybe this receptor protein is expressed on HSCs that engraft to the damaged liver? Another alternative is that biliary ducts/stromal cells express the stem cell chemoattractant ‘stromal derived factor-1’ (SDF-1), for which HSCs have the appropriate receptor known as CXCR4 [29].
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(loss of one differentiated phenotype and the acquisition of another) into hepatocytes [31]. In these experiments, continuous bromodeoxyuridine labelling ascertained that many exocrine pancreatic cells did not proliferate during this transition. This differentiation was associated with the induction of the transcription factor C/EBPb which was thought to accelerate fatty acid acyl CoA synthesis, which in turn bound to HNF4a, causing its translocation to the nucleus, where it activated genes such as alpha-fetoprotein and transthyretin, characteristic of early hepatocytic differentiation. Oncostatin M is a natural hepatocyte differentiation factor produced by haematopoietic cells in the fetal liver [32]. In turn, differentiating hepatocytes turn off the production of stimulatory haematopoietic cytokines, terminating extramedullary haematopoiesis, and haematopoiesis relocates to the bone marrow
Models of cell transplantation The shortage of donors for whole-liver transplantation [33] has driven the search for defining the conditions that are conducive to hepatocyte transplantation, thus allowing more patients to benefit from the scarce donor liver tissue. A number of models permit the near-total replacement of the liver parenchyma by donor cells and are all valuable for exploring the replication and functional potential of selected populations of liver cells, or indeed other cells (pancreatic, HSCs) with hepatocyte lineage potential.
Retrorsine model This model involves the prior administration of the DNA-binding pyrrolizidine alkaloid retrorsine (usually two injections of 30 mg/kg each, 2 weeks apart) to rats deficient in the bile canalicular enzyme dipeptidyl peptidase IV (DPPIVx). This blocks hepatocyte proliferation for a number of months and 4 weeks after the last injection, transplantation of 2r106 wild-type hepatocytes combined with a mitogenic stimulus such as partial hepatectomy or triiodothyronine (T3) injections at 10-day intervals leads to rapid replacement of DPPIVx cells by DPPIV+ donor cells [34,35]; even in the absence of a mitogenic stimulus, near-total replacement by donor cells occurs within 12 months [36].
Pancreatic cells There is no obvious cell trafficking between the pancreas and liver, but it is clear that pancreatic cells can readily differentiate into their embryologically related cell type, the hepatocyte. This was shown in vivo by Krakowski et al. [30], who generated insulin promoter-regulated keratinocyte growth factor (KGF) transgenic mice; within 6 months under the influence of KGF, numerous functional hepatocytes emerged within the islets of Langerhans. A combination of dexamethasone and oncostatin M is a very effective in vitro inducer of pancreatic exocrine cell transdifferentiation Copyright # 2002 John Wiley & Sons, Ltd.
The FAH-deficient mouse This model exerts a profoundly strong positive selection pressure on the transplanted cells, since FAHdeficient mice will die as neonates unless rescued by 2-(2-nitro-4-trifluoro-methylbenzoyl)-1,3-cyclohexanedione (NTBC), a compound that prevents the accumulation of toxic metabolites in the tyrosine catabolic pathway. The stem cell-like properties of hepatocytes have been impressively demonstrated in this model. When 104 normal hepatocytes from congenic male wild-type mice are intrasplenically injected into mutant J Pathol 2002; 197: 510–518.
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female mice, these cells will quickly colonize the mutant liver [37]. Moreover, serial transplantations from the colonized liver to other mutant livers indicated that at least 69 doublings would have been necessary from the original hepatocytes for six rounds of liver repopulation. This estimate is likely to be a minimal figure since it assumes that all injected hepatocytes migrate to the liver from the spleen and equally take part in the cycles of regeneration. In fact, probably at best only 15% of intrasplenically transplanted hepatocytes migrate to the liver and if all of these participated equally in repopulation, a minimum of 86 doublings would be required. This figure may be even higher if not all the cells that migrated to the liver actually took part in repopulation; the authors suggested that maybe there is a sub-population of hepatocyte stem cells, which they designated as regenerative transplantable hepatocytes (RTHs). Though most FAH-deficient mice withdrawn from NTBC treatment and transplanted with pancreatic cells will die, a small proportion do survive, with 50–90% replacement of the diseased liver by pancreatic cell-derived hepatocytes [38]. Given that animals fed a copper-deficient diet undergo pancreatic exocrine cell atrophy and that refeeding induces the surviving ducts to give rise to hepatocytes [39], it is surprising that pancreatic cell suspensions enriched for pancreatic ducts are poorer than unfractionated pancreatic cells at reconstituting the diseased FAH–/– liver with functional hepatocytes.
Urokinase-type plasminogen activator (uPA) transgenic mice In this model, uPA is targeted to the liver using a hepatocyte-specific promoter: toxicity is probably due to activation of the uPA substrate plasminogen to plasmin, thus inducing intracellular proteolytic damage. The livers of albumin-uPA transgenic mice can become largely replaced by healthy transplanted hepatocytes [40], and even long-term (32 months) cryopreserved hepatocytes [41] and polyploid or aged hepatocytes [42] will substantially colonize the livers of mouse major urinary protein (MUP-uPA) transgenic mice. The data in these papers also suggest that the estimates of the number of hepatocyte doublings in the FAH-deficient mouse are conservative. It was estimated that only one-third of engrafted hepatocytes actually participated in repopulation; 21% of splenically injected cells engrafted in the parenchyma, but only 7% of injected cells gave rise to proliferating hepatocyte foci. In an extension of the model, the albumin-uPA transgene has been incorporated into the immunotolerant nu/nu mouse, allowing the growth of hepatocytes from a different species, rat [43], raising the exciting possibility that the immunotolerant AlbuPA transgenic mice could support the growth of human hepatocytes for drug metabolism or carcinogenicity studies. Indeed, immunodeficient uPA/recombinant activation gene-2 (RAG-2) mice support the Copyright # 2002 John Wiley & Sons, Ltd.
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growth of human hepatocytes [44] and remain permissive for human hepatitis B virus infection.
Adeno-associated virus (AAV)–Bcl-2 transduction and Fas antibody treatment This is not strictly a model of transplantation, but exemplifies a strategy for expanding genetically modified cells. Direct injection of recombinant AAV (rAAV) into the liver only transduces about 2% of hepatocytes, but by incorporating into the construct a mini-gene encoding Bcl-2, and then preferentially inducing apoptosis in non-transduced cells by the systemic administration of Fas antibody, the proportion of transduced hepatocytes can be increased to 20% [45]. In a similar vein, bone marrow-derived hepatocytes can be selectively expanded in the liver if they are engineered to overexpress Bcl-2 and then the animals are subjected to a selective pressure of an anti-Fas antibody [46].
Enrichment of stem cells for transplantation In the diseased human liver, there may not be the substantial selective growth advantage for transplanted cells that is operative in the models described above and it therefore becomes of interest to determine whether it is possible to enrich for true stem cells that would continue to expand in the recipient liver in the absence of a major growth stimulus (see Table 1). Kubota and Reid [47] have described a population of bipotential progenitors from ED13 fetal rat liver that lacked expression of MHC class I and had modest ICAM-1 expression, features that may allow hepatoblasts to escape from the immune system when transplanted into an MHC-incompatible host. These cells were clonogenic in culture, with some descendants expressing phenotypic markers of hepatocytes (afetoprotein and albumin) and others of cholangiocytes (cytokeratin-19). In the fetal mouse liver (ED13.5), hepatocytes expressing the integrins a6 (CD49f) and b1 (CD29), but not c-kit, CD45 or Ter119 (erythroid precursor antigen), had the greatest colony-forming ability [48]; designated hepatic colony-forming units in culture (H-CFU-C), this sorting achieved a 35-fold enrichment of H-CFU-C over total fetal liver cells. In a recent development, further selection based on c-Met+ enriched for H-CFU-C and these cells could produce both hepatocytes (albumin-positive) and biliary cells (cytokeratin-19-positive) in culture [49]. EGFP-marked cells from these clonally derived H-CFU-C also produced hepatocyes and biliary cells when injected into mice and, more surprisingly, were found apparently to differentiate into pancreatic ducts and acini and duodenal mucosal cells when injected directly into these organs. Fetal liver epithelial progenitors (FLEPs) in rats (ED14) also appear more clonogenic than normal adult hepatocytes: injected into the DPPIVx rat, they are bipotential, proliferate for at least 6 months, and constitute 7% of the recipient liver at this J Pathol 2002; 197: 510–518.
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Table 1. Examples of strategies for hepatic stem cell enrichment Species ED13 rat ED14 rat Adult rat Adult rat Adult rat ED13 mouse Adult mouse Adult mouse
Basis of selection x
MHC class 1 ICAMlow+ Fetal liver epithelial progenitors (FLEPs) Allyl alcohol-induced oval cells Smallest sized hepatocytes Resistance to retrorsine after 2/3 PH CD49f+CD29+ c-Met+, but c-kitxCD45xTer119x Medium-sized hepatocytes KTLS bone marrow
time, compared with colonization of only 0.06% of the liver by wild-type adult hepatocyes [50]. Most models of oval cell activation have employed potential carcinogens to inhibit hepatocyte replication in the face of a regenerative stimulus [1–5]. Cells derived by such procedures are clearly not relevant to human studies, but Sell and co-workers [51] have demonstrated that it is possible to derive rat oval cells without resorting to mutagenic chemicals. Allyl alcohol causes periportal necrosis, and the resultant oval cells can be isolated and propagated, are capable of at least 100 doublings in the presence of a feeder layer of lethally irradiated fibroblasts, and are able to differentiate into hepatocytes and biliary cells after removal of feeder cells. In the adult animal, in terms of hepatocyte size there is no real consensus as to the most clonogenic cells; in the FAH-deficient mouse, medium-sized rather than the smallest hepatocytes were the most effective colonizers [52], whereas in the retrorsine-treated DPPIV mutant Fischer rat, the smallest DPPIV+ hepatocytes produced the largest colonies [53]. Indeed, if rats are treated with retrorsine and then subjected to a twothirds partial hepatectomy, within a month the liver parenchyma becomes almost completely composed of small hepatocytes apparently resistant to the mitoinhibitory effects of retrorsine [54] – are these hepatocytes the progeny of a stem cell sub-population? Perhaps this subpopulation is akin to the population of RTHs considered by Overturf et al. [37].
Therapeutic potential of stem cells Hepatic stem cells from whatever source, hepatocytes themselves, oval cells/cholangiocytes or HSCs, may be therapeutically useful for treating a variety of diseases that affect the liver. This would include a number of genetic diseases that produce liver disease, such as Wilson’s disease (copper accumulation), Crigler Najjar syndrome (lack of bilirubin conjugation activity), and tyrosinaemia, and cases where there is extrahepatic expression of the disease, e.g. Factor IX deficiency. For example, following on from an improvement in Copyright # 2002 John Wiley & Sons, Ltd.
Properties
Reference
Clonogenic Bipotential Superior to adult hepatocytes in colonizing the DPPIVx rat liver Clonogenic Bipotential Maximum colonization of retrorsine-treated DPPIVx rat liver Small hepatocytes selectively repopulate the liver Clonogenic Pluripotential? Maximum colonization of FAH–/– mouse Colonization of FAH–/– liver
47 50 51 53 54 49 52 24
bilirubin conjugation in Gunn rats (a model of Crigler Najjar syndrome) after transplantation with reversibly immortalized hepatocytes transduced with the bilirubinUDP-glucuronosyl-transferase (BUGT gene) [55], an infusion of isolated hepatocytes through the portal vein equivalent to 5% of the parenchymal mass to a patient with Crigler Najjar syndrome has achieved a mediumterm reduction in serum bilirubin and increased bilirubin conjugates in the bile [56]. Stem cells, particularly HSCs, might also prove to be ideal vehicles for delivering therapeutic genes to the liver. Their use is doubly attractive, since not only can HSCs be readily harvested from a patient’s own blood, but also in treating non-genetic diseases there would be no need for immunosuppression. For example, HGF transfection of skeletal muscle in a rat model of cirrhosis (induced by dimethylnitrosamine) resulted in a reduction in many of the pathological hallmarks of cirrhosis in the liver [57]. However, HGF is a mitogen for many tissues, so HGF-transduced HSCs could be expected to target the damaged liver, delivering higher concentrations of HGF than could be safely achieved through the systemic route. Liver stem cells might also be useful for delivering anti-inflammatory cytokines (e.g. IL-10) for autoimmune liver disease such as primary biliary cirrhosis (PBC), and indeed, Crosby et al. [58] have, on the basis of CD34+ or c-kit+, isolated cells from human liver that differentiate into biliary epithelial cells in vitro. Moreover, they observed in PBC that although CD34+ cells were commonly found surrounding bile ducts, only c-kit+ cells were found actually integrated into the biliary ducts – perhaps this represents lineage progression (differentiation) (Crosby, personal communication). Worldwide, over 350 million people are chronically infected with hepatitis B virus [59] and failure to clear the virus results in continuing hepatocyte destruction, leading to cirrhosis and liver cancer. Clinically, IFN-a is used systemically to treat persistent hepatitis B infection and leads to a 30% probability of virus elimination. However, systemic toxicity limits its use to the lower end of the dose–response curve. Thus, if J Pathol 2002; 197: 510–518.
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interferon was produced locally by ex vivo transduced liver stem cells, in particular HSCs, they might enjoy the same selective advantage that wild-type bone marrow has in the FAH null mouse (see Figure 4). In many renewing cell populations, tumourigenesis is believed to be initiated in a stem cell for the simple reason that these are the only cells sufficiently longlived to acquire the necessary genetic alterations to begin the neoplastic process. In the liver, we can only speculate on the identity of the founder cells for the common types of carcinoma; hepatocellular carcinoma commonly arises in a cirrhotic liver where there are regenerative hepatocyte nodules, while fluke infection that results in biliary hyperplasia is common in areas where there is a high incidence of cholangiocarcinoma. Insight can also be gained from experimental models of liver carcinogenesis where individual carcinogens can provoke quite different cellular reactions [25]. Hepatocyte transplantation may also prove useful for patients with acute liver failure, bridging them to recovery of their own liver or to whole-liver transplantation [60]. For example, in rats, the intrasplenic injection of reversibly immortalized hepatocytes equivalent to about 5% of the normal liver mass ensures shortterm survival from a 90% hepatectomy, an operation that usually results in death from acute liver failure [61]. There are now many experimental models demonstrating the success of encapsulated xenogeneic hepatocytes as an implantable bio-artifical liver (BAL) for the treatment of acute liver failure. Finally, approximately one-third of liver transplant patients can potentially be weaned off long-term immunosuppression [62] and recipient bone marrow cell engraftment to the liver could be a significant factor in this tolerization. The blood vessels of a transplanted organ are the major interface between donor and recipient, and consequently the endothelium of blood vessels is a major target for graft rejection. Recipient bone marrow-derived endothelium has been found in transplanted liver and kidney [63,64] and c-kit+ cells can be found fully integrated into bile ducts [58], observations that suggest that hepatic engraftment of cells from the bone marrow of a transplant recipient may well have a significant role in the clinical tolerance of liver allografts. At present, this is mere speculation, but a study of 27 sequential biopsies from nine transplant recipients found that whereas biliary epithelial chimerism was a consistent feature of most biopsies, hepatocyte chimerism was more prominent in those patients suffering recurrent hepatitis [65]. It seems logical that more severe organ damage promotes engraftment and perhaps in the long-term, these patients will benefit more in terms of immune tolerance – things have to get worse before they get better? From the foregoing discussion it is quite clear that HSCs do play a significant role in liver biology and experimental models suggest that the process is susceptible to manipulation. The next challenge will be to define the extracellular cues that drive this pathway of engraftment and differentiation. Copyright # 2002 John Wiley & Sons, Ltd.
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