Stem Cells of the Liver and

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If we believe that tumours have their origins in normal stem cells, or at least in cells with sufficient longevity to acquire multiple genetic mutations, then we need to appreciate normal cell turnover in the liver and intestine.

Hepatic stem cells

Foetal liver is a source of bipotential progenitor cells (hepatoblasts) as seen by their extensive colonisation of the diseased livers of rats after transplantation.8 In postnatal animals, hepatocytes are highly differentiated cells with multiple synthetic and metabolic functions; they are also the functional stem cells in the liver under most circumstances. In health, individual hepatocytes have a life expectancy of over a year. Therefore in the normal adult liver, there is little cell proliferation detectable with only 0.01% of hepatocytes in the cell cycle at any one time. However, in response to parenchymal cell loss, hepatocytes restore the liver mass by self-replication. This is a very efficient system and in rodents, when two-thirds of the liver is resected (partial hepatectomy, PH), the remaining remnant can re-grow to the original liver size in approximately ten days. This model has been intensively studied and has provided much data on the mechanisms controlling liver regeneration.9'10 In response to this stimulus, normally quiescent hepatocytes leave G0 to enter the cell cycle under the influence of many growth factors. Hepatocyte proliferation begins in the periportal region of the liver and spreads to the centrilobular region. This regeneration requires each hepatocyte to undergo, on average, less than two rounds of replication to restore the liver to its original size. This does not however mean that hepatocytes have a limited replication potential. Hepatocyte transplantation models in mice have shown that hepatocytes are capable of significant clonal expansion within the diseased livers of experimental animals. In the fumarylacetoacetate hydrolase (FAH)-deficient mouse, a model of hereditary type 1 tyrosinemia, there is strong positive selection pressure exerted on transplanted wild-type hepatocytes as host hepatocytes readily undergo cell death due to the cytoplasmic accumulation of fumarylacetoacetate (FAA). Without transplantation, the FAH null genotype is lethal unless the mice are protected by 2-(2-nitro-4-trifluoro-methylbenzoyl)-1,3-cyclohexanedione (NTBC), a compound that prevents the accumulation of cytotoxic FAA. When 104 normal hepatocytes from congenic male wild-type mice are intra-splenically injected into mutant female mice and the NTBC treatment is withdrawn, these cells colonise the mutant liver efficiently.11 Moreover, serial transplantations from these colonised livers to other mutant livers indicated that at least 69 hepatocyte doublings can occur, thereby confirming the clonogenic potential of hepatocytes, meeting one of the main criteria that defines stem cells.

The clonogenic ability of human hepatocytes in chronic hepatitis can be indirectly estimated. Using mathematical modelling of viral kinetics it has been estimated that in chronic HBV infection, between 0.3% and 3% of all hepatocytes are killed daily and therefore replaced to maintain a stable liver cell mass (this approximates to 109 of the liver's 2 x 1011 hepatocytes).12 This is in accordance with the hepatocyte proliferation levels in chronic hepatitis B and C, where proliferating cell nuclear antigen (PCNA) indices of 0.1% to 3.6% are found, and Ki-67 labelling indices in hepatitis C of 1% to 14%.13'14 In chronic hepatitis the parenchymal mass can therefore be maintained through prolonged hepatocyte self-replication, and such cells could be the target for DNA-damaging agents and thus initiation events.

The hepatocyte proliferation rate increases in hepatitis C with increasing histological damage until cirrhosis is reached when the proliferation rate falls.15 This fall in hepatocyte proliferation rate may represent hepato-cytes coming to the end of their division potential and undergoing replica-tive senescence,16 although other factors such as distortion of blood flow through the liver are also likely to play a part. Whatever the reason, the reduction in hepatocyte proliferation indices in chronic hepatitis occurs concurrently with the activation of a potential stem cell compartment located within the smallest branches of the intrahepatic biliary tree. This so-called ductular reaction in human liver is equivalent to the oval cell reaction seen in many rodent models of hepatocarcinogenesis. The development of an oval cell reaction in response to hepatocyte replicative senescence has been demonstrated in a transgenic mouse model of fatty liver and DNA damage. In this model, the mice developed fatty livers and a large number of senescent hepatocytes. A striking oval cell response developed in these mice which related to the number of senescent mature hepatocytes.17

Following extensive liver damage or in situations where hepatocyte regeneration after damage is compromised, a potential stem cell compartment located within the smallest branches of the intrahepatic biliary tree is activated. This "oval cell" or "ductular reaction" amplifies a biliary population of transit-amplifying cells that are at least bipotential, capable of differentiating into either hepatocytes or cholangiocytes. Most rodent models of oval cell activation have employed potential carcinogens to inhibit hepatocyte replication in the face of a regenerative stimulus. For example in the rat, protocols have included administering 2-acetylaminofluorene (2-AAF) to inhibit hepatocyte proliferation before creating a demand for hep-atocyte proliferation by PH or a necrogenic dose of carbon tetrachloride.18 The need to maintain parenchymal cell mass results in the development of an oval cell response in the liver that spreads from the edge of the portal tract to deep into the parenchyma (Fig. 1A). Oval cells are small cells with a large nuclear to cytoplasmic ratio, in which the nucleus has a distinctive ovoid shape. Because oval cells express some of the antigens traditionally associated with haematopoietic cells (c-kit, flt-3, Thy-1 and CD34), there was speculation that hepatic oval cells were directly derived from bone marrow precursor cells. However, most studies now concur that the location of a stem cell niche for oval cells is in the canals of Hering, which is

Fig. 1. (A) An oval cell reaction in the rat liver. Oval cells, highlighted by cytokeratin 19 immunoexpression, branch out from the portal tract (PT). (B) An extensive ductular reaction in a human liver in response to parenchymal necrosis. Both hepatocytes and ductular cells express cytokeratin 18, but the strongest expression is in the ductular cells.

Fig. 1. (A) An oval cell reaction in the rat liver. Oval cells, highlighted by cytokeratin 19 immunoexpression, branch out from the portal tract (PT). (B) An extensive ductular reaction in a human liver in response to parenchymal necrosis. Both hepatocytes and ductular cells express cytokeratin 18, but the strongest expression is in the ductular cells.

a transitional zone between the periportal hepatocytes and the biliary cells lining the smallest terminal bile ducts.

The human counterparts to the oval cells described in rodent models are often referred to as HPCs. These have been observed after severe hepatocellular necrosis, chronic viral hepatitis, alcoholic liver disease and nonalcoholic fatty liver disease. It is thought that activation of the potential stem cell compartment leads to the formation of reactive ductules, anastomosing cords of immature biliary cells with an oval nucleus and a small rim of cytoplasm (Fig.lB).

Differentiation towards the hepatocyte lineage occurs via intermediate hepatocytes, these are polygonal cells with a size and phenotype intermediate between progenitor cells and hepatocytes (Fig. 2).19 After submassive liver cell necrosis, reactive ductules, in continuity with intermediate hepatocytes, are seen at the periphery of the necrotic areas. In patients studied with sequential liver biopsies, intermediate hepatocytes become more numerous with time and extend further into the liver lobule. This sequence of changes suggests the gradual differentiation of human progenitor cells into intermediate hepatocytes, analogous with what is seen in rat models of chemical injury associated with impaired hepatocyte replication. Elegant three-dimensional reconstructions of serial sections of human liver immunostained for cytokeratin-19 have shown that the smallest biliary ducts, the canals of Hering, normally extend into the proximate third of the lobule (unlike those in rodents), and it is envisaged that these canals react to massive liver damage (akin to a trip-wire), proliferation and then differentiation into hepatocytes.20 Oval cell numbers in human liver rise with increasing severity of liver disease.21

There is also the possibility that some oval cells/hepatocytes could be derived from circulating bone marrow cells (BMCs), although the results to date have been highly variable ranging from non-existent in some studies of long-term liver allografts to over 40% of hepatocytes being derived from bone marrow. One suspects that this variation is partly due to differences in the severity of parenchymal damage, but equally well may be a reflection of the ability (or lack of it) of the intrahepatic stem/progenitor cells to mount an effective regenerative response to damage.8

The "proof of principle" demonstration that bone marrow could cure mice with a potentially fatal metabolic liver disease, hereditary tyrosine-mia type-1, was a landmark publication.22 In the setting of liver failure, wild-type bone marrow could apparently switch lineage determination and differentiate into hepatocytes expressing the enzyme "fumarylacetoacetate

Fig. 2. Ductular reaction in a case of massive hepatic necrosis due to acetaminophen toxicity, immunohistochemically stained for CK7. Note many intermediate cells are still with a biliary-type staining pattern (CK7+), but with a morphology midway between cholan-giocytes and hepatocytes (courtesy of Professor Tania Roskams with permission from John Wiley Publishers).

Fig. 2. Ductular reaction in a case of massive hepatic necrosis due to acetaminophen toxicity, immunohistochemically stained for CK7. Note many intermediate cells are still with a biliary-type staining pattern (CK7+), but with a morphology midway between cholan-giocytes and hepatocytes (courtesy of Professor Tania Roskams with permission from John Wiley Publishers).

hydrolase" (Fah), the component of the tyrosine catabolic pathway absent in tyrosinemic (fah-/-) animals. Such lineage switching has been called transdifferentiation or plasticity. However, it is now clear that the new functioning liver cells in the fah-/- mouse are each the result of cell fusion between a donor bone marrow-derived macrophage and an fah-/- hep-atocyte nucleus.23 From an oncology point of view, the interest in such heterokaryons is that many of these cells appeared to be genomically unstable, seemingly shedding chromosomes at random to become aneuploid.24 Although the exact significance of bone marrow-derived cells to liver disease is far from fully elucidated, the fact that damaged hepatocytes can alter the lineage commitment of haematopoietic stem cells (HSCs) towards that of hepatocytes without the occurrence of cell fusion25 and that a number of studies now report on the ability of human cord blood mononuclear cells to give rise to hepatocytes in the liver of NOD/SCID mice (reviewed in Alison et al.8), the possibility that primary liver tumours could be initiated in bone marrow-derived cells cannot be discounted.

Intestinal stem cells

The immature, relatively undifferentiated nature of gastrointestinal epithelial stem cells means that they are not directly identifiable and researchers in this field in the past have had to rely on ingenious indirect methods to identify their position and track their progeny.26 The putative stem cell compartment position varies according to the location in the digestive tract. Throughout the small and large intestine the luminal surface is clothed by a simple columnar epithelial cell layer, with glandular invaginations called crypts. Several of these crypts contribute epithelium to finger-like projections called villi in the small bowel. The cells of the intestinal epithelium are arranged hierarchically, becoming progressively more differentiated as they age and pass along the crypt-to-villus axis. The stem cell compartment is believed to be at the origin of this axis, the base of the colonic crypt and at cell positions 4 to 5 in the small bowel (reviewed by Brittan and Wright27). The number of stem cells within each crypt is debated but is generally believed to be between 4 and 6.28'29 Stem cells themselves divide infrequently and it is the first few generations of stem cell daughters, known as transit-amplifying cells, that proliferate in the lower part of the crypt.30 Stem cells reside within a stem cell compartment or "niche". This is a group of epithelial and mesenchymal cells and extracellular substrates, which provide an optimal microenvironment for stem cells to give rise to their differentiated progeny. In the intestinal crypts, this is formed by a fen-estrated sheath of surrounding mesenchymal cells which regulate stem cell behaviour through paracrine secretion of growth factors and cytokines.31 Functionally, a niche is characterised by its persistence on the removal of stem cells and the cessation of stem cell potential when cells are removed from this niche.32 The rapid turnover of the gastrointestinal epithelium means that differentiating cells are shed into the lumen and replaced every few days; thus, they do not have a sufficient lifespan to gather the multiple genetic defects required for malignant transformation. Therefore, the perpetual stem cell has long been considered the target of carcinogenic mutations.30,33,34

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