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.
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, atleast some hepatocytes can do this. Hepatocyte transplantation protocols, developed because of the shortage of livers for whole-organ transplantation, have shown that the transplanted cells are capable of significant clonal expansion within the diseased liver of a recipient (see below).
When either massive damage is inflicted upon the liveror 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 hepatocytes1–5. 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 lobule6 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 liver disease7 and this ductular reaction is widely accepted to be a stem cell response rather than a ductular metaplasia of ‘damaged’ hepatocytes. Non-parenchymal epithelial cells from adult porcine liver, essentially ductal epithelia, are also capable of differentiating into both biliary epithelia and hepatocytes in vitro8. Moreover, antigens traditionally associated with haematopoietic cells can also be expressed by oval cells, including c-kit, flt-3, Thy-1, and CD349–12. 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 damaged13, though we firmly believe that many/most oval cells are derived from the direct intrahepatic proliferation of cells already located within the biliary tree1–5.
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-chromosome-positive 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 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+lin− 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 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 C17. Subsequent human investigations with G-CSF mobilized CD34+ stem cells have shown that these cells arealso able to transdifferentiate into hepatocytes, with 4–7% of hepatocytes in female livers being Y-chromosome-positive after a bone marrow transplant from a male donor18.
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 cells19, 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 differentiation19. However, it should be noted that the frequency of hybrid cell formation was not increased by using the Sca1+Lin− 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 marrow-derived 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 liver20. 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. Post-partum exacerbation of thyroiditis is sometimes observed and could be due to transplacentally acquired fetal cells that cause an alloimmune disease previously regarded as an autoimmune disease21. 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-chromosome18.
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 replication22. 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 lethalirradiation23. We have also identified Y-chromosome-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 asnormal hepatocytes and contribute towards liver regeneration. Furthermore, in mice, the ability of bone marrow cells to cure a metabolic liver disease has been established24. 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-kithighThylowLin−Sca-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 2×105 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 (DPPIV−) female rat transplanted with male DPPIV+ bone marrow, and clusters of hepatocytes expressing DPPIV on their bile canalicular surface were seen to emerge13.
In another model of rat liver parenchymal damage, it is highly likely that the periductular cells that proliferate in response to allyl alcohol-induced periportal damage are haematopoietic in origin25. 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 (β2m−/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 co-culture 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 marrow-derived 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 2×104 female supportive haematopoietic progenitor cells, give rise to a variable proportion of epithelial cells in some organs: at 11 months, a surprisingly highproportion of pneumocytes were Y-chromosome-positive, but less than 1% of biliary cells and no hepatocytes were Y-chromosome-positive27. 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 CXCR429.
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 (loss of one differentiated phenotype and the acquisition of another) into hepatocytes31. 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/EBPβ which was thought to accelerate fatty acid acyl CoA synthesis, which in turn bound to HNF4α, 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 liver32. In turn, differentiating hepatocytes turn off the production of stimulatory haematopoietic cytokines, terminating extramedullary haematopoiesis, and haematopoiesis relocates to the bone marrow