Hepatic stem cells

HEPATOCYTE TRANSPLANTATION AND BIOARTIFICIAL LIVER

Hepatic Stem Cells M. Alison

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HIS ARTICLE will focus on three aspects of liver stem cell biology: (1) candidate cells, (2) models of cell transplantation, and (3) therapeutic potential of stem cells. CANDIDATE CELLS

Possibly because of its unique exposure to a host of potentially harmful foreign compounds, liver can call upon not just one, but three phenotypically distinct cell lineages to assist in 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 Go phase. However, even after a two-thirds partial hepatectomy, the remaining cells only have to cycle 2 to 3 times to restore preoperative cell number, and this fact led to the incorrect assumption that hepatocytes had only limited division potential. A crucial property of stem cells is their ability to give rise to a large family of descendants and at least 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 the recipient. 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 transdifferentiate into hepatocytes.1 In humans, the smallest biliary ducts, the canals of Hering, normally extend into the proximate third of the lobule,2 and it is envisaged that these canals “sense” massive liver damage (akin to a trip-wire), proliferating and differentiating into hepatocytes. Interestingly, antigens traditionally associated with haematopoietic cells also are expressed by oval cells, including c-kit, flt-3, Thy-1, and CD34. Bone Marrow

Within an adult tissue the 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. Oval cells/hepatocytes in the rat can be derived from circulating bone marrow cells. Petersen et al3 followed the fate of syngeneic male bone marrow cells transplanted into lethally irradiated female recipient animals whose livers were subFrom the Department of Histopathology, Imperial College School of Medicine, Hammersmith Hospital, London, United Kingdom. Address reprint requests to Malcolm Alison, DSc, FRCPath, Department of Histopathology, Imperial College School of Medicine, Hammersmith Hospital, London W12 ONN, United Kingdom. E-mail: [email protected]

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Transplantation Proceedings, 34, 2702–2705 (2002)

HEPATIC STEM CELLS

sequently injured by carbon tetrachloride in a scenario designed to cause oval cell activation. Moreover, using a similar gender mismatch bone marrow transplantation approach in mice to track the fate of bone marrow cells, Theise et al4 reported that during a 6-month period 1% to 2% of hepatocytes in the murine liver may be derived from bone marrow in the absence of any obvious liver damage, suggesting bone marrow contributes to normal “wear and tear” renewal. Alison et al5 and Theise et al6 have demonstrated that hepatocytes also can be derived from bone marrow cell populations in humans. Two approaches were adopted. First, the livers of female patients who previously had 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, localised using in situ hybridisation. Second, Y chromosome–positive cells were sought in female livers engrafted into male patients but that were later removed for recurrent disease. In both sets of patients, Y chromosome–positive hepatocytes were readily identified. Because bone marrow could be used either to increase the functional capability of an ailing liver or to deliver therapeutic genes (eg, 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 because polyploidization is an integral feature of hepatocyte differentiation.7 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. We also have identified Y chromosome–positive hepatocytes of both diploid and polyploid class within liver biopsy specimens both from a female who has received a bone marrow transplant from a male donor and from a male patient that had received a female orthotopic liver transplant. Moreover, the Y chromosome–positive hepatocytes were invariably 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, further indicating that they have the potential to function as normal hepatocytes and contribute to liver regeneration. In mice, the ability of bone marrow cells to cure a metabolic liver disease has been established.8 Mice deficient in the enzyme fumarylacetoacetate hydrolase (FAH⫺/⫺, a model of fatal hereditary tyrosinemia 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. Furthermore, only purified HSCs (c-kithigh Thylow Lin⫺Sca-1⫹) were capable of this functional repopulation. In a further development, it has been claimed that even a single cell from a male bone marrow population (lineagedepleted and enriched for CD34⫹ and Sca-1⫹ by in vivo homing to the bone marrow), can, when injected into

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female recipients along with 2 ⫻ 104 female supportive progenitor cells, give rise to a small proportion of epithelial cells in some organs (at 11 months ⬍ 1% of biliary cells but no hepatocytes).9 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 closely related cell type, the hepatocyte. Krakowski et al10 generated insulin promoter–regulated KGF transgenic mice and within 6 months numerous functional hepatocytes emerged within the islets of Langerhans. A combination of dexamethasone and oncostatin M is a very effective inducer of pancreatic exocrine cell transdifferentiation into hepatocytes.11 MODELS OF CELL TRANSPLANTATION

The shortage of donors for whole liver transplantation 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 and HSCs) with hepatocyte lineage potential. Retrorsine Model

The retrorsine model involves the prior administration of the pyrrolizidine alkaloid retrorsine to rats deficient in the bile canalicular enzyme dipeptidyl peptidase IV (DPP-IV-). This apparently blocks hepatocyte proliferation and transplantation of wild-type hepatocytes combined with a mitogenic stimulus, such as partial hepatectomy or T3, leads to rapid replacement by donor cells12,13; even in the absence of a mitogenic stimulus near total replacement by donor cells occurs within 12 months.14 The FAH-Deficient Mouse

This model exerts a profoundly strong positive selection pressure on the transplanted cells because FAH-deficient mice will die as neonates unless rescued by 2-(2-nitro-4trifluoro-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 female mice these cells will quickly colonise the mutant liver.15 Moreover, serial transplantations from the colonised liver to other mutant livers indicated that at least 69 doublings would have been necessary from the original hepatocytes for 6 rounds of liver repopulation. Although most FAH-deficient mice withdrawn from NTBC treatment and transplanted with pancreatic cells will die, a small

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proportion do survive with 50% to 90% replacement of the diseased liver by pancreatic cell– derived hepatocytes.16 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,17 and even long-term cryopreserved hepatocytes18 and polyploid hepatocytes19 will colonise the livers of mouse major urinary protein (MUP-uPA) transgenic mice. In an extension of the model, the albuminuPA transgene has been incorporated into the immunotolerant nu/nu mouse, allowing the growth of hepatocytes from a different species, rat,20 raising the exciting possibility that the immunotolerant Alb-uPA 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 growth of human hepatocytes21 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 is a strategy for expanding genetically modified cells. rAAV only transduces approximately 2% of hepatocytes when injected directly into mouse liver, but by incorporating into the construct a minigene encoding Bcl-2, and then preferentially inducing apoptosis in non-transduced cells by the systemic administration of Fas antibody, the proportion of transduced hepatocytes increased to 20%.22 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, therefore, it becomes of interest to determine if 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. Kubota and Reid 23 have described a population of bipotent progenitors from ED13 foetal 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. In the foetal mouse liver, hepatocytes expressing the integrins ␣6 (CD49f) and ␤1 (CD29) had the greatest colony-forming ability.24 In terms of hepatocyte size there is no real consensus; in the FAH-deficient mouse mediumsized rather than the smallest hepatocytes were the most effective colonisers,25 whereas in the retrorsine-treated DPP-IV mutant Fischer rat the smallest DPP-IV (⫹) hepatocytes produced the largest colonies.26 Indeed, if rats are treated with retrorsine and then subjected to a two-

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thirds partial hepatectomy, the liver parenchyma shortly becomes almost completely composed of small hepatocytes apparently resistant to the mitoinhibitory effects of retrorsine27—is this the progeny of a stem cell sub-population? 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, Crigler Najjar syndrome, tyrosinemia, and cases where there is extrahepatic expression of the disease, eg, Factor IX deficiency. For example, infusing through the portal vein the equivalent of 5% of the parenchymal mass into a patient with Crigler Najjar syndrome achieved a medium-term reduction in serum bilirubin and increased bilirubin conjugates in the bile.28 Stem cells, particularly HSCs, also might prove to be ideal vehicles for delivering therapeutic genes to the liver, particularly anti-inflammatory cytokines (interleukin-10) for autoimmune liver disease, anti-fibrotic cytokines (hepatocyte growth factor), and anti-viral cytokines (interferon-␣) for hepatitis B virus infection. Hepatocyte transplantation also may prove useful for patients with fulminant liver failure, bridging them to recovery of their own liver or to whole liver transplantation. Finally, approximately one third of liver transplant patients can be weaned off long-term immunosuppression, and recipient bone marrow cell engraftment to the liver could be a significant factor in this tolerisation: recipient bone marrow– derived endothelium has been found in transplanted liver,29 and c-kit–positive cells can be found fully integrated into bile ducts.30 From the foregoing discussion it is quite clear that HSCs do play a significant role in liver biology, and experimental models suggest the process is susceptible to manipulation. The next challenge will be to define the extracellular cues that drive this pathway. REFERENCES 1. Alison MR: Curr Opin Cell Biol 10:710, 1998 2. Theise ND, Saxena R, Portmann BC, et al: Hepatology 30:1425, 1999 3. Petersen BE, Bowen WC, Patrene KD, et al: Science 284: 1168, 1999 4. Theise ND, Badve S, Saxena R: Hepatology 31:235, 2000 5. Alison MR, Poulsom R, Jeffery R, et al: Nature 406:257, 2000 6. Theise ND, Nimmakalu M, Gardner R, et al: Hepatology 32:11, 2000 7. Gupta S: Semin Cancer Biol 10:161, 2000 8. Lagasse E, Connors H, Al-Dhalimy M, et al: Nature Medicine 6:1229, 2000 9. Krause DS, Theise ND, Collector MI, et al: Cell 105:369, 2001 10. Krakowski ML, Kritzik MR, Jones EM, et al: Am J Pathol 154:683, 1999 11. Shen CN, Slack JM, Tosh D: Nat Cell Biol 2:879, 2000 12. Laconi E, Oren R, Mukhopadhyay DK, et al: Am J Pathol 153:319, 1998 13. Oren R, Dabeva MD, Karnezis AN, et al: Hepatology 30:903, 1999

HEPATIC STEM CELLS 14. Laconi S, Pillai S, Porcu PP, et al: Am J Pathol 158:771, 2001 15. Overturf K, al-Dhalimy M, Ou CN, et al: Am J Pathol 151:1273, 1997 16. Wang X, Al-Dhalimy M, Lagasse E, et al: Am J Pathol 158:571, 2001 17. Rhim JA, Sandgren EP, Degen JL, et al: Science 263:1149, 1994 18. Jamal HZ, Weglarz TC, Sandgren EP: Gastroenterology 118:390, 2000 19. Weglarz TC, Degen JL, Sandgren EP: Am J Pathol 157: 1963, 2000 20. Rhim JA, Sandgren EP, Palmiter RD, et al: Proc Natl Acad Sci U S A 92:4942, 1995 21. Dandri M, Burda MR, Torok E, et al: Hepatology 33:981, 2001 22. Chen SJ, Tazelaar J, Wilson JM: Hum Gene Ther 12:45, 2001

2705 23. Kubota H, Reid LM: Proc Natl Acad Sci U S A 97:12132, 2000 24. Suzuki A, Zheng Y, Kondo R, et al: Hepatology 32:1230, 2000 25. Overturf K, Al-Dhalimy M, Finegold M, et al: Am J Pathol 155:2135, 1999 26. Katayama S, Tateno C, Asahara T, et al: Am J Pathol 158:97, 2001 27. Gordon GJ, Coleman WB, Hixson DC, et al: Am J Pathol 156:607, 2000 28. Fox IJ, Chowdhury JR, Kaufman SS, et al: N Engl J Med 338:1422, 1998 29. Gao Z, McAlister VC, Williams GM: Lancet 357:932, 2001 30. Crosby HA, Kelly DA, Strain AJ: Gastroenterology 120:534, 2001