iconstar paper   Hepatitis C Articles (HCV)  
c Back grey arrow rt.gif
Stem cell therapy for chronic liver disease-choosing the right tools for the job Commentaries
  Gut Feb 2008;57:153-155
Stuart J Forbes
Correspondence to:
Professor Stuart J Forbes, MRC/University of Edinburgh Centre for Inflammation Research, The Queen's Medical Research Institute, 47 Little France Crescent, Edinburgh EH16 4TJ, UK; stuart.forbes@ed.ac.uk
The liver has a fantastic regenerative capacity but, following chronic liver damage, this begins to fail, and then fibrosis, and eventually cirrhosis, develops.1 Currently the only curative treatment for advanced liver cirrhosis is liver transplant. Although liver transplant has become a procedure with a relatively good 5-year survival, organ donation has not kept up with demand, which has resulted in an increasing number of patients on the liver transplant waiting list waiting longer for a donor organ, which leads to increased morbidity and mortality.2 Furthermore it is estimated that over the next few years there will be a 5-fold increase in the need for liver transplantation in the UK.3 Although there is emerging evidence that extending the donor organ criteria may impact on this mortality rate,4 there is clearly still an urgent need to develop alternative strategies for the treatment of advanced liver disease, and numerically cirrhosis is the most important target. It is with this background that there has been understandable enthusiasm for the development of stem cell therapies for liver regeneration.
Bone marrow (BM) stem cells have been intensively investigated as a potential source of liver stem cells and as a means to regenerate the cirrhotic liver, and it is worth briefly outlining why they have attracted this attention. There are a population of intrahepatic progenitor cells, termed oval cells in rodents, which can take over liver regeneration when the usual mode of regeneration, via division of mature hepatocytes, begins to fail. It was suggested some time ago that these cells expressed the haematopoeitic stem cell marker THY-15 (though this is now disputed), and it was therefore postulated that these oval cells were originating from the BM. Indeed initial experiments supported this hypothesis.6 Furthermore, following analysis of both mouse BM transplant models and tissue from liver and BM transplant patients, it was suggested that the BM could contribute to the mature hepatocyte population.7 8 Real excitement resulted from the demonstration that BM transplant could rescue a mouse model of tyrosinaemia, a hereditary defect of a hepatocytic enzyme.9 The BM transplant resulted in repopulation of the liver with apparently normal hepatocytes. Given that this model was otherwise lethal to the mice, it was a powerful demonstration of the therapeutic potential of stem cells, and stimulated further studies using different animal models of liver disease. Unfortunately, these studies have mostly been negative and little evidence has been found for a significant repopulation of liver parenchyma by BM-derived cells.10-13 Furthermore, subsequent analysis of the tyrosinaemia model showed that the rescue was a result of cell fusion of BM-derived monocytes with the diseased hepatocytes, resulting in a form of cellular gene therapy. Because of the unique selection pressure seen in this model, the fused cells divided to repopulate the liver.14 A selection pressure of this strength is unlikely to be available for most clinical applications.
Mesenchymal stem cells (MSCs) can be derived from various tissues including bone, fat and dental tissue. Their definition includes the ability to differentiate into osteoblasts, adipocytes and chondroblasts.15 There are now several studies that show that MSCs can be coaxed in vitro into many cell types, including cells with "hepatocyte-like" properties, based on a combination of morphology, gene expression and metabolic/synthetic activity.16-20 The cells are often described as being "hepatocyte-like" as they often fall short of being identical to hepatocytes. Adipose tissue-derived MSCs (AT-MSCs) appear capable of differentiation into a hepatocyte phenotype in vitro.21-23 For example, Banas et al found that a particular fraction of human AT-MSCs (CD105+) could become hepatocyte-like, with gene expression, morphology and metabolic activity similar to hepatocytes.23 With the current excess of human adipose tissue, these cells could be a readily available source with many willing donors! Data on the in vivo capabilities of MSCs are less clear, with some studies assessing functional performance for as little as 24 h post-transplant,23 or using limited criteria to define hepatocyte-like cells. Of the studies where longer term engraftment was studied, the data are still conflicting. For example, Sgodda et al found that pre-differentiated rat AT-MSCs were capable of integrating into recipient livers for at least 10 weeks and appeared to form appropriate connections in the recipient liver.22 In an apparently robust study, Sato et al found that human MSCs could engraft immunosuppressed rat livers and form relatively large numbers of donor-derived cells with a hepatocyte phenotype based on gene expression and protein criteria.24 Furthermore, a recent study in fetal sheep used direct intrahepatic injection of clonal populations of MSCs to produce a significant number of human hepatocyte-like cells (12.5%) that were both long lasting (56-70 days) and able to secrete human albumin.25
Conversely, in an equally carefully conducted study, Popp et al found that MSCs did not differentiate into hepatocytes within the liver of rats undergoing prolonged liver injury.26 Unfortunately, such apparently contradictory results have been commonly seen in this field and probably reflect a combination of factors, including the use of varying cell derivation and differentiation protocols, and the fact that the models of liver injury are often differing and rarely repeated. Notwithstanding these reservations, it appears that MSCs can be induced to demonstrate at least some hepatocyte functions in vitro, but more in vivo research is definitely needed as it appears that both the exact derivation and pre-conditioning of the starting cells and the host liver environment will determine the resulting phenotype of the transplanted cells.
It is in this context that the current work by Valfre di Bonzo et al27 makes interesting reading (see page 10.1136/gut.2006.111617). Using the differentiation protocol published by Lee et al,16 they confirmed that cells could be found to express human albumin mRNA weakly. However, when tested in a NOD-SCID model, that importantly had chronic liver injury, very few human hepatocyte-like cells were identified. Worse still (ie, if you want to "make hepatocytes"), they found the transplanted MSCs had a propensity to form myofibroblast-like cells (scar-forming cells) in the areas of hepatic injury, and by the end of the study period the transplanted cells were >10 times more likely to form myofibroblasts than hepatocytes. This less optimistic but undoubtedly realistic study is, however, perhaps not too surprising. We and others have shown that BM-derived cells can form myofibroblasts within various damaged organs including the liver.28 Furthermore, the data from murine models have implicated MSCs as the major source of these BM-derived myofibroblasts.29
This report is particularly timely as clinical studies of stem cell therapy for cirrhosis and liver disease are now beginning to be undertaken in earnest. To date there are at least four studies reported of BM therapy for liver disease. The first was in patients undergoing resection for liver cancer; here autologous CD133+ BM stem cells were used to stimulate the liver's regenerative capacity.30 The protocol involved portal vein embolisation to induce contralateral lobe hypertrophy and thereby increase the size of the future remnant liver volume prior to an extensive partial hepatectomy. Autologous CD133+ BM cells were infused into the portal vein supplying the future remnant and appeared to stimulate the resulting liver regeneration. In another study by Gordon et al, a population of CD34+ cells was identified that in vitro appear to have multilineage potential. In a study of five cirrhotic patients, autologous CD34+ cells were isolated using leucopharesis and re-injected via the hepatic artery or portal vein. Again this largely appeared to induce an improvement in the liver function of these patients.31 Similarly, studies by Terai et al and Lyra et al have used autologous BM-derived monocytes.32 33 The cells were concentrated ex vivo and re-applied to the patient's hepatic artery or portal vein. All the protocols appear to be safe (including the use of granulocyte colony-stimulating factor (GCSF)), and indeed the data may even be encouraging; however, none of these studies was a definitive controlled study-which are likely to follow. The cell populations used in these clinical studies to date are either enriched for haematopoietic stem cells (HSCs) or-like monocytes-are derived from HSCs. This may seem odd given that the majority of the data cited suggest that it is the population of MSCs that are likely to be inducible into a hepatocyte-like phenotype, and there are few hard data to suggest HSCs have this potential to any useful degree. There is, however, logic to this approach as the monocyte-macrophage population appears to have the potential to remodel the scars of the cirrhotic liver. In a recent study into the mechanism of liver fibrosis formation and resolution, hepatic scar-associated macrophages were selectively deleted in either the phase of fibrosis progression during injury or the phase of fibrosis resolution post-injury.34 If they were deleted during the injury phase, there was less fibrosis formation-demonstrating they can have a pro-fibrogenic role; however if they were deleted during the phase of resolution, then there was less scar resolution-thereby demonstrating their role in scar resolution and tissue remodelling.
This report adds weight to the growing feeling that MSCs can have their downsides as well as upsides, especially if applied in vivo to damaged organs, and is a timely caution for anyone contemplating clinical trials of MSC therapy for liver cirrhosis. This is not to say that MSCs have no future in this area. It may be that if MSCs can be differentiated efficiently into a hepatocyte phenotype, then these cells can be used for ex vivo liver support, drug testing, etc. For transplantation protocols, one would need to be confident that the cells were pre-programmed either to differentiate into or to remain as hepatocytes, and it appears we are some way short of this mark. Finally, this paper highlights a key conceptual issue in the understanding of regenerative therapy for the cirrhotic liver. Is it likely to be fruitful to add hepatocyte-like cells to an already cirrhotic liver, where the environment is extremely harsh, and even professional hepatocytes find the going tough, or should one seek to modify the cellular and extracellular milieu, to allow the endogenous hepatocytes and progenitor cells to regenerate the liver? The latter may well be a more realistic medium-term goal for cell therapy. In other words, spending some time improving the soil may bring forth more regeneration than simply throwing more seeds onto rocky ground. In the end, the humble rake may be a better tool for this job than that fancy double-edged sword.
Competing interests: The author has been awarded a translational research grant by the Jules Thorn Trust: "Stem cell therapy for cirrhosis".
1. Fausto N. Liver regeneration and repair: hepatocytes, progenitor cells, and stem cells. Hepatology 2004; 39: 1477-87.[CrossRef][Medline]
2. Transplant activity in the UK. http://www.uktransplant.org.uk/ukt/statistics/transplant_activity_report/current_activity_reports/ukt/transplant_activity_uk_2005-2006_v2.pdf (accessed 7 Nov 2007)
3. Moore K, Thursz M, Mirza DF. National plan for liver services-specialised services for hepatology, hepatobiliary and pancreatic surgery. Report prepared for the British Association for the Study of the Liver. 2003. http://www.basl.org.uk/downloads/National%20Plan%20for%20Liver%20Services%20UK%20Final-May04.pdf (accessed 7 Nov 2007).
4. Barshes NR, Horwitz IB, Franzini L, et al. Waitlist mortality decreases with increased use of extended criteria donor liver grafts at adult liver transplant centers. Am J Transplant 2007; 7: 1265-70.[CrossRef][Medline]
5. Petersen BE, Goff JP, Greenberger JS, et al. Hepatic oval cells express the hematopoietic stem cell marker Thy-1 in the rat. Hepatology 1998; 27: 433-45.[CrossRef][Medline]
6. Petersen BE, Bowen WC, Patrene KD, et al. Bone marrow as a potential source of hepatic oval cells. Science 1999; 284: 1168-70.[Abstract/Free Full Text]
7. Theise ND, Badve S, Saxena R, et al. Derivation of hepatocytes from bone marrow cells in mice after radiation-induced myeloablation. Hepatology 2000; 31: 235-40.[CrossRef][Medline]
8. Alison MR, Poulsom R, Jeffery R, et al. Hepatocytes from non-hepatic adult stem cells. Nature 200020; 406: 257.
9. Lagasse E, Connors H, Al-Dhalimy M, et al. Purified hematopoietic stem cells can differentiate into hepatocytes in vivo. Nat Med 2000; 6: 1229-34.[CrossRef][Medline]
10. Kanazawa Y, Verma IM. Little evidence of bone marrow-derived hepatocytes in the replacement of injured liver. Proc Natl Acad Sci USA 2003; 100: (Suppl 1): 11850-3.[Abstract/Free Full Text]
11. Menthena A, Deb N, Oertel M, et al. Bone marrow progenitors are not the source of expanding oval cells in injured liver. Stem Cells 2004; 22: 1049-61.[Abstract/Free Full Text]
12. Vig P, Russo FP, Edwards RJ, et al. The sources of parenchymal regeneration after chronic hepatocellular liver injury in mice. Hepatology 2006; 43: 316-24.[CrossRef][Medline]
13. Thorgeirsson SS, Grisham JW. Hematopoietic cells as hepatocyte stem cells: a critical review of the evidence. Hepatology 2006; 43: 2-8.[CrossRef][Medline]
14. Willenbring H, Bailey AS, Foster M, et al. Myelomonocytic cells are sufficient for therapeutic cell fusion in liver. Nat Med 2004; 10: 744-8.[CrossRef][Medline]
15. Dominici M, Le Blanc K, Mueller I, et al. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy 2006; 8: 315-7.[CrossRef][Medline]
16. Lee KD, Kuo TK, Whang-Peng J, et al. In vitro hepatic differentiation of human mesenchymal stem cells. Hepatology 2004; 40: 1275-84.[CrossRef][Medline]
17. Kang XQ, Zang WJ, Song TS, et al. Rat bone marrow mesenchymal stem cells differentiate into hepatocytes in vitro. World J Gastroenterol 2005; 11: 3479-84.[Medline]
18. Kang XQ, Zang WJ, Bao LJ, et al. Fibroblast growth factor-4 and hepatocyte growth factor induce differentiation of human umbilical cord blood-derived mesenchymal stem cells into hepatocytes. World J Gastroenterol 2005; 11: 7461-5.[Medline]
19. Talens-Visconti R, Bonora A, Jover R, et al. Hepatogenic differentiation of human mesenchymal stem cells from adipose tissue in comparison with bone marrow mesenchymal stem cells. World J Gastroenterol 2006; 12: 5834-45.[Medline]
20. Snykers S, Vanhaecke T, Papeleu P, et al. Sequential exposure to cytokines reflecting embryogenesis: the key for in vitro differentiation of adult bone marrow stem cells into functional hepatocyte-like cells. Toxicol Sci 2006; 94: 330-41.[Abstract/Free Full Text]
21. Seo MJ, Suh SY, Bae YC, et al. Differentiation of human adipose stromal cells into hepatic lineage in vitro and in vivo. Biochem Biophys Res Commun 2005328: 258-64.
22. Sgodda M, Aurich H, Kleist S, et al. Hepatocyte differentiation of mesenchymal stem cells from rat peritoneal adipose tissue in vitro and in vivo. Exp Cell Res 2007; 313: 2875-86.[CrossRef][Medline]
23. Banas A, Teratani T, Yamamoto Y, et al. Adipose tissue-derived mesenchymal stem cells as a source of human hepatocytes. Hepatology 2007; 46: 219-28.[CrossRef][Medline]
24. Sato Y, Araki H, Kato J, et al. Human mesenchymal stem cells xenografted directly to rat liver are differentiated into human hepatocytes without fusion. Blood 2005; 106: 756-63.[Abstract/Free Full Text]
25. Chamberlain J, Yamagami T, Colletti E, et al. Efficient generation of human hepatocytes by the intrahepatic delivery of clonal human mesenchymal stem cells in fetal sheep. Hepatology. Published Online First: 17 August 2007. doi: 10.1002/hep.21899.
26. Popp FC, Slowik P, Eggenhofer E, et al. No contribution of multipotent mesenchymal stromal cells to liver regeneration in a rat model of prolonged hepatic injury. Stem Cells 2007; 25: 639-45.[Abstract/Free Full Text]
27. Valfre di Bonzo L, Ferrero I, Cravanzola C, et al. Human mesenchymal stem cells as a two-edged sword in hepatic regenerative medicine: engraftment and hepatocyte differentiation versus profibrogenic potential. Gut 2008;57:223-31.
28. Forbes SJ, Russo F, Rey V, et al. A significant proportion of myofibroblasts are of bone marrow origin in human liver fibrosis. Gastroenterology 2004; 126: 955-63.[CrossRef][Medline]
29. Russo FP, Alison MR, Bigger BW, et al. The bone marrow functionally contributes to liver fibrosis. Gastroenterology 2006; 130: 1807-21.[CrossRef][Medline]
30. am Esch JS, Knoefel WT, Klein M, et al. Portal application of autologous CD133+ bone marrow cells to the liver: a novel concept to support hepatic regeneration. Stem Cells 2005; 23: 463-70.[Abstract/Free Full Text]
31. Gordon MY, Levicar N, Pai M, et al. Characterization and clinical application of human CD34+ stem/progenitor cell populations mobilized into the blood by granulocyte colony-stimulating factor. Stem Cells 2006; 24: 1822-30.[Abstract/Free Full Text]
32. Terai S, Ishikawa T, Omori K, et al. Improved liver function in patients with liver cirrhosis after autologous bone marrow cell infusion therapy. Stem Cells 2006; 24: 2292-8.[Abstract/Free Full Text]
33. Lyra AC, Soares MB, da Silva LF, et al. Feasibility and safety of autologous bone marrow mononuclear cell transplantation in patients with advanced chronic liver disease. World J Gastroenterol 2007; 13: 1067-73.[Medline]
34. Duffield JS, Forbes SJ, Constandinou CM, et al. Selective depletion of macrophages reveals distinct, opposing roles during liver injury and repair. J Clin Invest 2005; 115: 56-65.[Abstract/Free Full Text]
  iconpaperstack View Older Articles   Back to Top   www.natap.org