QJM Advance Access published January 22, 2014
Review article
Title: Stem cells for liver regeneration Nwe Ni Than, Philip N Newsome
Keywords: Liver regeneration, hepatocytes, stem cells, oval cells, liver progenitor cells, bone marrow-derived stem cell, mesenchymal stem cells, haematopoietic stem cell
© The Author 2014. Published by Oxford University Press on behalf of the Association of Physicians. All rights reserved. For Permissions, please email: [email protected]
Downloaded from http://qjmed.oxfordjournals.org/ at Aston University on January 26, 2014
Centre for Liver Research & NIHR Biomedical Research Unit in Liver Diseases, University of Birmingham, United Kingdom
Abstract
The liver has a unique capacity to repair following injury which is largely achieved by proliferation of hepatocytes. However, in situations of chronic or overwhelming liver injury, additional repair mechanisms, namely liver progenitor or oval cells, are activated. These cells, located in the canals of Hering, express markers for both hepatocytes and biliary cells and have the capacity to differentiate down both hepatocyte and biliary lineages. Previous work has suggested that the administration of autologous or allogeneic cell therapies such as haematopoietic or mesenchymal stem cells can augment liver repair by either stimulating endogenous repair mechanisms or by suppressing ongoing damage. A better understanding of how cell therapies can promote liver regeneration will lead to the refinement of these therapeutic approaches and also develop new pharmacological agents for liver repair. Downloaded from http://qjmed.oxfordjournals.org/ at Aston University on January 26, 2014
Introduction
Under physiological conditions, there is remarkably little cell turnover in the liver, with as few as one in 300 hepatocytes dividing to maintain the liver mass. In response to simple injury such as partial hepatectomy, repair is mediated by proliferation of endogenous hepatocytes without need for any adjunctive repair pathways. However, if hepatocyte replication is compromised such as in chronic liver injury or overwhelming acute injury, endogenous liver stem cells known as hepatic progenitor or oval cells are activated and contribute to the repair process. To understand the role of endogenous stem cells in liver repair, or indeed exogenously administered stem cells, it is helpful to firstly review the mechanisms mediating liver regeneration in response to differing types of injury. Endogenous liver regeneration
there is complete restoration by three months. Liver regeneration in this non-toxic model of injury is a multi-step process with at least two important phases: transition of quiescent hepatocytes into the cell cycle (priming) and then their progression beyond the restriction point in the G1 phase of the cycle [1]. The priming step is reversible until the cells have crossed the G1 checkpoint (conversion of cyclin D to cyclin E), after which the cells are irreversibly committed to replication [1]. Control of this process depends on a complex interaction of cytokine and growth factors released in response to liver injury. Three main growth factors: Hepatocyte growth factor (HGF), Epidermal growth factor (EGF) and Transforming growth factor alpha (TGF-α) underpin normal hepatic regeneration through their potent mitogenic action on hepatocytes via stimulation of DNA synthesis. In addition, Tumour necrosis factoralpha (TNF-α) and Interleukin-6 (IL-6) are required for priming of the cell cycle [1, 2], whilst HGF and TGF-α are required for cell cycle progression. Termination of hepatocyte proliferation at the end of regeneration is an important part of this process which is regulated by Transforming growth factor- beta (TGF-β) and Activin which serve as negative feedback mechanisms. Termination of hepatocyte proliferation is regulated by the ratio of liver to body mass rather than liver mass per se, thus providing a remarkable check on the extent of liver regeneration [1]. Indeed in the setting of liver transplantation when a large liver is placed into a smaller recipient donor liver mass reduces in size to reach the optimal ratio [1]. During more extensive acute liver injury such as that seen in paracetamol toxicity, there is widespread necrosis and apoptosis with release of cytokines, which far exceeds the capacity of remaining healthy hepatocytes to replicate and restore liver function [2]. As a result, resident liver progenitor cells (LPC) within the canals of Hering are activated to support or take over the role of regeneration [3]. Similarly in chronic liver injury, a combination of replicative senescence in endogenous hepatocytes and the inhibitory effect of progressive fibrosis provide a stimulus for the activation LPC which can contribute to liver repair [3]. Hepatic oval cell (HOC) as they are termed in rodents or LPC as termed in humans are an intrahepatic population of bipotent progenitor cells, that have the ability to proliferate clonogenically as well as being capable of differentiating into both mature hepatocytes and bile duct epithelial cells [2]. The term ‘oval’ cell derives from their appearance as small, rounded cells with a large nuclear to cytoplasmic ratio. These cells reside in the terminal branches of the intrahepatic biliary tree (Canal of Hering) and have been demonstrated to
Downloaded from http://qjmed.oxfordjournals.org/ at Aston University on January 26, 2014
After partial hepatectomy in rodents, the original liver mass is restored within 7-10 days whereas in humans
support liver regeneration when hepatocyte proliferation is ineffective or deficient. In human liver damage, the number of liver progenitor cells found is strongly related to the severity of the underlying disease, supporting their potential role in aiding liver regeneration.
The oval cell response can be divided into 4 phases: activation, proliferation, migration of progenitor cells and differentiation, the final step leading to either hepatocytes or bile duct epithelial cells [4]. IL-6 appears to regulate activation and proliferation of oval cells, with the subsequent expansion of the LPC compartment taking place over 7 days [4, 5]. Differentiation of LPC into intermediate hepatocytes often requires an additional 7 days, which can be seen to be a much slower regenerative process compared to hepatocyte replication [5]. A range of growth factors TGF-alpha, EGF, HGF and Stem cell factor (SCF) have been shown to be important in stimulating oval cell growth, TWEAK/Fn14 important for their activation and Stromal derived factor/ CXC
attractant, appears to be up-regulated and localised to the biliary epithelium during more extensive or chronic liver injury [6]. Recent studies have elegantly defined the mechanism controlling HOC fate in response to liver damage. During HOC mediated liver regeneration an inflammatory niche forms around HOC [7] constituting the ductular reaction in human disease and murine models. This niche is populated by macrophages and myofibroblasts and requires the new synthesis of Extracellular Matrix (ECM) to facilitate appropriate HOC/ LPC expansion [7]. Activation of Notch and Wnt pathways by the surrounding niche has been shown to play a critical role in the lineage specification of HOC to hepatocytic and cholangiocytic fates [7]. Boulter et al demonstrated that during biliary regeneration, expression of Jagged 1 (a Notch ligand) by myofibroblasts promoted Notch signalling in HPC resulting in their adopting a biliary specification to cholangiocytes. However, during hepatocyte regeneration, macrophage phagocytosis of hepatocyte debris resulted in an increase in Wnt3a expression, which led to Wnt signalling in nearby HPC. This was demonstrated to result in an increased expression of Numb within these cells and hence their differentiation to hepatocytes. By these two pathways adult parenchymal regeneration during chronic liver injury is promoted [7]. Stem cells to augment liver regeneration
Two broad strategies have been employed to enhance liver regeneration; (1) Administration of Granulocyte stimulating factor (G-CSF) to enhance endogenous stem cell regeneration pathways, and (2) Infusions of exogenous stem cells to drive regeneration. Indeed, there remains much debate as to whether cellular therapies will serve as a means of identifying druggable targets, or whether they will persist as therapies in their own right. (1) Administration of G-CSF to enhance endogenous stem cell regeneration pathways Recent studies have shown that (G-CSF) may be effective in facilitating liver repair [8, 9], although it remains unclear whether G-CSF acts by mobilising BM cells or by acting locally within the liver microenvironment by facilitating endogenous repair pathways [9] . Of note, Petersen et al reported that oval cells express the receptor for G-CSF and that G-CSF administration significantly increased oval cell proliferation and migration in this model [8]. Most clinical studies have used G-CSF alongside cell infusions in patients with chronic liver disease,
Downloaded from http://qjmed.oxfordjournals.org/ at Aston University on January 26, 2014
receptor 4 (SDF1/CXCR4) important for their migration [1]. SDF-1 expression, which acts as a chemo-
although in a few studies it has been used alone [10] . Unequivocal demonstration of the efficacy of GCSF in liver disease is still outstanding [11] although a recent study indicated that its administration in patients with acute on chronic liver failure improved patient survival [12]. Another potential mechanism of action described, but not verified, in this recent study is the effect of GCSF in enhancing neutrophil function in patients with liver disease and hence reducing deaths from sepsis. (2) Infusions of exogenous stem cells to drive regeneration
Initial approaches with stem cells focussed on their ability to differentiate into hepatocytes, although this is now felt to represent a very minor component of their action. Moreover, most studies have examined the role of cell therapy in the setting of chronic liver disease where the presence of fibrosis is the main feature. To a lesser extent cell therapies have also been used in the setting of acute or chronic liver inflammation in the absence of
Haematopoietic stem cells (HSC) and macrophages
There are an accumulating number of rodent and clinical studies investigating the effects of BM stem cell therapy in patients with liver disease. HSC are the most studied adult stem cell as they can be readily isolated by their surface expression of c-Kit+Sca-1+Lineage− (KSL) in rodents or CD34 or CD133 in humans.
Sakaida et al investigated the effect of a mouse bone marrow cell (BMC) tail vein infusion half way through an eight week long mouse model of carbon tetrachloride (CCl4) induced liver fibrosis [13]. Mice receiving BMC infusions had reduced liver fibrosis and a significantly improved survival rate compared with control injured mice [13]. Mice receiving BMC had increased hepatic matrix metalloproteinase-9 (MMP 9) expression, with the suggestion being that BMC were adopting a macrophage phenotype [13], although the identity of the cell responsible for this action within the BMC suspension was not confirmed. Notably, Thomas et al demonstrated that infusion of syngeneic macrophages in a mouse model of CCl4-induced liver fibrosis also led to a reduction in fibrosis, which was in marked contrast to the increase seen with unfractionated BM cells [14]. This study also identified that there was a marked expansion in numbers of recipient macrophages and neutrophils in the host liver suggesting that donor cells may exert the majority of their effect by augmenting endogenous repair mechanisms [14].
There are many published studies in the human setting with either haematopoietic or mesenchymal stem cells in liver disease. Although many of these studies reported an improvement in liver function the results should be interpreted with caution due to their small size, lack of defined primary end-point and often lack of a control group [15]. One of the more compelling studies examined the use of autologous CD133+ HSC in the setting of patients needing liver regeneration prior to undergoing hepatic resection of metastases. Portal venous embolisation with chemotherapy alongside infusion of autologous CD133+ cells into the non-occluded portal vein resulted in dramatically increased expansion of the residual liver lobe [16] Compared with a control group, the increase in
Downloaded from http://qjmed.oxfordjournals.org/ at Aston University on January 26, 2014
fibrosis where the aim has been to reduce aberrant inflammation.
the liver volume was significantly higher in the CD133-treated group allowing for earlier surgery to resect hepatic metastases.
Mesenchymal stem cells (MSC) and regulatory T cells
Both MSC and T Regulatory (T Reg) cells have been considered as possible therapeutic agents due to their ability to suppress inflammation which is seen in many forms of chronic liver injury [17]. Pre-clinical studies from different laboratories have reported beneficial effects of rodent and human MSC in models of liver injury. These studies have used simple toxic models of liver injury such as carbon tetrachloride [18, 19] or galactosamine [20] , chemical-induced primary biliary cirrhosis [21], non-alcoholic fatty liver disease [22] or models of hepatic transplantation [21]. Use of rodent MSC in such models report improvements in liver damage
Of note studies with murine MSC also report a reduction in fibrosis [23, 24] when infused in models of chronic liver damage with carbon tetrachloride (CCl4). This effect would appear to be mediated by blockade of Dlk1 activation and hence reduction in activation of hepatic stellate cells [23] , along with increased MMP13 activity promoting fibrinolysis within the liver. Human MSC infusions have been reported to have similar beneficial actions in CCl4 injury [18] although the mechanism of action was not well explored with the exception of a reduction in oxidative stress [18]. Infusion of the CM-MSC reduced lymphocytic ingress to the injured liver; with a secretome analysis suggesting the effect may be chemokine dependent. Route of administration and location of action Many differing populations of stem cells have been administered via a range of routes including systemic infusion, intrahepatic injection, portal vein injection and intra-splenic injection. Whilst every route has its own advantages and disadvantages, none has been shown to be more effective and hence the focus will be on practical routes of administration such as systemic administration via a peripheral vein. This does raise the question as to where do infused cells need to migrate to function and is this a critical determinant of their efficacy in clinical studies. Whilst intuitively hepatic migration is required for anti-fibrotic therapies studies suggest that for anti-inflammatory therapies there may be extra-hepatic actions which represent a major component of their action [25]. Conclusion
Tailored cell therapies offer great promise in the management of patients with liver disease, although their efficacy needs to be demonstrated in large clinical trials whilst also ensuring that the cost of such interventions is affordable. Alongside this the challenge will be to develop a better understanding of their mechanism of action in patients as well as in pre-clinical studies.
Acknowledgements: NNT and PN are funded by NIHR (National Institute for Health Research) Conflict of interest: None
Downloaded from http://qjmed.oxfordjournals.org/ at Aston University on January 26, 2014
[19] [17] which appears to be in part mediated by a reduction in oxidative stress [19] and cellular infiltrates [22].
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Kung, J.W. and S.J. Forbes, Stem cells and liver repair. Current opinion in biotechnology, 2009. 20(5): p. 568-74.
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Duncan, A.W., C. Dorrell, and M. Grompe, Stem cells and liver regeneration. Gastroenterology, 2009. 137(2): p. 466-81. Best, J., Dolle, L., Manka, P., Coombes, J., van Grunsven, L.A., Syn, W.K, Role of liver progenitors in acute liver injury. Frontiers in physiology, 2013. 4: p. 258.
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Kollet, O., Shivtiel, S., Chen, Y.Q., Suriawinata, J., Thung, S.N., Dabeva, M.D., et al., HGF, SDF-1, and MMP-9 are involved in stress-induced human CD34+ stem cell recruitment to the liver. J Clin Invest, 2003. 112(2): p. 160-9.
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Boulter, L., Govaere, O., Bird, T.G., Radulescu, S., Ramachandran, P., Pellicoro, A., et al., Macrophage-derived Wnt opposes Notch signaling to specify hepatic progenitor cell fate in chronic liver disease. Nature medicine, 2012. 18(4): p. 572-9.
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Pi, L., Oh, S.H., Shupe, T., Petersen, B.E, Role of connective tissue growth factor in oval cell response during liver regeneration after 2-AAF/PHx in rats. Gastroenterology, 2005. 128(7): p. 2077-88.
9.
Yannaki, E., Athanasious, E., Xagorari, A., Constantinou, V., Batsis, I., Kaloyannidis, P., et al., GCSF-primed hematopoietic stem cells or G-CSF per se accelerate recovery and improve survival after liver injury, predominantly by promoting endogenous repair programs. Experimental hematology, 2005. 33(1): p. 108-19.
10.
Gaia, S., Smedile, A., Omede, P., Olivero, A., Sanavio, F., Balzolo, F., et al., Feasibility and safety of G-CSF administration to induce bone marrow-derived cells mobilization in patients with end stage liver disease. Journal of Hepatology, 2006. 45(1): p. 13-9.
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Houlihan, D.D. and P.N. Newsome, Critical review of clinical trials of bone marrow stem cells in liver disease. Gastroenterology, 2008. 135(2): p. 438-50.
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Garg, V., Garg, H., Khan, A., Trehanpati, N., Kumar, A., Sharma, B.C., et al., Granulocyte colonystimulating factor mobilizes CD34(+) cells and improves survival of patients with acute-on-chronic liver failure. Gastroenterology, 2012. 142(3): p. 505-512 e1.
13.
Sakaida, I., Terai, S., Yamamoto, N., Aoyama, K., Ishikawa, T., Nishina, H., et al., Transplantation of bone marrow cells reduces CCl4-induced liver fibrosis in mice. Hepatology, 2004. 40(6): p. 1304-11.
14.
Thomas, J.A., Pope, C., Wojtacha,D., Robson, A.J., Gordon-Walker, T.T., Hartland, S., et al., Macrophage therapy for murine liver fibrosis recruits host effector cells improving fibrosis, regeneration, and function. Hepatology, 2011. 53(6): p. 2003-15.
15.
Forbes, S.J. and P.N. Newsome, New horizons for stem cell therapy in liver disease. J Hepatol, 2012. 56(2): p. 496-9.
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Fürst, G., Schulte, a.E.J., Poll, L.W., Hosch, S.B., Fritz, L.B., Klein, M.,et al., Portal vein embolization and autologous CD133+ bone marrow stem cells for liver regeneration: initial experience. Radiology, 2007. 243(1): p. 171-9.
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Eksteen, B., Afford, S.C., Wigmore, S.j., Holt, A.P., Adams, D.H., Immune-mediated liver injury. Semin Liver Dis, 2007. 27(4): p. 351-66.
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Kuo, T.K., Hung, S.P., Chuang, C.H., Chen, C.T., Shih, Y.R., Fang, S.C., et al., Stem cell therapy for liver disease: parameters governing the success of using bone marrow mesenchymal stem cells. Gastroenterology, 2008. 134(7): p. 2111-21, 2121 e1-3.
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Cho, K.A., Woo, S.Y., Seoh, J.Y., Han, H.S., Ryu, K.H., Mesenchymal stem cells restore CCl4induced liver injury by an antioxidative process. Cell Biol Int, 2012. 36(12): p. 1267-74.
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van Poll, D., Parekkadan, B., Cho, C.H., Berthiaume, F., Nahmias, Y., Tilles, A.W., et al.,
vitro and in vivo. Hepatology, 2008. 47(5): p. 1634-43. 21.
Wan, C.D., Cheng, R., Wang, H.B., Liu, T., Immunomodulatory effects of mesenchymal stem cells derived from adipose tissues in a rat orthotopic liver transplantation model. Hepatobiliary Pancreat Dis Int, 2008. 7(1): p. 29-33.
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Ezquer, M., Ezquer, F., Ricca, M., Allers, C., Conget, P., Intravenous administration of multipotent stromal cells prevents the onset of non-alcoholic steatohepatitis in obese mice with metabolic syndrome. J Hepatol, 2011. 55(5): p. 1112-20.
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Pan, R.L., Wang, P., Xiang, L.X., Shao, J.Z., Delta-like 1 serves as a new target and contributor to liver fibrosis down-regulated by mesenchymal stem cell transplantation. J Biol Chem, 2011. 286(14): p. 12340-8.
24.
Rabani, V., Shahsavani, M., Gharavi, M., Priyaei, A., Azhdari, Z., Baharvand, H., Mesenchymal stem cell infusion therapy in a carbon tetrachloride-induced liver fibrosis model affects matrix metalloproteinase expression. Cell Biol Int, 2010. 34(6): p. 601-5.
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Zanotti, L., Sarukhan, A., Dander, E., Castor, M., Cibella, J., Soldani, C., et al., Encapsulated mesenchymal stem cells for in vivo immunomodulation. Leukemia, 2013. 27(2): p. 500-503.
Downloaded from http://qjmed.oxfordjournals.org/ at Aston University on January 26, 2014
Mesenchymal stem cell-derived molecules directly modulate hepatocellular death and regeneration in
Review article
Title: Stem cells for liver regeneration Nwe Ni Than, Philip N Newsome
Keywords: Liver regeneration, hepatocytes, stem cells, oval cells, liver progenitor cells, bone marrow-derived stem cell, mesenchymal stem cells, haematopoietic stem cell
© The Author 2014. Published by Oxford University Press on behalf of the Association of Physicians. All rights reserved. For Permissions, please email: [email protected]
Downloaded from http://qjmed.oxfordjournals.org/ at Aston University on January 26, 2014
Centre for Liver Research & NIHR Biomedical Research Unit in Liver Diseases, University of Birmingham, United Kingdom
Abstract
The liver has a unique capacity to repair following injury which is largely achieved by proliferation of hepatocytes. However, in situations of chronic or overwhelming liver injury, additional repair mechanisms, namely liver progenitor or oval cells, are activated. These cells, located in the canals of Hering, express markers for both hepatocytes and biliary cells and have the capacity to differentiate down both hepatocyte and biliary lineages. Previous work has suggested that the administration of autologous or allogeneic cell therapies such as haematopoietic or mesenchymal stem cells can augment liver repair by either stimulating endogenous repair mechanisms or by suppressing ongoing damage. A better understanding of how cell therapies can promote liver regeneration will lead to the refinement of these therapeutic approaches and also develop new pharmacological agents for liver repair. Downloaded from http://qjmed.oxfordjournals.org/ at Aston University on January 26, 2014
Introduction
Under physiological conditions, there is remarkably little cell turnover in the liver, with as few as one in 300 hepatocytes dividing to maintain the liver mass. In response to simple injury such as partial hepatectomy, repair is mediated by proliferation of endogenous hepatocytes without need for any adjunctive repair pathways. However, if hepatocyte replication is compromised such as in chronic liver injury or overwhelming acute injury, endogenous liver stem cells known as hepatic progenitor or oval cells are activated and contribute to the repair process. To understand the role of endogenous stem cells in liver repair, or indeed exogenously administered stem cells, it is helpful to firstly review the mechanisms mediating liver regeneration in response to differing types of injury. Endogenous liver regeneration
there is complete restoration by three months. Liver regeneration in this non-toxic model of injury is a multi-step process with at least two important phases: transition of quiescent hepatocytes into the cell cycle (priming) and then their progression beyond the restriction point in the G1 phase of the cycle [1]. The priming step is reversible until the cells have crossed the G1 checkpoint (conversion of cyclin D to cyclin E), after which the cells are irreversibly committed to replication [1]. Control of this process depends on a complex interaction of cytokine and growth factors released in response to liver injury. Three main growth factors: Hepatocyte growth factor (HGF), Epidermal growth factor (EGF) and Transforming growth factor alpha (TGF-α) underpin normal hepatic regeneration through their potent mitogenic action on hepatocytes via stimulation of DNA synthesis. In addition, Tumour necrosis factoralpha (TNF-α) and Interleukin-6 (IL-6) are required for priming of the cell cycle [1, 2], whilst HGF and TGF-α are required for cell cycle progression. Termination of hepatocyte proliferation at the end of regeneration is an important part of this process which is regulated by Transforming growth factor- beta (TGF-β) and Activin which serve as negative feedback mechanisms. Termination of hepatocyte proliferation is regulated by the ratio of liver to body mass rather than liver mass per se, thus providing a remarkable check on the extent of liver regeneration [1]. Indeed in the setting of liver transplantation when a large liver is placed into a smaller recipient donor liver mass reduces in size to reach the optimal ratio [1]. During more extensive acute liver injury such as that seen in paracetamol toxicity, there is widespread necrosis and apoptosis with release of cytokines, which far exceeds the capacity of remaining healthy hepatocytes to replicate and restore liver function [2]. As a result, resident liver progenitor cells (LPC) within the canals of Hering are activated to support or take over the role of regeneration [3]. Similarly in chronic liver injury, a combination of replicative senescence in endogenous hepatocytes and the inhibitory effect of progressive fibrosis provide a stimulus for the activation LPC which can contribute to liver repair [3]. Hepatic oval cell (HOC) as they are termed in rodents or LPC as termed in humans are an intrahepatic population of bipotent progenitor cells, that have the ability to proliferate clonogenically as well as being capable of differentiating into both mature hepatocytes and bile duct epithelial cells [2]. The term ‘oval’ cell derives from their appearance as small, rounded cells with a large nuclear to cytoplasmic ratio. These cells reside in the terminal branches of the intrahepatic biliary tree (Canal of Hering) and have been demonstrated to
Downloaded from http://qjmed.oxfordjournals.org/ at Aston University on January 26, 2014
After partial hepatectomy in rodents, the original liver mass is restored within 7-10 days whereas in humans
support liver regeneration when hepatocyte proliferation is ineffective or deficient. In human liver damage, the number of liver progenitor cells found is strongly related to the severity of the underlying disease, supporting their potential role in aiding liver regeneration.
The oval cell response can be divided into 4 phases: activation, proliferation, migration of progenitor cells and differentiation, the final step leading to either hepatocytes or bile duct epithelial cells [4]. IL-6 appears to regulate activation and proliferation of oval cells, with the subsequent expansion of the LPC compartment taking place over 7 days [4, 5]. Differentiation of LPC into intermediate hepatocytes often requires an additional 7 days, which can be seen to be a much slower regenerative process compared to hepatocyte replication [5]. A range of growth factors TGF-alpha, EGF, HGF and Stem cell factor (SCF) have been shown to be important in stimulating oval cell growth, TWEAK/Fn14 important for their activation and Stromal derived factor/ CXC
attractant, appears to be up-regulated and localised to the biliary epithelium during more extensive or chronic liver injury [6]. Recent studies have elegantly defined the mechanism controlling HOC fate in response to liver damage. During HOC mediated liver regeneration an inflammatory niche forms around HOC [7] constituting the ductular reaction in human disease and murine models. This niche is populated by macrophages and myofibroblasts and requires the new synthesis of Extracellular Matrix (ECM) to facilitate appropriate HOC/ LPC expansion [7]. Activation of Notch and Wnt pathways by the surrounding niche has been shown to play a critical role in the lineage specification of HOC to hepatocytic and cholangiocytic fates [7]. Boulter et al demonstrated that during biliary regeneration, expression of Jagged 1 (a Notch ligand) by myofibroblasts promoted Notch signalling in HPC resulting in their adopting a biliary specification to cholangiocytes. However, during hepatocyte regeneration, macrophage phagocytosis of hepatocyte debris resulted in an increase in Wnt3a expression, which led to Wnt signalling in nearby HPC. This was demonstrated to result in an increased expression of Numb within these cells and hence their differentiation to hepatocytes. By these two pathways adult parenchymal regeneration during chronic liver injury is promoted [7]. Stem cells to augment liver regeneration
Two broad strategies have been employed to enhance liver regeneration; (1) Administration of Granulocyte stimulating factor (G-CSF) to enhance endogenous stem cell regeneration pathways, and (2) Infusions of exogenous stem cells to drive regeneration. Indeed, there remains much debate as to whether cellular therapies will serve as a means of identifying druggable targets, or whether they will persist as therapies in their own right. (1) Administration of G-CSF to enhance endogenous stem cell regeneration pathways Recent studies have shown that (G-CSF) may be effective in facilitating liver repair [8, 9], although it remains unclear whether G-CSF acts by mobilising BM cells or by acting locally within the liver microenvironment by facilitating endogenous repair pathways [9] . Of note, Petersen et al reported that oval cells express the receptor for G-CSF and that G-CSF administration significantly increased oval cell proliferation and migration in this model [8]. Most clinical studies have used G-CSF alongside cell infusions in patients with chronic liver disease,
Downloaded from http://qjmed.oxfordjournals.org/ at Aston University on January 26, 2014
receptor 4 (SDF1/CXCR4) important for their migration [1]. SDF-1 expression, which acts as a chemo-
although in a few studies it has been used alone [10] . Unequivocal demonstration of the efficacy of GCSF in liver disease is still outstanding [11] although a recent study indicated that its administration in patients with acute on chronic liver failure improved patient survival [12]. Another potential mechanism of action described, but not verified, in this recent study is the effect of GCSF in enhancing neutrophil function in patients with liver disease and hence reducing deaths from sepsis. (2) Infusions of exogenous stem cells to drive regeneration
Initial approaches with stem cells focussed on their ability to differentiate into hepatocytes, although this is now felt to represent a very minor component of their action. Moreover, most studies have examined the role of cell therapy in the setting of chronic liver disease where the presence of fibrosis is the main feature. To a lesser extent cell therapies have also been used in the setting of acute or chronic liver inflammation in the absence of
Haematopoietic stem cells (HSC) and macrophages
There are an accumulating number of rodent and clinical studies investigating the effects of BM stem cell therapy in patients with liver disease. HSC are the most studied adult stem cell as they can be readily isolated by their surface expression of c-Kit+Sca-1+Lineage− (KSL) in rodents or CD34 or CD133 in humans.
Sakaida et al investigated the effect of a mouse bone marrow cell (BMC) tail vein infusion half way through an eight week long mouse model of carbon tetrachloride (CCl4) induced liver fibrosis [13]. Mice receiving BMC infusions had reduced liver fibrosis and a significantly improved survival rate compared with control injured mice [13]. Mice receiving BMC had increased hepatic matrix metalloproteinase-9 (MMP 9) expression, with the suggestion being that BMC were adopting a macrophage phenotype [13], although the identity of the cell responsible for this action within the BMC suspension was not confirmed. Notably, Thomas et al demonstrated that infusion of syngeneic macrophages in a mouse model of CCl4-induced liver fibrosis also led to a reduction in fibrosis, which was in marked contrast to the increase seen with unfractionated BM cells [14]. This study also identified that there was a marked expansion in numbers of recipient macrophages and neutrophils in the host liver suggesting that donor cells may exert the majority of their effect by augmenting endogenous repair mechanisms [14].
There are many published studies in the human setting with either haematopoietic or mesenchymal stem cells in liver disease. Although many of these studies reported an improvement in liver function the results should be interpreted with caution due to their small size, lack of defined primary end-point and often lack of a control group [15]. One of the more compelling studies examined the use of autologous CD133+ HSC in the setting of patients needing liver regeneration prior to undergoing hepatic resection of metastases. Portal venous embolisation with chemotherapy alongside infusion of autologous CD133+ cells into the non-occluded portal vein resulted in dramatically increased expansion of the residual liver lobe [16] Compared with a control group, the increase in
Downloaded from http://qjmed.oxfordjournals.org/ at Aston University on January 26, 2014
fibrosis where the aim has been to reduce aberrant inflammation.
the liver volume was significantly higher in the CD133-treated group allowing for earlier surgery to resect hepatic metastases.
Mesenchymal stem cells (MSC) and regulatory T cells
Both MSC and T Regulatory (T Reg) cells have been considered as possible therapeutic agents due to their ability to suppress inflammation which is seen in many forms of chronic liver injury [17]. Pre-clinical studies from different laboratories have reported beneficial effects of rodent and human MSC in models of liver injury. These studies have used simple toxic models of liver injury such as carbon tetrachloride [18, 19] or galactosamine [20] , chemical-induced primary biliary cirrhosis [21], non-alcoholic fatty liver disease [22] or models of hepatic transplantation [21]. Use of rodent MSC in such models report improvements in liver damage
Of note studies with murine MSC also report a reduction in fibrosis [23, 24] when infused in models of chronic liver damage with carbon tetrachloride (CCl4). This effect would appear to be mediated by blockade of Dlk1 activation and hence reduction in activation of hepatic stellate cells [23] , along with increased MMP13 activity promoting fibrinolysis within the liver. Human MSC infusions have been reported to have similar beneficial actions in CCl4 injury [18] although the mechanism of action was not well explored with the exception of a reduction in oxidative stress [18]. Infusion of the CM-MSC reduced lymphocytic ingress to the injured liver; with a secretome analysis suggesting the effect may be chemokine dependent. Route of administration and location of action Many differing populations of stem cells have been administered via a range of routes including systemic infusion, intrahepatic injection, portal vein injection and intra-splenic injection. Whilst every route has its own advantages and disadvantages, none has been shown to be more effective and hence the focus will be on practical routes of administration such as systemic administration via a peripheral vein. This does raise the question as to where do infused cells need to migrate to function and is this a critical determinant of their efficacy in clinical studies. Whilst intuitively hepatic migration is required for anti-fibrotic therapies studies suggest that for anti-inflammatory therapies there may be extra-hepatic actions which represent a major component of their action [25]. Conclusion
Tailored cell therapies offer great promise in the management of patients with liver disease, although their efficacy needs to be demonstrated in large clinical trials whilst also ensuring that the cost of such interventions is affordable. Alongside this the challenge will be to develop a better understanding of their mechanism of action in patients as well as in pre-clinical studies.
Acknowledgements: NNT and PN are funded by NIHR (National Institute for Health Research) Conflict of interest: None
Downloaded from http://qjmed.oxfordjournals.org/ at Aston University on January 26, 2014
[19] [17] which appears to be in part mediated by a reduction in oxidative stress [19] and cellular infiltrates [22].
References 1.
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Mesenchymal stem cell-derived molecules directly modulate hepatocellular death and regeneration in
