Review Series Stem Cell Research

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1 Q J Med 2014; 107: doi: /qjmed/hcu013 Advance Access Publication 22 January 2014 Review Series Stem Cell Research Stem cells for liver regeneration N.N. THAN and P.N. NEWSOME From the Centre for Liver Research & NIHR Biomedical Research Unit in Liver Diseases, University of Birmingham, Birmingham, UK Address correspondence to Dr N.N. Than, Mindelsohn Way, Edgbaston, Birmingham B15 2WB, UK. sophiathan@nhs.net Summary 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. 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 After partial hepatectomy in rodents, the original liver mass is restored within 7 10 days whereas in humans there is complete restoration by 3 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 G 1 phase of the cycle. 1 The priming step is reversible until the cells have crossed the G 1 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! The Author Published by Oxford University Press on behalf of the Association of Physicians. All rights reserved. For Permissions, please journals.permissions@oup.com

2 418 N.N. Than and P.N. Newsome (EGF) and transforming growth factor-alpha (TGF-a) underpin normal hepatic regeneration through their potent mitogenic action on hepatocytes via stimulation of DNA synthesis. In addition, tumour necrosis factor-alpha and interleukin-6 (IL-6) are required for priming of the cell cycle, 1,2 whereas HGF and TGF-a 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 TGF-b 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 provides a stimulus for the activation LPC which can contribute to liver repair. 3 Hepatic oval cell (HOC) as termed in rodents or LPC as known in human is an intrahepatic population of bipotent progenitor cells, which 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 support liver regeneration when hepatocyte proliferation is ineffective or deficient. In human liver damage, the number of LPC 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 four 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 with hepatocyte replication. 5 A range of growth factors TGF-a, EGF, HGF and stem cell factor have been shown to be important in stimulating oval cell growth, TWEAK/Fn14 important for their activation and stromal-derived factor/cxc receptor 4 (SDF-1/CXCR4) important for their migration. 1 SDF-1 expression, which acts as a chemo-attractant, appears to be up-regulated and localized 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: (i) administration of granulocyte stimulating factor (G-CSF) to enhance endogenous stem cell regeneration pathways and (ii) 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.

3 Stem cells for liver regeneration 419 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 mobilizing Bone marrow (BM) cells or by acting locally within the liver microenvironment by facilitating endogenous repair pathways. 9 It has been 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 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 G-CSF in enhancing neutrophil function in patients with liver disease and hence reducing deaths from sepsis. 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 fibrosis where the aim has been to reduce aberrant inflammation. Haematopoietic stem cells 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. Haematopoietic stem cells (HSC) are the most studied adult stem cell as they can be readily isolated by their surface expression of c-kit + Sca-1 + Lineage 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 8-week long mouse model of carbon tetrachloride (CCl 4 ) 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 was 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 CCl 4 -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 (MSC) 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 endpoint 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 embolization 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 the liver volume was significantly higher in the CD133-treated group allowing for earlier surgery to resect hepatic metastases. 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 Preclinical studies from different laboratories have reported beneficial effects of rodent and human MSC in models of liver injury. These studies have used 18,19 simple toxic models of liver injury, such as CCl 4 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 reports improvements in liver damage 17,19 which appears to be in part mediated by a reduction in oxidative stress 19 and cellular

4 420 N.N. Than and P.N. Newsome infiltrates. 22 Of note studies with murine MSC also report a reduction in fibrosis 23,24 when infused in models of chronic liver damage with CCl 4. 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 MMP-13 activity promoting fibrinolysis within the liver. Human MSC infusions have been reported to have similar beneficial actions in CCl 4 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. Although 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. Although 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 N.N.T. and P.N.N. are funded by NIHR (National Institute for Health Research). Conflict of interest: None declared. References 1. Fausto N. Liver regeneration and repair: hepatocytes, progenitor cells, and stem cells. Hepatology 2004; 39: Newsome PN, Hussain MA, Theise ND. Hepatic oval cells: helping redefine a paradigm in stem cell biology. Curr Top Dev Biol 2004; 61: Kung JW, Forbes SJ. Stem cells and liver repair. Curr Opin Biotechnol 2009; 20: Duncan AW, Dorrell C, Grompe M. Stem cells and liver regeneration. Gastroenterology 2009; 137: Best J, Dolle L, Manka P, Coombes J, van Grunsven LA, Syn WK. Role of liver progenitors in acute liver injury. Front Physiol 2013; 4: Kollet O, Shivtiel S, Chen YQ, Suriawinata J, Thung SN, Dabeva MD, 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: Boulter L, Govaere O, Bird TG, Radulescu S, Ramachandran P, Pellicoro A, et al. Macrophage-derived Wnt opposes Notch signaling to specify hepatic progenitor cell fate in chronic liver disease. Nat Med 2012; 18: Pi L, Oh SH, Shupe T, Petersen BE. Role of connective tissue growth factor in oval cell response during liver regeneration after 2-AAF/PHx in rats. Gastroenterology 2005; 128: Yannaki E, Athanasious E, Xagorari A, Constantinou V, Batsis I, Kaloyannidis P, et al. G-CSF-primed hematopoietic stem cells or G-CSF per se accelerate recovery and improve survival after liver injury, predominantly by promoting endogenous repair programs. Exp Hematol 2005; 33: 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. J Hepatol 2006; 45: Houlihan DD, Newsome PN. Critical review of clinical trials of bone marrow stem cells in liver disease. Gastroenterology 2008; 135: Garg V, Garg H, Khan A, Trehanpati N, Kumar A, Sharma BC, et al. Granulocyte colony-stimulating factor mobilizes CD34(+) cells and improves survival of patients with acute-on-chronic liver failure. Gastroenterology 2012; 142: e 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: Thomas JA, Pope C, Wojtacha D, Robson AJ, Gordon-Walker TT, Hartland S, et al. Macrophage therapy for murine liver fibrosis recruits host effector cells improving fibrosis, regeneration, and function. Hepatology 2011; 53: Forbes SJ, Newsome PN. New horizons for stem cell therapy in liver disease. J Hepatol 2012; 56: Fürst G, Schulte Am Esch J, Poll LW, Hosch SB, Fritz LB, Klein M, et al. Portal vein embolization and autologous CD133+ bone marrow stem cells for liver regeneration: initial experience. Radiology 2007; 243:171 9.

5 Stem cells for liver regeneration Eksteen B, Afford SC, Wigmore SJ, Holt AP, Adams DH. Immune-mediated liver injury. Semin Liver Dis 2007; 27: Kuo TK, Hung SP, Chuang CH, Chen CT, Shih YR, Fang SC, et al. Stem cell therapy for liver disease: parameters governing the success of using bone marrow mesenchymal stem cells. Gastroenterology 2008; 134: , 2121.e Cho KA, Woo SY, Seoh JY, Han HS, Ryu KH. Mesenchymal stem cells restore CCl4-induced liver injury by an antioxidative process. Cell Biol Int 2012; 36: van Poll D, Parekkadan B, Cho CH, Berthiaume F, Nahmias Y, Tilles AW, et al. Mesenchymal stem cellderived molecules directly modulate hepatocellular death and regeneration in vitro and in vivo. Hepatology 2008; 47: Wan CD, Cheng R, Wang HB, 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: 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: Pan RL, Wang P, Xiang LX, Shao JZ. 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: 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: Zanotti L, Sarukhan A, Dander E, Castor M, Cibella J, Soldani C, et al. Encapsulated mesenchymal stem cells for in vivo immunomodulation. Leukemia 2013; 27:500 3.