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Potential of regenerative medicine techniques in canine hepatology a

a

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Baukje A. Schotanus , Louis C. Penning & Bart Spee a

Department of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands Published online: 14 Jan 2014.

To cite this article: Baukje A. Schotanus, Louis C. Penning & Bart Spee (2013) Potential of regenerative medicine techniques in canine hepatology, Veterinary Quarterly, 33:4, 207-216, DOI: 10.1080/01652176.2013.875240 To link to this article: http://dx.doi.org/10.1080/01652176.2013.875240

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Veterinary Quarterly, 2013 Vol. 33, No. 4, 207–216, http://dx.doi.org/10.1080/01652176.2013.875240

REVIEW ARTICLE Potential of regenerative medicine techniques in canine hepatology Baukje A. Schotanus, Louis C. Penning* and Bart Spee Department of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands

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(Received 31 October 2013; accepted 10 December 2013) Liver cell turnover is very slow, especially compared to intestines and stomach epithelium and hair cells. Since the liver is the main detoxifying organ in the body, it does not come as a surprise that the liver has an unmatched regenerative capacity. After 70% partial hepatectomy, the liver size returns to normal in about two weeks due to replication of differentiated hepatocytes and cholangiocytes. Despite this, liver diseases are regularly encountered in the veterinary clinic. Dogs primarily present with parenchymal pathologies such as hepatitis. The estimated frequency of canine hepatitis depends on the investigated population and accounts for 1%–2% of our university clinic referral population, and up to 12% in a general population. In chronic and severe acute liver disease, the regenerative and replicative capacity of the hepatocytes and/or cholangiocytes falls short and the liver is not restored. In this situation, proliferation of hepatic stem cells or hepatic progenitor cells (HPCs), on histology called the ductular reaction, comes into play to replace the damaged hepatocytes or cholangiocytes. For unknown reasons the ductular reaction is often too little and too late, or differentiation into fully differentiated hepatocytes or cholangiocytes is hampered. In this way, HPCs fail to fully regenerate the liver. The presence and potential of HPCs does, however, provide great prospectives for their use in regenerative strategies. This review highlights the regulation of, and the interaction between, HPCs and other liver cell types and discusses potential regenerative medicine-oriented strategies in canine hepatitis, making use of (liver) stem cells. Keywords: dog; canine; stem cells; liver; cell therapy; regeneration; regenerative medicine; stellate cell; Kupffer cells

1. Introduction The histopathology of canine hepatitis has been extensively characterized and shows features highly similar to their human counterpart. In both species, hepatitis is associated with hepatocyte apoptosis and necrosis, an inflammatory infiltrate, and potentially fibrosis, depending on the type and duration of liver disease (Van den Ingh et al. 2006). In both species, liver fibrosis ultimately results in irreversible cirrhosis (Bonis et al. 2001; Friedman 2007). Also on molecular level canine and human hepatitis are highly similar. Molecular characterization of canine hepatitis and parallel investigation with human liver diseases revealed that the cellular pathways of liver regeneration, and the cells and signals involved in liver fibrosis, are the same in man and dog (Leveille & Arias 1993; Gressner et al. 2002; Spee et al. 2005; Spee et al. 2006; Spee et al. 2007; Mortensen & Revhaug 2011). In human medicine, the development of new therapeutic strategies for liver disease has gained much attention. This is required because of the insufficient availability of donor organs for liver transplantation goals. For most canine liver diseases, no curative treatment option is available. Canine hepatology would therefore greatly benefit from the development of new therapeutic strategies. This review will highlight the different cellular components involved in liver disease and regeneration and discuss the potential regenerative medicine-oriented strategies in canine hepatitis. Liver development has been reviewed in great detail and is therefore not included (Lemaigre & Zaret 2004; Zhao & Duncan 2005; Lemaigre 2009; Si-Tayeb et al. 2010).

*Corresponding author. Email: [email protected] Ó 2014 Taylor & Francis

2. Liver diseases in dogs, prevalence and aetiology Liver disease is frequently encountered in the veterinary clinic. Dogs primarily present with parenchymal pathologies such as hepatitis (Watson 2004; Center 2009). The estimated frequency of canine hepatitis depends on the investigated population and accounts for 1%–2% of our university clinic referral population (Poldervaart et al. 2009), and up to 12% in a general population (Watson et al. 2010). Potential causes of canine hepatitis include micro-organisms, toxins and drugs, and immune-mediated reactions. Disorders of copper metabolism account for roughly 30% of chronic canine hepatitis cases (Favier 2009). In most cases, however, the cause of canine hepatitis is not known. This is despite large efforts to define suspected viral aetiologies (Boomkens et al. 2004, 2005; van der Heijden et al. 2012). Recently, canine hepacivirus was described to be involved in canine hepatitis. However, its causal role remains enigmatic (Kapoor et al. 2011). Treatment of canine liver disease is mostly symptomatic. This partly relates to the unknown cause of canine hepatitis, but is also due to the current lack of treatment options. Regenerative medicine as a field might hold great potential for treating currently untreatable liver diseases in dogs.

3. Pathways of regeneration The liver is an important organ in body homeostasis due to its central role in many metabolic processes. Being the first tissue to be exposed to toxins entering the body, the liver is often the main site of cell damage. The central

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guardian function of the liver can only be fulfilled due to two unique characteristics of the organ: the enormous reserve capacity and the ability to regenerate. The mechanisms responsible for the strict regulation of liver mass and size are not well understood. However, there is increasing insight into the understanding of the mechanisms underlying the huge regenerative capacity of the liver (Taub 2004; Michalopoulos 2007; Riehle et al. 2011). Under healthy conditions, hepatocytes are in a G0 phase and are quiescent (not proliferating). They have a life span of 200–300 days, and as part of normal homeostasis they display a very slow turnover rate. In response to injury or partial hepatectomy, hepatocyte replication is strongly increased. The capacity of hepatocytes to replicate is vast; transplantation and repopulation studies have shown that hepatocytes can replicate at least 34 times (Overturf et al. 1997, 1999; Taub 2004). Their proliferative activity is evoked by inflammatory cytokines from outside and inside the liver which induce cell cycle progression. Examples of non-liver-derived hepatocyte proliferation signals are norepinephrine and triiodothyronine. Another important factor is lipopolysaccharide, derived from the gut. This bacterial component activates the hepatic stellate cells (HSCs) and Kupffer cells in the liver. Once activated these non-parenchymal liver cells release cytokines and growth factors such as interleukin 6, tumour necrosis factor, hepatocyte growth factor, and epidermal growth factor which in turn stimulate the regenerative response. Transforming growth factor-b signalling inhibits hepatocyte proliferation and is blocked during regeneration (Russell et al. 1988). Soon after the activation of hepatocyte proliferation, the stromal compartment and cholangiocytic cells start to proliferate. The cells work in concert to obtain full liver regeneration. Although most information on normal liver regeneration is derived from rodent studies, the process is highly similar in other species including humans and dogs (Mortensen & Revhaug 2011). In fact, one of the first species where the regenerative capacity of the liver was investigated after partial hepatectomy was the dog (Mortensen & Revhaug 2011). Species similarity is further exemplified by the common hepatocyte replication response upon (massive) hepatocyte necrosis due to ischemia, toxins, viruses or immunemediated causes (Kiss et al. 2001; Taub 2004; Mortensen & Revhaug 2011). In dogs, this type of liver regeneration is most apparent upon surgical correction of a congenital portosystemic shunt (CPSS). A CPSS forms a direct circulatory communication between the portal venous system and the systemic circulation, causing the nutrient- and trophic factor-rich blood to bypass the liver. This shunt results in an underdeveloped liver and liver dysfunction (Berent & Tobias 2009). Upon successful surgical attenuation of the shunt, the liver grows out to the size determined by the functional need of the body (Pascher et al. 2002). In the course of chronic progressive liver diseases or due to toxins that inhibit the replicative capacity of hepatocytes, the proliferative potential of hepatocytes is exhausted or impaired. This leads to a strong impairment

of hepatocyte-dependent liver regeneration. In this situation another cell compartment is activated to regenerate the liver: the hepatic progenitor cell (HPC) compartment. These adult stem cells of the liver are considered a backup system when normal liver regeneration through hepatocytes fails. Although activated HPCs can be found in virtually all types of liver diseases (Schotanus et al. 2009; IJzer et al. 2010), the activation is thought to be too little and too late, leading to an unsuccessful liver regeneration. Unravelling the key signals that proliferate and differentiate HPCs might therefore hold the key to successful liver regeneration and will be important for the development of HPC-based regenerative strategies. 4. Cellular components of the liver To exploit liver regeneration for approaches in regenerative medicine, insight into the cellular make-up of the liver is essential. The liver is built up of functional liver units, hepatic lobules, structured around the central vein. At each corner of the hexagonal lobules, there is a portal triad harbouring a hepatic artery, portal vein, bile duct, lymph vessel and nerve. The lobule consists of parenchymal hepatocytes and biliary epithelium, and in addition non-parenchymal cells such as HSCs, Kupffer cells, endothelial cells and progenitor cells (Figure 1). The composition of the different cell types in the liver is highly organized and is a prerequisite for the liver to exert its critical physiological functions. The role of the different cell types in the liver is further discussed in the next section. HPCs will be discussed in detail in a following section. Hepatocytes are the principle functional cell type of the liver and make up around 75%–80% of the total liver volume (Si-Tayeb et al. 2010). They are epithelial, polarized cells structured in one-cell-thick plates. Their apical sides align the bile canaliculi, which transport the hepatocyte-derived bile to the larger bile ductules in the portal area. The basolateral side of the hepatocellular plates faces the space of Disse, separated from the sinusoids by endothelial cells and HSCs (Figure 1). The nutrient- and oxygen-rich blood enters the liver through the portal vein and the hepatic artery, respectively. It is transported through the sinusoids, where exchange of metabolites occurs to the central vein where it drains into the hepatic vein. Hepatocytes function in a large variety of catabolic and anabolic processes such as lipid, carbohydrate and protein metabolism, production and secretion of bile, and detoxification of exogenous toxins. Hepatocytes are also responsible for the acute phase response during inflammation and produce most coagulation factors. The second important functional entity of the liver is formed by the intra- and extrahepatic bile ducts and the gallbladder. Bile ducts are built up of cholangiocytes, cuboidal epithelial cells that participate in cell signalling and transport of water, ions and solutes, thereby contributing to bile secretion (Hollman et al. 2012). They transport bile to the gallbladder where the bile is concentrated, and subsequently to the duodenum, regulated by, e.g. cholecystokinin, produced in the small intestinal tract (Tietz &

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Figure 1. Cellular components of the liver. Schematic overview of the cellular components of the liver. Blood enters the liver lobe via the portal vein or portal artery and flows towards the central vein through sinusoids. Sinusoids are lined with endothelial cells forming fenestrae. Between the hepatocytes and the endothelial cells is the space of Disse, which harbours the hepatic stellate cells and the macrophages. Bile, produced by the hepatocytes and excreted in the bile canaliculae, flows in the opposite direction of blood towards the bile ducts in the portal area. The smallest ramifications of the biliary three, the canals of Hering, harbour the progenitor cells which, after stimulation, can proliferate and differentiate into hepatocytes and/or cholangiocytes. PA: portal artery; BD: bile duct; PV: portal vein; COH: canal of Hering; BC: bile canaliculus; CV: central vein; SD: space of Disse.

Larusso 2006). The smallest branches of the intrahepatic biliary tree are formed by the canals of Hering (CoH). The CoH consist of small cholangiocytes and hepatocytes forming an anatomical transitional area where the bile canaliculus enters the bile duct. Important supporting cells of the liver are HSCs (or previously called Ito cells), located in the peri-sinusoidal space of Disse (Geerts 2001). In the healthy liver, HSCs are quiescent and store high levels of vitamin A in lipid droplets. They may contribute to the regulation of the sinusoidal blood flow of the hepatic lobules and produce a multitude of cytokines and growth factors such as hepatocyte growth factor, which activates hepatocyte proliferation (Winau et al. 2007). When the liver is damaged HSCs are activated due to paracrine signalling by neighbouring cells including necrotic or apoptotic hepatocytes and Kupffer cells. Factors involved in their activation include for instance transforming growth factor-b, plateletderived growth factor and epidermal growth factor (Friedman 2008). Upon activation, HSCs undergo myofibroblastic transformation: they lose their lipid droplets, proliferate, increasingly produce extracellular matrix

(ECM) and become fibrogenic. In the healthy situation, ECM deposition by HSCs is balanced by matrix degradation through matrix metalloproteinases (MMPs) produced by, e.g. macrophages. The increased number of activated HSCs and enhanced production of ECM, together with an impaired matrix degradation during disease leads to liver fibrosis. Chronic fibrosis leads to end-stage cirrhosis (Guyot et al. 2006; Friedman 2007). The contractile HSCs are a major determinant of increased portal blood pressure (portal hypertension) during cirrhosis. Other important non-parenchymal cell types in the liver are Kupffer cells. Kupffer cells are located in the space of Disse, predominantly in the periportal area, which allows an optimal role in phagocytosis of foreign particles and infecting micro-organisms. Upon liver injury local Kupffer cells proliferate and additional macrophages are recruited from the bone marrow. Kupffer cells are important producers of cytokines including the tumour necrosis factor, interleukins and MMPs (Smedsrod et al. 1994; Duffield et al. 2005). Depending on the state of liver disease and regeneration, recruited macrophages can obtain a different functional profile and as such can be, e.g. pro-inflammatory,

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pro-fibrogenic or restorative in nature (Mosser & Edwards 2008; Ramachandran et al. 2012). A third type of non-parenchymal cell we will address here is the liver sinusoidal endothelial cell (LSEC). LSECs form a fenestrated lining of the hepatic sinusoids. The absence of a basement membrane allows for direct contact of blood plasma with the hepatocytes, and provides a large area for absorption of nutrients from the blood plasma (Smedsrod et al. 1994; Grisham 2009). LSECs produce prostanoids such as prostaglandin-E2 and thromboxane-B2, which effect haemodynamcis of the liver and modulate the immune response. Endothelial cells, Kupffer cells and HSCs are important antigenpresenting cells in the liver (Taub 2004; Fainboim et al. 2007) and are very important in the regulation of HPC activation (Boulter et al. 2013). This role of the nonparenchymal cells in liver regeneration and more specifically in HPC activation points out the importance of taking these cells into account when designing new regenerative therapies. 5. Stem cells for functional recovery of liver diseases Within the field of regenerative medicine, the regenerative process which is intrinsic to many tissues is utilized to replace or regenerate damaged cells in order to establish normal function. Biomedical approaches within the regenerative medicine field encompass (stem) cell therapies, immunomodulation therapy or tissue engineering. This next segment will highlight stem cells considered for clinical use in hepatology today, and subsequently their potential applications. 5.1.

Hepatic progenitor cells

One of the candidates for stem cell-therapy or -transplantation during liver disease is the previously mentioned adult stem cell of the liver, the HPC. For a long time, the presence of an adult stem cell in the liver was not anticipated (Sell 2001; Falkowski et al. 2003; Fausto 2004). The inexhaustible replication of terminally differentiated hepatocytes did not imply a need for such adult stem cells. Currently it is clear that HPCs, residing in the smallest branches of the biliary tree, are quiescent under situations of hepatocyte proliferation, yet proliferate and differentiate in those situations where hepatocyte or cholangiocyte replication is hampered. These two features, self-renewal (the capacity to form new progenitor cells after replication) and differentiation (in this case towards hepatocytes or cholangiocytes), characterize (adult) stem cells. At histology, the pattern of HPC activation is called a ‘ductular reaction’, and is positively stained for cytokeratin-7 or -19 with immunohistochemistry. A limited number of publications describe the canine HPC niche and its regulation with emphasis on HPCs, stellate cells and their activation status within various liver diseases (Boisclair et al. 2001; Ide et al. 2001; Yoshioka et al. 2004; IJzer et al. 2006; Schotanus et al. 2009; IJzer et al. 2010). Moreover, it was shown that within a healthy dog liver HPCs can be isolated using the side-population technique, which allows

the isolation of a sub-population of cells that exhibit stem cell-like characteristics (Arends et al. 2009b). Finally, HPCs were cultured from healthy canine livers (Arends et al. 2009a). Although these data clearly describe the presence and regulation of HPCs, no reports exist on the (pre-)clinical applications of these cells in canine hepatitis. Clinical application of these cells could involve transplantation or, more elegant, intrahepatic manipulation during disease. Especially transplantation of HPCs may have come one step closer since these stem cells can be grown for prolonged period of time (over one year) once they grow in three-dimensional (3D) cultures as so-called organoids, at least for mouse livers (Huch et al. 2013). The culture of canine 3D liver organoids has not been described yet. 5.2.

Mesenchymal stem cells

In contrast to the bi-potent HPCs within the liver, mesenchymal stem cells (MSCs, or mesenchymal stromal cells) are extrahepatic multi-potent stem cells, with a strong anti-inflammatory capacity. As such they are potentially relevant stem cells for treatment of liver diseases where inflammation is abundant. These cells can be acquired from, amongst other sources, bone marrow, adipose tissue, yolk sac, umbilical cord or amniotic fluid (Seo et al. 2009; Zucconi et al. 2010; Filioli Uranio et al. 2011; Wenceslau et al. 2011; Park et al. 2012; Chio et al. 2013). Which origin suits best for the various potential applications is still a matter of debate. As for HPCs, no reports on the clinical application in canine hepatitis are published yet. Marker expression and differentiation of canine MSCs has been described in several reports. But importantly, fully mature hepatocytic differentiation into cytochrome P450-expressing cells is not yet achieved for MSCs (Chio et al. 2013). A latest discovery in the MSC field are the liver-specific MSCs (LMSCs), cultured from human liver grafts (Pan et al. 2011). These cells are believed to have hepatocyte differentiation potential in addition to their immunomodulatory effects. This renders them potentially more interesting for use in regenerative strategies. It remains to be seen whether such cells are a peculiarity of human liver transplants or whether LMSCs are also present in dog livers. 5.3. Induced pluripotent stem cells A major breakthrough came in August 2006, when Yamanaka’s group showed the production of induced pluripotent stem (iPS) cells (Takahashi & Yamanaka 2006). By lentiviral introduction of four transcription factors into adult fully differentiated mouse skin fibroblasts, stem cells were created with the ability to form all cell types of the body. In this way, the use of embryonic stem cells was circumvented and therewith the ethical hurdles related to it. This approach was awarded the Nobel Prize in 2012. Within one year human iPS cells were created, and slightly later the first reports on canine iPS cells were published (Luo et al. 2011; Whitworth et al. 2012; Koh et al. 2013). iPS cells hold mainly potential for disease

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modelling purposes, and also for cell transplantation goals. Although the conversion from a canine skin fibroblast into a pluripotent cell seems feasible, differentiation into a fully active hepatocyte has not yet been achieved, a situation similar to the MSC-derived hepatocyte-like cells (Haraguchi et al. 2012).

6. Potential for stem cell therapy/transplantation The aforementioned stem cells can be considered a source for cell therapy or could be used for disease modelling purposes. Adult liver stem cells or HPCs, especially when cultured in the form of highly proliferative organoids, are an interesting source for cell transplantation, e.g. during acute liver disease or in the treatment of metabolic diseases such as copper storage deficiencies. The use of autologous cells is preferred in view of the lack of nonself rejection. Gene therapy could be applied ex vivo before cells are expanded and transplanted back into the patient, potentially upon differentiation to hepatocytes (Figure 2). Genetically healthy cells will have growth advantage in the metabolically diseased liver and will enhance the success of engraftment and repopulation of the liver upon transplantation. One other and potentially most elegant option would be to stimulate the resident adult stem cells of the liver to repopulate the liver during

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disease. Depending on the type of disease the focus should be on an enhancement of proliferation, such as during acute fulminant hepatitis, or on induction of differentiation. For example, chronic hepatitis or even more so lobular dissecting hepatitis cases show sufficient HPC proliferation but lack adequate differentiation to hepatocytes to restore liver function. When designing therapies to stimulate the endogenous HPC compartment it is important to improve tissue homeostasis but prevent reactions of cell types that could further deteriorate liver integrity and function. Pathways that in the liver exclusively activate HPCs should ideally be targeted. Growth factors such as TNF-related weak inducer of apoptosis (TWEAK) have been described to regulate the number of HPCs specifically (Jakubowski et al. 2005; Tirnitz-Parker et al. 2013). These growth factors have not found their way to the clinics yet. We believe that the ongoing research in this area, in both the human and veterinary field, will yield compounds able to activate the endogenous stem cells of the liver for clinical purposes. A recent and highly interesting development is the use of HPCs in tissue engineering. Liver tissue engineering is based on bioreactor systems (i.e. biocompatible scaffolds) providing the most optimal environment for cell growth and differentiation. These systems can be applied either in vitro (drug toxicity screening or liver support devices) or in vivo (implantable

Figure 2. Possible treatment modalities for hepatic progenitor cells (HPCs). HPCs can be isolated from the canine liver and cultured as organoids. After ex vivo expansion these cells can be differentiated into hepatocyte-like cells for cell transplantation in dogs with liver disease or disease modelling purposes. In case of autologous cell therapy, inherited gene defects causing metabolic disease can be corrected by gene therapy. Stem cell therapy can also be used to stimulate the endogenous HPCs in vivo to aid in the regeneration of a diseased liver. HPCs, alone or with other non-parenchymal liver cells, can also be used in tissue engineering where three-dimensional bioreactor systems are used in vitro (drug toxicity screening or liver support devices) or in vivo (implantable devices). Dotted lines indicate less likely possibilities.

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devices) (Ananthanarayanan et al. 2011). Since 2D static culture conditions poorly recapitulate the in vivo cellular environment, 3D bioreactor systems are under development (Palakkan et al. 2013). HPCs are an ideal cell source for these bioreactor systems since adult stem cells do not have the risk of teratoma formation and their use avoids many ethical issues compared with embryonic stem cells. Moreover, these systems allow a co-culture with nonparenchymal liver cells which greatly mimics the cellular make-up of the liver. This aids in the functionality of the bioreactor systems (Nahmias et al. 2006). In addition to HPCs, MSCs are considered highly effective for treatment of liver disease (Figure 3). MSCs are of interest because of their immunomodulatory properties and can therefore be used as a source for allogeneic stem cell transplantation in acute types of liver disease. Although the application of MSCs during chronic types of liver diseases is ill advised due to their potential contribution to ECM producing cells (Baertschiger et al. 2009; Meier et al. 2013), combination therapy with other stem cells has been shown to have great potential (Keramaris et al. 2012). Last but certainly not least is the potential of iPS cells to be used in the treatment of canine liver disease (Figure 4). The current state of iPS cell research renders them mainly beneficial for disease modelling purposes, to contribute to the development of new, safer and better drugs to treat liver disease. When the technique of acquiring iPS cells has been proven safe and effective, tissue

engineering and transplantation of iPS cells to repopulate the liver are also viable options for regenerative hepatology. In summary, given the various advantages and shortcomings of each type of stem cell, different liver diseases might benefit from a different type of stem cell.

7. Challenges and pitfalls for stem cell therapy/ transplantation The presence and differentiation potential of HPCs gave rise to high expectations regarding their application in a regenerative setting. There is, however, a possible downside to the HPCs and other stem cells (Harding et al. 2013). The first identification of these cells was in a precancerous environment (Kinosita 1937), and some of the experimental regimens used ultimately led to tumour formation (Hacker et al. 1992; Knight et al. 2000). In addition, human hepatocellular and cholangiocellular tumours are thought to have, at least in part, their origin in the HPC (Libbrecht & Roskams 2002; Shachaf et al. 2004; Durnez et al. 2006; Lee et al. 2006; Komuta et al. 2008). In fact, a recent publication indicated that 21% of canine hepatocellular tumours of the dog exhibited HPC characteristics (van Sprundel et al. 2013). In both man and dog, these HPC characteristics are linked to an increased malignancy (Govaere et al. 2013; van Sprundel et al. 2013). This potential cancerous development of HPCs/ stem cells, especially upon persistent stimulation where the balance of regeneration and cancer development is

Figure 3. Possible treatment modalities for mesenchymal stromal cells (MSCs). Due to the immunomodulatory capacity of MSCs and the lack of allorecognition, allogeneic MSCs can be transplanted in dogs with liver disease where inflammation is abundant. MSCs can be differentiated into hepatocyte-like cells for cell transplantation purposes or disease modelling. Moreover, a co-transplantation with other stem cell types, such as progenitor cells, might increase the chances of a successful engraftment of cells. Dotted lines indicate less likely possibilities.

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Figure 4. Possible treatment modalities for induced pluripotent stem (iPS) cells. iPS cells can be generated by over-expression of the so-called Yamanaka factors (OCT4, KLF4, SOX2 and c-MYC) in isolated (skin) fibroblasts. Due to the possible teratoma formation these cells need to be differentiated before cell therapy with these cells can be applied in dogs with liver disease. These differentiated iPS cells can also be useful for disease modelling. In case of autologous cell therapy, inherited gene defects causing metabolic diseases can be corrected by gene therapy. Dotted lines indicate less likely possibilities.

challenged, must be taken into serious consideration when their therapeutic possibilities in regenerative medicine are further exploited. This does not only account for the application of stem cells in canine hepatology, but also, and maybe even more so, for the application of putative MSCs in other veterinary applications. Another important consideration in the design of regenerative therapies is the cause of disease and the pathological state of the liver at the time of treatment. Cell therapy may not be curative if the cause of the insult is not known or treatable. In addition, active inflammation and fibrosis may need to be resolved to obtain long-term success of regenerative treatments. The combined transplantation of immunomodulatory cells and HPCs with potential to repopulate the liver, could be highly interesting from this point of view (Leveille & Arias 1993). A practical hurdle in stem cell therapy is the very low engraftment and hepatocyte repopulation efficiency of transplanted stem cells, as displayed by many rodent studies to date (Dorrell et al. 2011; Huch et al. 2013). Although the route of injection will be more direct in larger animals, such as the dog, the key to success of the treatment may be to optimize both liver and cell conditions before transplantation. Many advances have been made in the last year to improve the engraftment of cells after injection. One of the methods in use today in the human field is the irradiation of (a part of) the liver. In this way the regenerative response of the liver is highly

activated due to the induction of hepatocyte senescence. This has shown to provide a beneficial environment to transplanted cells and increase their engraftment (Krause et al. 2011; Zhou et al. 2012). The current golden standard of cell therapy for liver diseases in man is hepatocyte transplantation derived from non-heart-beating donors (Jorns et al. 2012). An important question is whether a further degree of differentiation of the cells towards hepatocytes is beneficial for success of stem cell transplantation. If this is the case, and that may depend on the type of liver disease, efforts are needed to improve differentiation techniques to obtain fully mature hepatocytes in culture. Classical approaches of hepatocyte maturation include adapting media components, co-cultures or the addition of ECM components to the differentiation media (Fraczek et al. 2013). More innovative approaches of hepatocyte maturation are the over-expression of liver transcription factors or fine-tuning of the epigenetic state of differentiating cells (Fraczek et al. 2013). These strategies aim to generate long-term fully functional hepatocytes in vitro. Currently, however, none of these approaches have been successful in reproducing the entire spectrum of liver functions in vitro. This said, it could be expected that transplanted cells will differentiate into fully maturated cells when they get into contact with liver-specific cells composing their natural niche. The fact that signalling from non-parenchymal cells in the liver is important in HPC biology is corroborated by a recent publication

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where a ‘liver bud’ was created by co-culturing endothelial cells, together with a mesenchymal cell type, and hepatocytes derived from stem cells (Takebe et al. 2013). These liver buds showed a high degree of self-organization and even blood flow connection when transplanted into the abdomen of mice. In conclusion, it may not be one single stem cell type that will be able to restore liver function. More likely the regenerative crosstalk of a combination of different cell types, which may be specific for the various types of liver disease, is needed to obtain full liver regeneration.

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8. Epilogue The field of regenerative medicine is rapidly progressing with breakthrough discoveries. Many of these techniques have high potential for application in the field of veterinary medicine. When considering stem cell transplantation and/or stem cell therapy for canine liver disease, the presence and state of the different cell types in the liver are important. They form the direct environment for clinically applied stem cells, and influence the success or failure of the treatment. Since this environment will be dissimilar in different types of liver diseases it is expected that different approaches will be needed for the different diseases. Ultimately it remains to be seen whether these novel regenerative medicine techniques will be able to reverse more advanced stages of hepatitis such as cirrhosis. But the clinical application of stem cells in acute and metabolic liver diseases is nearby. The liver evidently lacks behind the application of stem cells in veterinary orthopaedics. On the long run it will be clear whether the step-by-step approach as taken in liver stem cell research will be clinically highly beneficial. Acknowledgements Part of this work was supported by the Netherlands Research Council under grant numbers 92003538 and 116004121.

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