Therapeutic implications of mesenchymal stem cells in liver injury

Hindawi Publishing Corporation Journal of Biomedicine and Biotechnology Volume 2011, Article ID 860578, 8 pages doi:10.1155/2011/860578 Review Article Therapeutic Implications of Mesenchymal Stem Cells in Liver Injury Maria Ausiliatrice Puglisi,1 Valentina Tesori,1 Wanda Lattanzi,2 Anna Chiara Piscaglia,1 Giovanni Battista Gasbarrini,3 DomenicoM. D’Ugo,4 and Antonio Gasbarrini1 1 GI & Liver Stem Cell Research Group (GILSteR), Department of Internal Medicine and Gastroenterology, Gemelli Hospital, Largo A. Gemelli 8, 00168 Rome, Italy 2 Institute of Anatomy and Cell Biology, Catholic Univeristy of the Sacred Heart, Largo F. Vito 1, 00168 Rome, Italy 3 Medical Research Foundation ONLUS, Galleria falcone Borsellino 2, Bologna, Italy 4 Department of Surgical Sciences, Gemelli Hospital, Largo A. Gemelli 8, 00168 Rome, Italy Correspondence should be addressed to Maria Ausiliatrice Puglisi, ausiliapuglisi@yahoo.it Received 15 July 2011; Revised 17 October 2011; Accepted 17 October 2011 Academic Editor: Ken-ichi Isobe Copyright © 2011 Maria Ausiliatrice Puglisi et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Mesenchymal stem cells (MSCs), represent an attractive tool for the establishment of a successful stem-cell-based therapy of liver diseases. A number of different mechanisms contribute to the therapeutic effects exerted byMSCs, since these cells can differentiate into functional hepatic cells and can also produce a series of growth factors and cytokines able to suppress inflammatory responses, reduce hepatocyte apoptosis, regress liver fibrosis, and enhance hepatocyte functionality. To date, the infusion of MSCs or MSC- conditioned medium has shown encouraging results in the treatment of fulminant hepatic failure and in end-stage liver disease in experimental settings. However, some issues under debate hamper the use of MSCs in clinical trials. This paper summarizes the biological relevance of MSCs and the potential benefits and risks that can result from translating theMSC research to the treatment of liver diseases. 1. Introduction The liver has a remarkable regenerative capacity in response to acute injury. Mature hepatocytes can reenter the cell cycle and undergo several cell divisions to restore the hepatic mass. However, following chronic liver damage, the regenerative ability of hepatocytes is lost. In such conditions, the liver is unable to maintain its functional mass; this is clinically mirrored by the so-called “liver failure.” Currently, orthotopic liver transplantation (OLT) represents the most suitable therapeutic option for patients with advanced liver diseases and hepatic failure. Nevertheless, only a minority of candidates undergo OLT, given the organ shortage. Hence, alternative strategies for the treatment of decompensated liver diseases are needed to be developed [1]. Cell-based therapy has been proposed as a potential alternative to OLT. Indeed, it has been known for more than 30 years that hepatocytes isolated from a donor liver and infused intraportally in animal models of liver damage can be engrafted into the recipient hepatic parenchyma and express metabolic activity. These results have encouraged clinical trials using hepatocytes transplantation to treat a variety of liver diseases [2]. The best outcome of allogeneic hepatocytes transplantation was reported for the treatment of acute liver failure, in which hepatocytes infusion provides the rapid metabolism of liver toxins and the stabilization of hemodynamic parameters. However, transplantation of liver cells provides serious practical problems: donor scarcity, risk of rejection, low hepatocyte viability (only 30% of hepatocytes survive transplantation) and inability maintain and amplify cell cultures [3, 4]. Given this background, a growing enthusiasm has greeted the development of stem-cell-based therapies for liver diseases. In particular, transplantation of hematopoietic bone marrow (BM) stem cells and mesenchymal stem cells (MSCs) has been extensively investigated as potential sources for liver regeneration. In 1999, Petersen et al. first showed that liver stem cells might be derived from BM, in a rat model of liver injury [5], and it was suggested that BM could contribute to the mature 2 Journal of Biomedicine and Biotechnology hepatocyte population. Subsequent studies have shown that BM-derived hepatocytes might arise from cell fusion and not only by direct differentiation [6] and that BM cells give a limited contribution to the hepatocyte population, under physiological conditions or in response to mild injury [7]. MSCs represent another promising candidate for liver stem cell therapy. Several studies have demonstrated that MSCs can differentiate in vitro along the hepatogenic lineage [8, 9]. To date, studies on animal models reported the benefi- cial effect of MSCs in promoting hepatic tissue regeneration. Kuo et al. have shown that both MSC-derived hepatocytes andMSCs, transplanted by either intrasplenic or intravenous route, can be engrafted into the recipient liver and differenti- ate into functional hepatocytes. Intravenous transplantation was more effective in rescuing liver failure than intrasplenic transplantation. Moreover, MSCs were more resistant to reactive oxygen species in vitro, reduced oxidative stress in recipient mice, and accelerated repopulation of hepatocytes after liver damage, suggesting a possible role for paracrine effects [10]. These results have been confirmed also by Banas et al., who evaluated the therapeutic potential of MSCs for the treatment of liver failure and postulated that the beneficial effects of human MSC transplantation were due at least in part to the cells’ ability to produce a large number and volume of bioactive factors [11]. To date, only a few clinical trials have been performed in patients with end- stage liver disease caused by hepatitis B, hepatitis C, and alcoholic fibrosis. The results of these studies have shown that MSC injection can be used for the treatment of end- stage liver diseases, with satisfactory tolerability and clinically relevant effects [12]. Nonetheless, these studies have not provided definitive evidence that MSCs have a capability to differentiate into functional hepatocytes in vivo [13], because the observed improvements could be attributed to the secretion of soluble growth factors by MSCs, rather than to their transdifferentiation into hepatocytes [7]. MSC cells have also emerged as promising candidate cells for immunomodulation therapy, especially in the setting of liver transplantation, given their ability to interact at various levels with the immune system [14, 15]. Overall, a number of different mechanisms contribute to the therapeutic effects exerted by MSCs, which can differentiate into functional hepatic cells and also produce a series of growth factors and cytokines that can sup- press inflammatory responses, reduce hepatocytes apoptosis, regress liver fibrosis, and enhance hepatocytes functionality [16]. 2. MSC Properties MSCs were first described by Friedenstein in the early 1990s, as an adherent, fibroblastoid cell population that showed inherent osteogenic properties [17]. Numerous studies have demonstrated that MSCs have a high degree of plasticity, as they differentiate into cells of the mesenchymal lineage, but they can also transdifferentiate into neurons, splenocytes, and various epithelial cells, including lung, liver, intestine, and kidney cells. BM was originally considered the reference source for MSC isolation, although they have been isolated from a multitude of adult tissues, including muscle, adipose tissue, connective tissue, trabecular bone, synovial fluid, along with perinatal tissues, such as umbilical cord, amniotic fluid, and placenta [18]. In particular, adipose tissue (AT) has several advantages compared to other adult tissues as a source of MSCs. Indeed, AT is abundant and can be easily removed by simple lipoaspirate. Moreover, adipose- tissue-derived MSCs (AT-MSCs) can be maintained longer in culture and possess a higher proliferation capacity than BM-derived MSCs. Thus, AT may be an ideal source of large numbers of autologous stem cells [19]. MSCs do not express the hematopoietic surface markers CD34 and CD45, but stain positive for CD44, CD29, CD105, CD73, and CD166 [20]. Moreover, MSCs express human leukocyte antigen (HLA) class I, but not HLA class II, and secrete several extracellular matrix (ECM) molecules, such as collagen, fibronectin, laminin, and proteoglycans. For this reason it has been postulated that MSCs might play a central role in ECM organization. We performed a high- throughput molecular analysis of BM- and AT-MSCs. The gene expression profile analysis has revealed that they share 190 coherentlymodulated transcripts, whichmight represent the molecular “MSC stemness signature.” Among them, we found several genes involved in basic biologic mechanisms, such as embryogenesis, organogenesis, signal transduction, cell adhesion, stress response, and transcription regulation. In particular, a key role in determining the outcome of MSC fate determination is played by KLF4, highlighting the specific binding of KLF4 to regulatory sequences of genes involved in adult stem cell maintenance [19]. BM-derivedMSCs are known to naturally support hema- topoiesis by secreting a number of trophic molecules, includ- ing soluble extracellular matrix glycoproteins, cytokines, and growth factors [21, 22]. Recent studies have demonstrated that MSCs can produce some antiapoptotic cytokines such as stromal-cell-derived factor-1 and vascular endothelial growth factor, which efficiently reduce the apoptosis of recip- ient cells via the stromal cell-derived factor-1/CX chemokine receptor-4 axis. The antiapoptotic effects of MSCs have been observed in liver injury models [23–26]. Furthermore, MSCs can secrete several cytokines such as hepatocyte growth factor (HGF), epidermal growth factor, IL-6, and TNF-α; in turn, these cytokines stimulate hepatocyte proliferation and maintain hepatocyte function, as indicated by the high levels of albumin and urea secretion granted upon MSC transplantation [27]. Finally, MSCs can produce a series of cytokines and signal molecules that can potentially suppress inflammatory responses such as IL-1 receptor antagonists and can upregulate anti-inflammatory cytokines such as IL- 10 [25]. 3. MSC Plasticity Given their wide differentiation potential and their self- renewal capacity, MSCs have been considered a promising candidate for cell-based therapy and tissue engineering. Moreover, these cells have the ability to proliferate to an extensive but finite degree, an important characteristic that Journal of Biomedicine and Biotechnology 3 should reduce concerns about potential tumorigenicity upon in vivo transplantation. The high degree of plasticity of MSCs has been widely demonstrated during the last decade [28–31]. In particular, in vitro models, using culture medium supplemented with a cocktail of growth factors, were used to successfully induce the transdifferentiation of MSCs into hepatic cells with functional properties, such as the production of albumin and urea, along with glycogen storage [32]. Moreover, the in vivo transdifferentiation of MSCs into hepatic cells has been described in rats [33], mice [34], and humans [35]. Seo et al. first reported that human AT-MSCs injected into SCID mice, following toxic liver damage, were able to differentiate into hepatocyte-like cells [36]. Several reports have confirmed the possibility of generating hepatocyte-like cells from AT-MSCs [37, 38]. In particular, in a xenogeneic transplantation model of liver regeneration, the engraftment of AT-MSCs predifferentiated in vitro to hepatocyte-like cells was significantly more efficient versus undifferentiated AT- MSCs, and AT-MSCs were better candidates than BM-MSCs for cell therapies [39]. We confirmed that AT-MSCs can transdifferentiate in vitro into hepatocyte-like cells, using a two-step protocol with sequential addition of growth factors. Under this regimen, spindle-shaped fibroblastoid cells differentiated to a layer of compact polygonal epithelial cells. These cells acquired specific liver functions, as shown by their ability to store glycogen and to express hepatic-associated genes and proteins. Moreover, the comparative high-throughput molecular analysis of AT-MSCs, before and after hepato- genic conversion, allowed the identification of a complex interplay between cell receptors, signaling pathways, and transcription factors, responsible for tissue cross-lineage conversion through the mesenchymal-epithelial transition (MET). Our study showed that the AT-MSC plasticity is dependent on MET and suggested that subtle regulations of the canonical pathways of BMP, WNT, and TGF-β may be important to allow MSCs to transdifferentiate into other lineages [40]. The pivotal role that MET plays in determining AT- MSCs transdifferentiation in hepatocytes was also confirmed in an interesting article by Yamamoto and colleagues [41]. The authors compared the transcriptomes of three cell populations, undifferentiated AT-MSCs, AT-MSC-derived hepatocytes (AT-MSC-Hepa) and human primary hepato- cytes, and human liver tissue, using microarray analysis. The results indicated that AT-MSC-Hepa and hepatocytes displayed a similar gene expression profile, while undif- ferentiated AT-MSCs showed a different pattern. The list of genes upregulated in AT-MSC-Hepa, liver cells, and tissue comprised, in particular, genes encoding hepatocyte- specific metabolic enzymes and markers [41]. Interestingly, the microarray data indicated the downregulation of two regulators of the epithelial-mesenchymal transition (EMT), Twist and Snail, along with the upregulation of epithelial markers, such as E-cadherin and a-catenin, in AT-MSC- Hepa. In contrast, the expression of mesenchymal markers, such as N-cadherin and vimentin, was downregulated. These findings support the notion that MET is activated during the hepatic differentiation of AT-MSCs, representing a pivotal step for stem cell transdifferentiation [41]. 4. MSCs and Immune System MSCs express fewHLA class I and no HLA class II molecules, allowing them to evade allogeneic immune response. This is the so-called “immunoprivilege,” an interesting feature in MSC biology, which makes these cells extremely suitable for both autologous and allogeneic transplantation [42]. Moreover, several studies have established that MSCs exert a generally suppressive effect on a wide variety of cells belonging to both adaptive and innate immunity, including T and B lymphocytes and natural killer cells (NKs). This immunomodulatory effect provides a rational basis for the application of MSCs in the treatment of immune-mediated diseases, such as graft-versus-host disease (GVHD). To date, the mechanisms underlying this immunoregulation remain unclear: some investigators suggested a cell-to-cell contact- mediated suppression, while others hypothesized a soluble- factor-mediated mechanism [43]. MSCs can suppress the activity of CD8+ cytotoxic T lymphocytes both directly by inhibiting their proliferation following antigen stimulation and indirectly by increasing the relative proportion of CD4+ T helper-2 (TH2) lym- phocytes and CD4+ regulatory T lymphocytes [44]. Since B-lymphocyte activation is largely T cell dependent, the influence of MSCs on T lymphocytes may also indirectly suppress B-cell functions [45]. Additionally, MSCs exert a direct influence on B-lymphocytes via cell-cell contact and through secretion of paracrine molecules [46]. MSCs exert significant effects on the innate immune system cells, including monocytes, dendritic cells (DCs), macrophages, NKs, and neutrophils. The mechanisms by which MSCs exert their inhibitory effect on DC maturation is still poorly defined. Spaggiari et al. have shown in vitro that MSCs inhibit the early stages of the progression from monocytes to immature DCs, induced by interleukin-4 (IL- 4) and granulocyte-macrophage colony-stimulating factor (GM-CSF). The authors have shown that different soluble factors mediate the inhibitory effect exerted by MSCs, and they provided a convincing evidence of the pivotal role of prostaglandin E2 (PGE2) [47]. MSCs have a profound inhibitory effect on NK function, suppressing the IL-2- induced cell proliferation, their cytolytic activity, and the production of cytokines. MSCs can inhibit NK-cell function via the production of soluble factors, including indoleamine 2,3-dioxygenase (IDO) and PGE2 [48]. Lastly, an in vitro study demonstrated that MSCs inhibit apoptosis, expression of adhesion molecules, and migration capability of neu- trophils. These results are consistent with the hypothesis that, within the BMniche,MSCs protect neutrophils of the storage pool from apoptosis, preserving their effector functions. Moreover, MSCs reduce intensity of the respiratory burst preventing the excessive or inappropriate activation of the oxidative metabolism. This may be a critical mechanism through which MSCs can limit the severity of tissue damage following ischemic and ischemia/reperfusion (I/R) injury [49]. 4 Journal of Biomedicine and Biotechnology 5. Therapeutic Implications of MSC-Based Treatments of Liver Diseases The therapeutic potentialities ofMSCs are also based on their inherent ability to home in sites of inflammation following tissue injury when injected intravenously. This involves their capability of migrating across endothelial cell layers and being attracted to and retained in the ischemic tissue but not in the remote or intact tissue. Although the mechanisms driving this property are not fully understood, it is likely that injured tissues express specific receptors or ligands that facilitate trafficking, adhesion, and infiltration of MSCs to the damaged site, similarly to leukocytes [50, 51]. It is well known that chemokines are released after tissue damage and that migratory direction follows the chemokine density gradient. In this regard, it has been recently demonstrated that MSCs express chemokine receptors and ligands that are involved in leukocyte migration during inflammation, including the stromal-derived factor-1 (SDF-1) chemokine receptor (chemokine (C-X-Cmotif) receptor 4, CXCR4) that stimulates the recruitment of progenitor cells to the site of tissue injury [52–55]. MSCs also express several adhesion molecules that respond to SDF-1, as well as chemokines, such as CX3CL1, CXCL16, CCL3, CCL19, and CCL21 [56–58]. Hence, the increase of inflammatory chemokine concentration at the site of inflammation is a key mediator of MSC trafficking to the site of injury [52]. In addition, many integrins, selectins, and chemokine receptors involved in the tethering, rolling, adhesion, and transmigration of leukocytes have also been reported to be expressed on MSCs. In particular, E- and P-selectin, CD44, and VCAM-1, which function in leukocyte adhesion, have been shown to be functionally important in the adhesion of MSCs to the endothelium [59–61]. The therapeutic role of MSCs has been investigated us- ing either autologous or allogeneic transplantation of cells, which were previously expanded in culture and then intro- duced intravenously or directly into the tissue of interest. To date, infusion of MSCs has shown encouraging results in the treatment of several immune- and inflammatory-mediated conditions including GVHD, diabetes, and ulcerative colitis and in the protection of solid organ grafts from rejection [62]. Recent experimental studies have shown the successful application of MSC transplantation in the treatment of fulminant hepatic failure (FHF), end-stage liver disease (ESLD), and inherited metabolic disorders (IMDs). These studies have shown that MSC tran