The transplantation of primary hepatocytes represents a potential cell-based therapy for liver diseases, including metabolic disorders and acute liver failure [5]. However, human hepatocytes have been shown to be unsuitable as the availability of human livers as a source of cells is limited, and it is difficult to maintain hepatocyte viability and function during cryopreservation of the liver. In addition, hepatocytes are difficult to culture in vitro [6]. Moreover, while the safe transplantation of -5% of the total recipient liver cell mass (a maximum of 1-2×10⁸ hepatocytes/body weight [kg]) has been demonstrated in human clinical trials [7], it has been reported that replacement of at least 10% of the hepatocellular mass is needed to compensate for missing or inactive proteins in genetic inborn errors of metabolism [8].

The infusion of unsorted BMCs in patients with cirrhosis has been reported to be safe and feasible. Moreover, the infusion of unsorted BMCs increases the serum albumin level and improves liver function [9]. However, although it has been reported that the infusion of unsorted BMCs elevates the levels of matrix metalloproteinases MMP-2, MMP-9, and MMP-14, reduces liver fibrosis, and improves survival rate [10], the therapeutic mechanism by which BMC infusion ameliorates liver damage remains unclear.

Hematopoietic stem cells from the bone marrow or peripheral blood, obtained by administration of G-CSF, which induces the mobilization of CD34+ cells into the peripheral blood, can be isolated, amplified, and differentiated into hepatocyte-like cells [11]. The infusion of hematopoietic stem cells improves serum albumin levels as well as Child-Pugh scores [12]. Although infused hematopoietic stem cells can differentiate into hepatocytes, through cell fusion or paracrine effects [13], further studies are necessary to define the role of hematopoietic stem cell therapy in liver disease patients.

MSCs can be isolated by plastic adherence from adipose tissue, brain, dermis, liver, lung, peripheral blood, skeletal muscle, umbilical cords, and cord blood [1415]. MSCs are potentially useful therapeutic agents for the treatment of liver diseases, because of their potential to differentiate into hepatocytes, as well as their immunomodulatory properties and ability to secrete trophic factors. These properties, putative therapeutic mechanisms, and injection routes of MSCs are discussed in detail in a later section.

ESCs, which are isolated from the inner cell mass of blastocyst-stage embryos, have pluripotent differentiation potentials, including the ability to differentiate into hepatocyte-like cells. However, although ESCs have been shown to improve liver function in CCl4-treated mice [3], several issues are associated with their use, such as teratoma formation, ethical issues, and immune rejection problems [16].

The use of iPSCs, produced from patient-specific cells, allows ethical and immune rejection-related issues to be circumvented. IPSCs have been shown to differentiate into hepatocyte-like cells, and human iPSC-derived hepatocyte-like cells, from different origins, have been demonstrated to successfully repopulate liver tissue in mice with liver cirrhosis [4]. Although Liu H and colleagues reported that tumor formation was not observed in any organs of the transplanted mice, up to 7 months after transplantation of iPSC-derived hepatocyte-like cells [4], tumor formation must be carefully monitored, as an increased risk of teratoma formation is associated with the transplantation of these cells.

In this review, we focus only on MSCs, as these cells can be obtained in sufficient numbers for investigation in clinical studies, without ethical or immune rejection-related issues. We summarize the properties of MSCs, their therapeutic mechanisms of action, and injection routes, with the aim of providing an improved understanding of the potential of MSCs in regenerative medicine, including the treatment of liver disease.

MSCs exposed to growth factors and cytokines (i.e. hepatocyte growth factor [HGF], fibroblast growth factor [FGF]-2/-4, epidermal growth factor [EGF], oncostatin M [OSM], and/or leukemia inhibitory factor [LIF]), and chemical compounds (i.e. dexamethasone [Dex], insulin-transferrin-selenium [ITS], and/or nicotinamide [NTA]) can differentiate into hepatocyte-like cells, which express HNF-3, GATA4, CK19, transthyretin, -fetoprotein, albumin, and CK18 [21]. Hepatocyte-like differentiation of MSCs can also be induced by co-culture with liver cells [22] and by pellet culture [23]. Additionally, in vivo hepatic differentiation of MSCs was achieved in allylalcohol (AA)-treated rat livers, by directly xenografting human bone marrow-derived MSCs into these AA-treated rat livers, resulting in most of the human MSCs differentiating into hepatocyte-like cells without fusion, as revealed by positive immunostaining for human specific AFP, albumin, CK19, CK18, and receptor [24]. Although hepatic-differentiated MSCs exhibit hepatocyte-specific cuboidal morphology and express hepatocyte marker genes in vitro and in vivo, it is still debatable as to whether transplanted MSCs can completely regenerate hepatocytes in vivo. As trans-differentiation of MSCs into hepatocytes is rarely observ-ed (less than 1% of the total liver mass) in animal models, recently published data suggest that the therapeutic potential of MSCs in the treatment of CLDs would be primarily based on their ability to secrete trophic factors, and their immunosuppressive effects, rather than trans-differentiation into hepatocytes [2526].

MSCs migrate towards damaged areas in response to homing signals. Many studies have shown that MSCs are able to migrate to injured tissues from blood, and exert therapeutic effects at these sites of injury [27]. Various factors, such as chemokines, cytokines, and growth factors, released upon injury, play roles as migratory cues for systemically or locally transplanted MSCs [28]. Specific receptors and ligands that are upregulated in injured tissues facilitate trafficking, adhesion, and infiltration of MSCs at sites of injury [27]. Moreover, these tissues provide MSCs with the appropriate microenvironment for self-renewal and maintenance of multi-potentiality [27]. Integrins, selectins, and chemokine receptors [29] expressed on MSCs play key roles in their migration across the endothelium. In addition, MSCs are passively arrested in capillaries or microvessels, including arterioles and post-capillary venules, where they interact directly with accessory cells and release a wide array of soluble growth factors and trophic cytokines [30].

MSCs can suppress immune cells, such as B-cells, T-cells, dendritic cells (DCs), and natural killer cells (NKs) and promote the generation of regulatory T (Tregs) cells through the production of soluble factors such as nitric oxide (NO), prostaglandin (PGE2), indoleamine 2,3-dioxygenase (IDO), IL-6, IL-10, and human leukocyte antigen (HLA)-G [31]. The production of NO by inducible nitric-oxide synthase (iNOS) in murine MSCs is suggested to play a major role in T-cell proliferation inhibition [32]. PGE2 acts as a powerful immunosuppressant, modulating production of IL-10, proliferation, and differentiation of T-cells, macrophages, and mo-nocytes, respectively [3334]. Moreover, IDO and HLA-G suppress the proliferation of B- and effector T-cells, inhibit the maturation of DCs, and reduce the cytotoxicity of natural killer (NK) cells.

In addition to immunosuppression mediated by the secretion of soluble factors, MSCs can make direct contact with immune cells, thereby suppressing their activation. MSCs can inhibit T-cell proliferation by inducing apoptosis of effector T-cells, through engagement of programmed death 1 (PD-1) protein to its ligands. The complexes, PD-L1 and PD-L2, thus formed are able to render T-cells anergic by downregulating the expression of co-stimulatory molecules CD80 and CD86 on antigen presenting cells [35].

The immune-privileged status of the liver can be disrupted by unbalanced immune cell populations, or immune cell infiltration into the liver, resulting in liver injury. Therefore, the immunosuppressive potential of MSCs plays an important role in the treatment of CLDs.

Various trophic factors can play important therapeutic roles in regenerative medicine. MSCs express various trophic factors, such as growth factors (i.e. brain-derived neurotrophic factor [BDNF], EGF, FGF-2, FGF-4, FGF-7, FGF-9, and FGF-17, glial cell-derived neurotrophic factor [GDNF], HGF, insulin-like growth factor [IGF]-1, nerve growth factor [NGF], platelet-derived growth factor [PD-GF], and vascular endothelial growth factor [VEGF]), cytokines (i.e IFN-γ, IL-1α, IL-1β, IL-2, IL-6, IL-8, IL-10, IL-12, and IL-13, TNF-α, and transforming growth factor [TGF]-β), and chemokines (i.e. various C-C motif ligand [CCLs], and C-X-C motif ligand [CXCLs]), which are known to not only reduce inflammation, apoptosis, and fibrosis of damaged tissues, but also stimulate angiogenesis and regeneration of injured cells [36]. These trophic factors, which are stimulated by local signals from damaged sites, such as inflammatory cytokines, ligands of Toll-like receptors, and hypoxia, modify the local environment in the liver [37], and facilitate the survival of living and dying hepatocytes via anti-apoptotic factors (i.e. stromal cell-derived factor [SDF]-1, HGF, IGF-1, and VEGF), mitogens (i.e. EGF, HGF, NGF, and TGF-α), and angiogenic factors (i.e. VEGF) [38].

After damage of healthy tissue, the repair process started to replace dead and damaged cells and then lead to substantial remodeling of the extracellular matrix (ECM), known as fibrosis. During liver fibrosis, HSCs are responsible for deposition of ECM proteins in liver. The anti-fibrotic activities of MSCs have been reported in various fibrotic animal models including heart, liver, kidneys, lungs, peritoneum, pancreas, skin, and rectum. The molecular mechanism underlying the anti-fibrotic properties of MSCs can mainly reside in the high expression levels of matrix metalloproteinases (MMPs), especially MMP-9, which may directly degrade the extracellular matrix and lead to apoptosis of HSCs [39]. In several fibrosis models, MSCs have been shown to increase the expression of MMPs (i.e., MMP-2, MMP-9, MMP-13, and MMP-14) [40] or to decrease tissue inhibitors of MMPs (TIMP)-1 expression [41], and these alterations are generally associated with resolution of fibrosis. Moreover, MSCs can alleviate the chronic inflammation via their immunosuppressive properties and then diminish tissue fibrosis. MSCs can also suppress the proliferation of activated HSCs and collagen synthesis through indirect or direct cell-cell contact models [42].

Excessive reactive oxygen species (ROS) induces liver diseases such as liver fibrosis, cirrhosis, viral hepatitis, hepatocellular carcinoma (HCC) and others through oxidative disease [43]. Carbon tetrachloride (CCl4) and thioacetamide (TAA) are toxins used to generate liver injury in animal models [44]. These toxins stimulate ROS production, which result in hepatocyte damage through lipid peroxidation and the alkylation of proteins, nucleic acids, and lipids [4445]. MSCs have been shown to overcome CCL4- and TAA-induced oxidative stress in vitro and to reduce liver injury through anti-oxidant activities in vivo [44,46]. The upregulation of ROS in CCl4-treated liver cells has been reported to be attenuated by co-culturing with MSCs via an increase in superoxide dismutase (SOD) activity and the induction of AREs, which represents a cytoprotective response in the injured liver [44]. Additionally, MSCs protect hepatocytes by reducing ROS damage that is induced by TAA both in vivo and in vitro [46].