Hepatocyte differentiation of mesenchymal stem cells

Xu-Bo Wu and Ran Tao

Shanghai, China

Hepatocyte differentiation of mesenchymal stem cells

Xu-Bo Wu and Ran Tao

Shanghai, China

BACKGROUND:Liver cell transplantation and bioartificial liver may provide metabolic support of liver function temporary and are prospective treatments for patients with liver failure. Mesenchymal stem cells (MSCs) are expected to be an ideal cell source for transplantation or liver tissue engineering, however the hepatic differentiation of MSCs is still insufficient for clinical application.

DATA SOURCES:A PubMed search on "mesenchymal stem cells", "liver cell" and "hepatocyte differentiation" was performed on the topic, and the relevant articles published in the past ten years were reviewed.

RESULTS:Hepatocyte-like cells differentiated from MSCs are a promising cell source for liver regeneration or tissue engineering. Although it is still a matter of debate as to whether MSC-derived hepatocytes may efficiently repopulate a host liver to provide adequate functional substitution, the majority of animal studies support that MSCs can become key players in liver-directed regenerative medicine. However the clinical application of human stem cells in the treatment of liver diseases is still in its infancy.

CONCLUSIONS:Future studies are required to improve the efficacy and consistency of hepatic differentiation from MSCs. It is necessary to better understand the mechanism to achieve transdifferentiation with high efficiency. More clinical trials are warranted to prove their efficacy in the management of patients with liver failure.

(Hepatobiliary Pancreat Dis Int 2012;11:360-371)

hepatocyte; differentiation; mesenchymal stem cells

Introduction

Avariety of etiologies such as toxic injury, viral infections, autoimmune or genetic disorders may cause severe liver dysfunction resulting in chronic liver disease and/or acute liver failure. Orthotopic liver transplantation remains the last resort for the treatment of acute liver failure and end-stage liver diseases. However, it is not applicable for many desperate patients due to the worldwide shortage of donor organs and high costs; on the other hand, the longterm survival can be impeded by rejection, recurrence of the original diseases and the inevitable side-effects of life-long immunosuppression. Therefore, it is rational to develop alternative approaches for the treatment of liver failure. Hepatocyte transplantation and bioartificial liver are prospective ways for liver failure and can provide metabolic support of liver function temporarily. Unfortunately, limitation of cell sources, immune rejection, short-term viability and rapid phenotypic dedifferentiation of hepatocytes are the major obstacles for clinical application. In the recent two decades, stem cells have attracted lots of attention because of their self-renewal capability and differentiation potential. It gradually becomes a very promising cell source in liver tissue engineering and regeneration.

The bone marrow is the largest reservoir of pluripotent stem cells in adults, and the bone marrow stem cells can give rise to most known adult cell lineages. The bone marrow contains two major stem cell populations: hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs). HSCs can differentiate into all cell types in the blood and are well characterized. On the contrary, many properties of MSCs are not well understood even after 10 years of research. We now realize that MSCs originally isolated from bone marrow can in fact be propagated from almost every tissue in the body, and can be expanded for more than 80 generationsin vitro. These multipotent cells can differentiate into cells of mesenchymal originin vitroandin vivo, such as bone, fat, and cartilage.[1]Furthermore, under someexperiment conditions, these cells can differentiate into a wider variety of cell types. Upon systemic administration,ex vivoexpanded MSCs preferentially home to the damaged tissues and participate in the regenerative process through their diverse biological properties. Besides their tissue differentiation ability, MSCs are capable of providing the stromal supporting scaffold via secretion of crucial cytokines.[2]Given the ease of isolation, expansion as well as their differentiation potential, MSCs may be ideal cell sources for clinical use in a variety of entities. A great deal of interests has been generated for their potential use in regenerative medicine and tissue engineering. In addition, numerous studies suggest that MSCs have low inherent immunogenicity and are capable of modulating/suppressing immunologic responses through interaction with various immune cells. They exert immunomodulatory effects by inhibition ofin vitroT-cell proliferation upon alloantigen/mitogens stimulation and preventing the development of cytotoxic T cells, they also prolong skin allograft survival after adoptive transfer. Possible clinical applications include therapy-resistant severe acute graft-versus-host diseases, tissue repair, treatment of allograft rejection and autoimmune disorders.[3]

Stem cells are involved in liver homeostasis and tissue repair after injury. Minor and transient injury to the liver usually results in mitotic division of hepatocytes. Worse and repeated insult causes activation and proliferation of resident stem cells within the liver, such as oval cells in rodents and liver progenitor cells in humans. Moreover severe and persistent injury results in transdifferentiation (epithelial-to-mesenchymal transition) or engraftment of stem cells within the liver (HSCs and MSCs) as a final attempt to restore liver homeostasis.[4]Thus it's rational to augment this natural process and apply MSCs in transplantation or liver tissue engineering. In 1999, Petersen et al[5]first reported that bone marrow stem cells could develop into hematopoietic lineagein vivo. In the next 10 years, it had been proven that both HSC and MSC populations in the bone marrow can differentiate into hepatocyte-like cells.[6-9]Both mouse and human BMMSCs have been shown to develop into hepatocyte-like cellsin vitroandin vivo.[10-13]The past decade witnessed the vigorous development of technologies to induce differentiation of MSCs into cells that possess liver functions (hepatocyte-like cells).[14]MSCs are derived from somatic cells and carry superiority in terms of ethics and safety in the treatment of liver diseases.[15]Apart from their hepatocyte differentiation potency, MSCs possess pleiotropic features including modulation of immunity, anti-inflammation, anti-apoptosis as well as pro-proliferative impact at the site of liver lesions. They migrate to the liver along chemoattractive gradients and contribute to the humoral and cellular response in tissue repair. Such versatile biological features render MSCs an ideal cell resource for the treatment of a variety of liver entities. In contrast to obtaining hepatocytes from cadaveric donors, they can provide a relatively easy source of stem cells for therapy, also they may obviate the need of life-long use of immunosuppressive medications. Another potential advantage of using these MSCs involves the relative resistance to hepatotropic virus infection.[16]

So far the efficiency of hepatic differentiation of MSCs is still insufficient for clinical application in bioartificial liver or cell transplantation, which might be improved by modifying culture conditions or adding various growth factors/cytokines. It is necessary to better understand the mechanism to achieve transdifferentiation with high efficiency.[1]We hereby review the literatures in English about hepatocyte differentiation of MSCs published in the past 10 years, then discuss the present achievements and prospective development.

Sources of MSCs differentiation into hepatocyte-like cells

MSCs were first discovered and defined in the bone marrow, and they were described as non-hematopoietic, undifferentiated, fibroblast-like and pluripotent progenitor cells. Apart from the bone marrow, MSCs reside in most other organs or tissues such as adipose tissue, cartilage and muscle. MSCs serve as a major source of dormant stem cells for tissue maintenance and regeneration. Although MSCs can be isolated from adipose tissue, peripheral blood, fetal liver, lung, amniotic fluid and umbilical cord blood, the bone marrow-derived MSCs are most prevalently used. Bone marrow is the largest reservoir of MSCs which can differentiate into hepatocyte-like cells. However, different background of BM-MSCs may have varied potential for hepatogenic differentiation and their various phenotypic markers and differentiation characteristics have been extensively studied. For example, Shu et al[17]isolated MSCs and HSCs (Thy-1.1+ Cells) from Sprague-Dawley rat bone marrow and found both types of cells demonstrated hepatic differentiation ability. However, only MSCs could differentiate into hepatocyte-like cells.[17]Baertschiger et al[18]compared the expansion and hepatogenic differentiation capacity between pediatric and adult BM-MSCs, they found the pediatric ones were more competent, however neither pediatric nor adult BM-MSCs expressed hepatocyte markersin vivo.

Adipose tissues are more readily available thanbone marrow. Seo et al[19]used leftover adipose tissues from donors undergoing elective abdominoplasty to isolate adipose tissue-derived stromal cells. They clearly showed that human adipose tissue-derived stromal cells can differentiate into functional hepatocyte-like cells by the treatment of cytokine mixturesin vitroor by transplantation into CCl4-induced liver injury model in nonobese diabetes-SCID mice. Sgodda et al[20]isolated MSCs from rat peritoneal adipose tissue and cultured in the hepatocyte growth medium. After 28 days, cells were strongly positive for glycogen, indicating the gain of hepatocyte-like phenotype. Studies on adipose tissuederived stromal cells differentiation into hepatocyte phenotype may be particularly useful in providing an autologous stem cell pool for liver tissue engineering.[21]

Besides these two common sources, MSCs from other tissues can also be induced into hepatocyte-like cells. In the past decade, human umbilical cord blood (UCB) was found to contain hematopoietic and mesenchymal components. Since BM-derived and UCB-derived MSCs are known to share some common stem cell properties, researchers postulated that UCB-MSCs might exhibit comparable hepatocyte differentiation potential. MSCs isolated from human UCB could differentiate into hepatocyte-like cells after 4 weeks of treatment with hepatocyte growth factor (HGF) plus oncostatin M (OSM)[22]or fibroblast growth factor-4 (FGF-4)[23]in vitro. As well as UCB-MSCs, Ling et al[24]found that MSCs from fetal lung were similar to adult BM-MSCs and could transdifferentiate into hepatocyte-like cells in the presence of HGF, epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF).

It's still a matter of debate as to which stem cell population is most effective in the regeneration of injured liver tissues. Although a pioneer study[5]suggested that HSCs and only the purified KTLS population (c-kithighThylowLin-Sca-1+) could give rise to hepatocytes and rescue the fumarylacetoacetate hydrolase (FAH)-deficient mouse (an animal model of tyrosinemia type 1), subsequent studies had questioned such findings. Another study found that compared with mononuclear cells and HSCs, MSCs show the highest differentiation potential into hepatocytes when co-cultured with injured liver cells.[25]

MSCs differentiation into hepatocyte-like cellsin vitro

In order to induce MSC differentiation into mature hepatocytesin vitro, it is essential to have adequate stimuli for the maintenance of cellular function, such as growth hormones, cytokines, extracellular matrix or co-culture with other cell types. Severalin vitromodels for hepatocyte development have been well established, however the degree of differentiation in these models is generally inadequate. Therefore researchers are keen to explore novel means to facilitate MSC differentiation into hepatocytes.

Hormones and cytokines

Numerous cytokines and growth factors are known to have certain effects on liver cell growth and differentiationin vitro,[26,27]including HGF, OSM, EGF, transforming growth factor (TGF), bFGF, insulin, insulin-like growth factor (IGF) and leukemia inhibitory factor (LIF), etc. In addition, chemical compounds such as dexamethasone (Dex), retinoic acid, sodium butyrate, nicotinamide (NTA), norepinephrine, and dimethylsulfoxide might play roles in promoting hepatic differentiation.

HGF and FGF are linked to endodermal commitment during embryonic development. Originally identified and cloned as a potent mitogen for hepatocytes, HGF has been found to be a pleiotropic cytokine of mesenchymal origin, acting with c-met receptor which is a transmembrane protein possessing an intracellular tyrosine kinase domain. Moreover, HGF plays an essential role in the development and regeneration of the liver, especially in the early stages of hepatogenesis.[28]So far most investigations using growth factors to induce MSC differentiation into hepatocyte-like cells involved HGF. FGF-4 is a mitogen for fibroblasts and endothelial cells. Mouse embryonic stem cells grown in medium containing FGF-4 can differentiate into cells expressing hepatocyte-related genes and antigens.[29]Chivu et al[30]studied the role of cytokines using different combinations and showed that FGF induces hepatic differentiation at the initial stage of endodermal patterning. OSM is an interleukin-6 (IL-6) subfamily member and was originally identified by its ability to inhibit the growth of A375 melanoma cells. OSM is produced by HSCs during the early stages of embryogenesis and plays an important role in the progression of hepatocyte development towards maturation despite the fact that it fails to induce a differentiated hepatocyte-like phenotype by itself.[31]LIF is another IL-6 subfamily member and shares signaling pathways with OSM. Lysy et al[32]observed that LIF can induce BM-MSC differentiation into the hepatic identity, similar to the exposure to OSM.

Chemical compounds such as insulin-transferrinselenium (ITS), NTA, and Dex have all been reported to contribute to the process of hepatocyte differentiation. ITS has proven to be effective in promoting the proliferation and survival of primary hepatocytes. Dex promotes the expression of the hepatocyte phenotypeby suppressing cell division.[30]Sato et al[33]reported that NTA can promote the proliferation of primary hepatocytes, and is important for the appearance of small hepatocyte colonies.

Different cytokines and growth factors may have varied effects on the differentiation of MSCs. On the other hand, MSC-derived hepatocyte-like cells may exhibit various phenotypic characteristics upon culture with different cytokines. Snykers et al[26]used sequential addition of several factors in a time-dependent manner mimicking the secretion pattern during hepatic embryogenesis and showed such a strategy enhances the differentiation efficacy of MSCsin vitro. Chivu et al[30]compared the differentiation efficacy of various liver-specific factors (HGF, ITS, Dex and NTA) used for stem cell differentiation into hepatocyte-like cells either individually or in combination. They found the HGF and NTA have the greatest hepatogenic potential.

Co-culture

Co-culture with hepatocytes or non-parenchymal liver cellsin vitrocan also induce MSCs differentiation into hepatocyte-like cells. Zhang et al[34]co-cultured GFP-transduced MSCs with freshly-isolated rat hepatocytes. Compared to those treated with HGF, such a co-culture system is more efficient in inducing hepatic differentiation. Lange et al[35]cultured GFP-labeled rat MSCs with fetal liver cells (FLCs) and the experiment indicated that such a co-culture system not only provides a favorable environment for hepatocyte differentiation of rat MSCs, but also promotes the expansion and differentiation of FLCs. Alternatively, Chen et al[36]cultured mouse bone marrow stromal stem cells in conditioned medium of hepatocytes and found that these stem cells were induced into hepatocytelike cells. Baertschiger et al[18]co-cultured MSCs with Huh-7 (a human hepatoma cell line) in hepatogenic differentiation medium in a transwell system to avoid direct cell-cell contacts. Albumin expression in MSCs was found in this condition, but not in the absence of Huh-7. These experiments suggest the critical importance of certain growth factors or cytokines secreted by hepatocytes in the differentiation of MSCs into hepatocyte-like cells. Non-parenchymal liver cells may have similar effects when co-cultured with MSCs. Yamazaki et al[37]cultured BMCs from mice transgenic for green fluorescence protein (GFP) with non-parenchymal liver cells and sera from patients with liver failure. Simultaneously, some GFP-BMCs were treated with 5-azacytidine (5-AZA). They found that mouse BMCs can transdifferentiate into hepatocytes by interacting with non-parenchymal liver cells, soluble factors in the sera of patients with liver failure and a demethylating agent. Deng et al[38]co-cultured primary rat BM-MSCs with hepatic stellate cells at different activation stages. Their experiments indicated that only fully-activated but not the quiescent hepatic stellate cells modulate differentiation of MSC into hepatocyte-like cells.

These studies clearly reveal a hepatogenic differentiation role of hepatocytes, non-parenchymal liver cells or certain cytokines. In fact, the liver itself can be considered as an ideal resident site for the hepatic differentiation of MSCs. However, the mechanism has not been fully elucidated.[39]

Three-dimensional (3-D) culture

Many studies have shown that 3-D co-culture of hepatocytes with different cell types, including liver-derived or non-liver-derived mesenchymal cells, improve hepatocyte viability and functionin vitro.[40,41]A 3-D scaffold can improve the ability in the preservation of membrane polarity and cell structure, maintain the functional properties, and suppress markers of dedifferentiation.[42,43]Research related to 3-D culture in hepatocyte differentiation of MSCs is rare. Kazemnejad et al[44]fabricated a 3-D nanofiber scaffold with poly(ε-caprolactone)/collagen/polyethersulfone to culture human BM-MSCs. Compared to the 2-D culture system, higher production levels of albumin, urea and alanine aminotransferase in differentiated cells were reported. The mechanism was not elucidated but it is possible that the biomimetic nanofibers enhance the biological activity of growth factors and cytokines for inducing differentiation. Lin et al[45]used a 3-D alginate scaffold to culture BM-MSCs and also found that the 3-D scaffolds are highly biocompatible with BM-MSCs and induced their differentiation into hepatocyte-like cells.

Transcription factors

Although the exact mechanism of hepatogenic differentiation has not been fully elucidated, certain transcription factors have been exploited in inducing MSC transdifferentiation into hepatocytes. HNF3β is a member of the forkhead box transcription factor family and is reported to regulate expression of more than 100 genes expressed in the liver, pancreas, intestine, and lung during early embryogenesis. HNF3β is a crucial transcription factor for liver development during mouse embryogenesis and also a key player in hepatogenesis. Ishii et al[46]established a tetracycline (Tet)-regulated expression system for HNF3β in UE7T-13 BM-MSCs. Approximately 80% of the cells became albuminpositive after treatment with Tet and bFGF, indicatingthat HNF3β induces efficient differentiation of UE7T-13 human BM-MSCs.

MSCs differentiation into hepatocyte-like cellsin vivo

Since MSCs had been shown to differentiate into hepatocyte-like cellsin vitro, the features of their differentiationin vivowere further investigated. Numerous studies using MSCs transplantation in rodent models of liver failure or fibrosis have confirmed theirin vivodifferentiation into hepatocytes, heralding a novel therapeutic modality for the management of liver diseases (Table).

Sato et al[47]examined the differentiation ability ofhuman BM-MSCs into hepatocytesin vivoby directly inoculating them into rat livers with chronic damage by allylalcohol treatment. Their results, in line with other findings, strongly indicated that hMSCs can differentiate into hepatocytes without fusion. The transplantation of autologous or allogeneic BM-MSCs holds great promise in the treatment of liver cirrhosis which is commonly considered to be an irreversible pathological process. MSCs can differentiate into hepatocytesin vivo, stimulate the regeneration of endogenous parenchymal cells, and enhance the fibrous matrix degradation.[48]Interestingly, undifferentiated BM-derived MSCs are more potent than adipogenic or hepatogenic cells in the suppression of CCl4-induced liver fibrosis. Expression levels of matrix metalloproteinase-2 (MMP-2) and MMP-9 are also the highest in undifferentiated MSCs.[49]

Table. MSCs differentiate into hepatocyte-like cells in vivo

In addition to theirin vitroandin vivoability to differentiate into hepatocytes under certain conditions, MSCs have some anti-inflammatory effects. They not only provide hepatocyte function but also produce secretory molecules to inhibit hepatocyte apoptosis and modulate the acute-phase response. These two properties make MSCs a particularly attractive cell population in the treatment of acute liver failure. A therapeutic trial of liver-assistance devices containing co-cultures of MSCs and hepatocytes showed a significant survival benefit compared to other co-culture and monocellular liverassistance devices.[50,51]

Characterization of hepatocyte-like cells differentiated from MSCs

Characterization of the cells afterin vitroorin vivodifferentiation is the crucial step, and generally includes morphological, phenotypical and functional characterization. Under conditions favoring hepatocyte differentiation, change from a fibroblast-like morphology to the polygonal shape typical of epithelial cells can be seen. Phenotypically, several liver transcription factors and cytoplasmic proteins that are selectively expressed during the differentiation of the liver can be identified. Microarray analysis of the differentiated hepatocyte-like cells from MSCs show a dramatic change in the gene expression profile, which represents features of both adult and fetal hepatocytes.[52]During the early stage of MSC-to-hepatocyte differentiation, these cells express RNA transcripts like nestin (stem cell-specific marker), CX43 (liver stem cell-specific gap junction protein), CK19 (epithelial cell marker) and several early hepatocyte differentiation markers (such as AFP, CK7, GATA4, HNF4, HNF3β, HNF1α, and C/EBPα).[53]In contrast, in the late stage of MSC differentiation, they express cell markers and proteins similar to mature hepatocytes, such as FoxM1 (forkhead transcription factor which plays a critical role in hepatocyte differentiation), cytokeratin 18 (CK18, epithelial cell marker), connexin 32 (CX32, hepatocyte-specific gap junction protein), and E-cadherin; secretory plasma proteins like albumin, fibrinogen, transferrin, and cell-cell adhesion molecules; and enzymes typically produced by well-differentiated hepatocytes including the xenobiotic metabolism enzyme cytochrome P450 subtypes 3A4 and 1A1, gluconeogenic phosphoenolpyruvate carboxykinase 1, the entry enzyme of urea synthesis carbamoylphosphate synthetase (CPS), as well as the ectopeptidase dipeptidylpeptidase type IV (CD26).[20,27]Human hepatocyte-like cells may also express Heppar1, a well-defined marker for human hepatocytes. Meanwhile, differentiated hepatocytes gradually lose the expression of mesenchymal cell markers such as α-SMA.[54,55]In addition to these well-characterized methods, more delicate molecular biological approaches have been exploited recently in the assessment of hepatocyte-like cell differentiation from MSCs. Sgodda et al[20]constructed a reporter gene plasmid expressing GFP or firefly luciferase under the control of the hepatocyte-specific gene phosphoenolpyruvate carboxykinase 1 promoter. Following culture in hepatocyte differentiation medium, rat adiposetissues-derived MSCs were transiently transfected with the plasmid, and hepatocyte differentiation was precisely identified by GFP expression with fluorescence microscopy/Western blotting or luminescence detection using the standard dual luciferase transfection assay.[20]

In order to study the differentiation of MSCs into hepatocytesin vivo, several techniques have been developed to identify and track this adoptively transferred population. They inclucle construction of bone marrow chimeric mice and analysis of the expression of human transcripts and antigens in the chimeric liver;[8]use of sex-mismatched donor and recipient combinations and tracking of male donor cells by using fluorescentin situhybridization of Y chromosome sequences; and tagging of donor-derived MSCs with fluorescence or β-galactosidase and follow-up with fluorescence microscopy or immunohistochemical staining. After adoptive transfer via the peripheral or portal vein, the majority of MSCs are found to move to the damaged liver.[11]

The majority ofin vivoMSC differentiation studies have been done in rodent models. Compared with thein vitrohepatocyte differentiation system,in vivophenotype characterization is much more complicated. Depending on the type of injury to the liver, stem cells may display various phenotypes. In a model of biliarycirrhosis induced by bile duct ligation, engrafted cells assume an activated fibroblast or myofibroblast-like phenotype with expression of fibroblast-specific protein and α-SMA. Liver injury caused by CCl4injection appears to direct the bone marrow cells to take on either a hepatocyte-like or a sinusoidal endothelial phenotype (CD146+). However, after treatment with alkyl alcohol, bone marrow cells contribute to the regeneration of hepatic vascular endothelium.[56-59]

Major liver functions include glycogen storage, detoxification, and lipid metabolism. The common liver functional tests like glycogen deposition (detected by periodic acid-Schiff staining), detoxification of ammonia through urea synthesis (urea formation determined via the colorimetric diacetyl monoxime method), uptake of low-density lipoprotein, and phenobarbital-inducible cytochrome P450 activity have been widely used to test the function of hepatocyte-like MSCs. Other tests such as the cellular GSH and GST catalytic activity toward 1-chloro-2, 4-dinitrobenzene are also used to discriminate hepatocyte-like cells differentiated from MSCs as opposed to undifferentiated MSCs.[60]

Mechanistic insights for MSC differentiation into hepatocyte-like cells

The detailed mechanism of MSC transdifferentiation into hepatocyte-like cells remains obscure. Mesenchymalto-epithelial transition (MET) is the reverse process of epithelial-to-mesenchymal transition (EMT), which is a crucial step in cancer progression and embryonic development. Ochiya et al[15]examined the gene expression profile of AT-MSC-derived hepatocytes (ATMSC-Hepa) using microarray analysis. They found that the expression levels of Twist and Snail, both regulators of the EMT, are down-regulated during the differentiation process. Meanwhile, epithelial signatures such as E-cadherin and α-catenin are up-regulated in AT-MSCHepa. In contrast, the expression of mesenchymal markers like N-cadherin and vimentin was down-regulated. These findings suggested that an MET occurs in the process of hepatic differentiation from AT-MSCs.[14,61]

Wnt signaling is known to be involved in many aspects of animal embryonic development. In recent years, it has also been shown to play major roles in selfrenewal as well as the proliferation of hematopoietic stem and progenitor cells. Ke et al[62]induced BMMSCs to differentiate into hepatocytes and showed that the Wnt signaling pathway is involved in this process. Blocking Wnt signaling promotes the differentiation of BMSCs towards hepatocytes.

Epigenetic modification such as DNA methylation and histone acetylation may also contribute to the differentiation of MSCs. DNA methyltransferase inhibitors (DNMTis), either alone or in combination with histone deacetylase inhibitors (HDACis), have been shown to alter cell fate in hepatocyte differentiation. Basically, DNMTis function as preconditioning agents before hepatic differentiation, whereas HDACis act as stimulants during or after differentiation.[20,26,37,52,63]Snykers et al[64]found that addition of trichostatin A to human MSCs cultures pretreated with hepatogenic-stimulating agents for 6 days trigger their "transdifferentiation'' into cells with phenotypic and functional characteristics similar to those of primary hepatocytes. In general, modulating chromatin remodeling seems to be a promising strategy to overcome cell fate and induce lineage-specific differentiation.

Immunological considerations

The immunogenicity of stem cells is a serious and controversial issue which may directly affect the efficacy of stem cell-based treatment; this is particularly true if we use allogeneic stem cell transplants in most circumstances. Allogeneic MSCs are proposed as cell therapies for degenerative, inflammatory, and autoimmune diseases. The rationale of allogeneic MSC therapy rests heavily on the concept that these cells are capable of avoiding or actively suppressing the immunological responses which normally cause rejection of most allogeneic cells and tissues. One comparison study showed that allogeneic BM-MSCs exhibit trivial immunogenicity and they are as efficient as syngeneic cells in the engraftment and enhancement of wound healing.[65]The phenotype of allogeneic MSCs is described as high expression of MHC classialloantigens, but minimal expression of class II alloantigens and costimulatory molecules (CD80, CD86 and CD40); purified T cells do not respond to allogeneic MSCs.[66]Although MSCs have long been considered as immunoprivileged and failed to elicit immune responsesin vitro, a recent study[67]suggest that they retain a degree of immunogenicity in some circumstances which might limit MSC longevity and attenuate their beneficial effects. An animal study[68]showed that allogeneic and xenogeneic MSCs are eventually rejected by the recipients. They are also capable of inducing memory T-cell responses after injection into immunocompetent hosts. Recent studies[69,70]showed that allogeneic MSCs are susceptible to lysis by cytotoxic CD8+T and NK cells, even autologous MSCs can still trigger lymphocyte infiltration and inflammation and are prone to NK cell-mediated lysis. Moreover NK cellmediated lysis is inversely correlated with the expressionof HLA classimolecules on MSCs. The concern of MSC immunogenicity is further complicated by the question of whether MSCs retain their immunosuppressive properties after trans-differentiation. As reported, the long-term ability of allogeneic MSCs to preserve function in the infarcted heart is largely limited by the transition from an immunoprivileged to an immunogenic state after differentiation, characterized by increased MHC-Ia and -II expression and reduced MHC-Ib expression.[71]Another example is adipose tissue-derived stem cells, which show both immunogenic (up-regulation of HLA-DR) and immunosuppressive (expression of immunosuppressive HLA-G and IL-10) features after chondrogenic differentiation.[72]These results indicate that the immunogenicity of adult stem cell-derived cells or tissues should be tested individually in animal models before they can be considered for clinical trials. Novel techniques have been developed recently to modify the immunogenicity of allogeneic MSCs in order to boost their efficacy. hMSCs encapsulated in alginate-PLL microcapsules are reported to be significantly hypoimmunogenic, leading to a 3-fold decrease in cytokine expression in comparison to entrapped cell lines.[73]Transduction of hMSCs with several viral invasion proteins results in strong inhibition of MHC classisurface expression and obviation of xenogeneic MSC rejection.[74]

MSCs are not only immunoprivileged but more importantly, display immunomodulatory capacities. Their immunomodulatory effects have been extensively studiedin vitroandin vivousing various animal models.[75]MSCs efficiently inhibit the maturation, cytokine production and T-cell priming capacity of dendritic cells (DCs). The potential mechanism involves the induction of mature DC differentiation into a novel Jagged-2-dependent regulatory DC population and the escape from their apoptotic fate.[76]A recent study also suggested that MSCs can impair immune synapse formation by altering DC actin distribution.[77]Furthermore, they markedly impair the proliferation, cytokine secretion and cytotoxic potential of NK cells and T lymphocytes. These effects are complemented by the induction of divisional arrest in T cells and by stem cell production of soluble immunomodulatory factors, including IFN-γ, IL-10, HGF, PGE2, TGF-β1, IDO and NO.[78,79]MSCs are also capable of inhibiting the proliferation and antibody production of B cells.[80]Therefore, MSCs have attracted great interest in immune-mediated therapies. Patients with lifethreatening acute or chronic graft-versus-host diseases not responding to conventional immunosuppressive therapy can be successfully treated with MSCs.[81]MSCs have also been used in the prevention of acute allograft rejection as well as the induction of transplant tolerance in conjugation with low-dose rapamycin, the potential mechanism involves the induction of tolerogenic DC and Foxp3+regulatory T cells.[82]Several phase III clinical trials using MSCs for the treatment of Crohn's disease are currently under way taking advantage of their immunoregulatory properties.[83]

Clinical trials

Clinical application of human stem cells in the treatment of liver diseases is still in its infancy. In a phaseitrial, four patients with decompensated liver cirrhosis received infusion of a mean 31.73×106MSCs propagated from bone marrow. The model for endstage liver disease scores of 2 patients improved by 4 and 3 points, and the quality of life of all four patients improved by the end of follow-up. No side-effect was observed.[84]Despite these early encouraging results, a lot of work is needed before we can finally use MSCs in the management of human liver diseases.

Safety

As far as ethical and safety issues are concerned, MSCs derived from somatic cells are generally considered to be superior,[14]differentiated MSCs seem to be more stable and safer than undifferentiated MSCs because of their lineage commitment to hepatocytes. Although evidence is lacking, there have been concerns regarding the safety of using these cells since they possess a self-renewal capability mimicking cancer cells. Sawada et al[85]evaluated the safety of tissueengineered medical devices using normal hMSCs. They found that the proliferation rate of hMSCs gradually decreased and cellular senescence was observed in about 3 months. The expression profile of the genes that regulate cellular proliferation in hMSCs are significantly different from those of cancer cells.[85]Despite the fact that many studies support the hypothesis that MSCs play a positive role in the regeneration of cirrhotic liver, Russo et al[86]showed that bone marrow transplantation might actually enhance liver fibrosis. Another study indicated that MSCs derived from human bone marrow permanently reside in liver tissue, retain a mesenchymal morphology, and do not express any hepatic markers. Furthermore, MSCs colocalize with collagen deposition suggesting their transdifferentiation into myofibroblasts with the development of fibrous tissue when transplanted into an injured or regenerating liver.[18]These untoward effects need to be kept in mind when applying MSCs in the treatment of liver diseases.

Summary

Hepatocyte-like cells differentiated from MSCs are promising sources for liver regeneration and tissue engineering. Although it is still a matter of debate as to whether MSCs-derived hepatocytes can efficiently repopulate a host liver to provide adequate functional substitution, most of the animal studies support the point that MSCs can be key players in liver-directed regenerative medicine. In addition to the hepatocyte replacement function, the unique properties involving immunoregulation and anti-fibrosis by MSCs may not only help the engraftment of MSCs and their longevity, but also benefit the treatment of several immunemediated liver diseases, such as autoimmune hepatitis, primary biliary cirrhosis and primary sclerosing cholangitis. Future studies are needed to improve the efficacy and consistency of hepatic differentiation from MSCs, and more importantly, large clinical trials need to be initiated to validate their therapeutic potential in human liver diseases.

Acknowledgements:We thank that Professor Cheng-Hong Peng gives us guidance and help in this field of liver tissue engineering.

Contributors:WXB and TR proposed the study. WXB retrieved and summarized the relevant literatures, and wrote the main body of the article under the supervision of TR. TR revised the review. TR is the guarantor.

Funding:This study was supported by a grant from the Science and Technology Commission of Shanghai (10411968500).

Ethical approval:Not needed.

Competing interest:No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.

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October 3, 2011

Accepted after revision February 6, 2012

Author Affiliations: Department of General Surgery, Central Hospital of Minhang District, Ruijin Medical Group, Shanghai 201199, China (Wu XB); Center for Organ Transplantation, Department of General Surgery, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200025, China (Tao R)

Ran Tao, MD, Center for Organ Transplantation, Department of General Surgery, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200025, China (Tel: 86-21-64370045ext666705; Fax: 86-21-64373909; Email: taohdac9@yahoo.com)

? 2012, Hepatobiliary Pancreat Dis Int. All rights reserved.

10.1016/S1499-3872(12)60193-3