Therapeutic potential of stem cell in liver regeneration

Abstract

Liver transplantation is the only life-saving procedure for patients with end-stage liver disease. However, its potential benefits are hampered by many disadvantages, such as the relative shortage of donors, operative risks, and high costs. These issues have prompted the search for new alternative therapies for irreversible liver disease. Stem cell therapy, with the ability for self-renewal and potential for multilineage differentiation, is a promising alternative approach. Several studies have demonstrated that transplantation of hepatic stem/progenitor cells or hepatocyte-like cells derived from multipotent stem cells leads to donor cell-mediated repopulation of the liver and improved survival rates in experimental models of liver disease. However, a registered clinical application based on stem cell technology will take at least an additional 5 to 10 years because of some limitations; e.g. the lack of suitable cell sources and risk of teratoma formation. This review summarizes the general understanding of the therapeutic potentials of stem cells in liver disease, including the sources, mechanisms, and delivery methods of hepatic stem cells in liver regeneration, and discusses some challenges for their therapeutic application.

Keywords

stem cell / liver disease / regenerative medicine

Introduction

The liver is a complex organ system composed of highly diverse cells that originate from both parenchymal and nonparenchymal cells. The main functions of the liver are bile excretion, synthesis of coagulation factors and other proteins, and metabolism of intracorporeal decomposition products. End-stage liver disease (ESLD) is the final stage of acute or chronic liver damage and is irreversibly associated with liver failure. Currently, liver transplantation is the only life-saving procedure for patients with ESLD. However, its potential benefits are hampered by many diasdvantages, such as the relative shortage of donors, operative risks, post-transplant rejection, recidivism of the pre-existing liver disease, and high costs [1]. These issues have prompted the search for new alternative therapies for intractable liver disease. In recent years, increasing attention has been directed toward stem cell therapy for liver disease.

Stem cells are generally defined as cells exhibiting two properties: a capacity for self-renewal and potency for multilineage differentiation. They can be divided into adult, fetal, or embryonic types based on the source from where they are isolated. Adult stem cells can be found in the blood, bone marrow, skin, adipose tissue, muscle, salivary glands, pancreas, spleen, liver, and brain. In the fetus, they can also be found in placental and umbilical cord blood. Embryonic stem cells (ESCs) are truly totipotent, having the ability to produce all differentiated cells in an organism. Increasing studies on the therapeutic potential of stem cells in liver disease and regeneration have demonstrated that stem cells in vitro or in vivo may become mature hepatocyte-like cells bearing hepatic functions, such as albumin production, urea metabolism, and so on [2-5]. These findings have prompted the new drive to seek the therapeutic potential of stem cells in liver diseases. This review focuses on the general view of the therapeutic potentials of stem cells in liver disease, including the sources, mechanisms, and delivery methods of hepatic stem cells in liver regeneration, and some challenges for their therapeutic application.

Sources of hepatic stem cells

Endogenous hepatic stem cells

Adult hepatic stem/progenitor cells

Under physiologic conditions, the liver does not need any external source of cells to repair injury because resting hepatocytes have the ability to re-enter the cell cycle rapidly and efficiently after an injury has occurred [6]. However, this capacity is overwhelmed during massive or chronic injury, and facultative hepatic progenitors (called oval cells in rodents), which are thought to reside in the terminal branches of the intrahepatic biliary tree (e.g. the canals of Hering), are activated [7]. These cells are bipotential and can give rise to both hepatocytes and biliary epithelia [8]. A previous study had indicated they may have a role in carcinogenesis and can be a potential source of liver tumors [9]. However, the lack of an exclusive oval cell marker makes this cell population elusive and this has aroused much speculation. Recent data show that bipotential epithelial colony-forming cells can be successfully isolated from fetal and adult mouse liver by immunoselection for epithelial cell adhesion molecule (EpCAM) [10,11]. This is an encouraging development as EpCAM-positive cells are putative hepatic progenitors in fetal and adult human livers that are capable of forming colonies in culture [12]. Although whether EpCAM-positive cells in mice and humans represent cells of comparable functional phenotype remains questionable, rodent studies using cells of the same histochemical marker had inferred that the cells could be extrapolated to facilitate the production of clinically usable material.

Fetal hepatic progenitor cells

During embryogenesis, the so-called hepatoblasts appear once the hepatic endoderm has been specified and the liver bud is growing. Hepatoblasts are bipotential and are capable of differentiating into hepatocytes or cholangiocytes. A number of studies showed that fetal liver progenitors might be a superior source of liver cells for transplantation, since they have the advantage of being highly proliferative, less apoptotic following transplantation, and less immunogenic [13,14]. In rat, cells positive for delta like kinase-1 (Dlk-1) exhibited cell culture characteristics and a gene expression profile expected for hepatic progenitors and were capable of repopulating the normal adult liver [15]. Further investigation on xenotransplantation studies demonstrated that fetal human progenitors possessed liver-repopulating activity [16]. However, fetal progenitors have limited availability due to ethical issues and shortage of sources.

Extrahepatic stem cells

Bone marrow stem cells

Hematopoietic stem cells (HSCs) are one of the main stem cell types in the bone marrow and give rise to all mature blood lineages [17]. The first report to suggest that bone marrow stem cells have a role in liver repair was published in the late 1990s by Petersen et al. [18]. Currently, many studies have found that both rodent and human HSCs can be induced to differentiate into hepatocytes in vitro and in vivo [19-23]. Most in vitro protocols for inducing CD34⁺ HSC differentiation into hepatocytes concluded that a growing medium conditioned with growth factors and mitogens (e.g. hepatocyte growth factors (HGF), fibroblast growth factor (FGF) and oncostatin M) and culture layers specific for hepatocyte growth, like matrigel. Although these studies showed “transdifferentiation” of some HSCs into hepatocytes, the reported percentage of hepatocytes derived from HSCs did not exceed 5%. Thus, HSCs exhibit a limited differentiation potential that make them non-optimal candidates for tissue regeneration purposes. The cost of repeated cultures needed to obtain sufficient amounts of hepatocytes from HSCs would presumably be too high for cell therapy-based applications.

Mesenchymal stem cells (MSCs), another type of bone marrow-derived stem cells, were first identified in 1970, but their ability to differentiate into a hepatocyte-like phenotype was first described in 2004 [24,25]. MSCs can be obtained from the bone marrow, cord blood, adipose tissue, salivary glands, pancreatic tissue, and the umbilical cord [26,27]. MSC-derived hepatocytes can effectively rescue immunodeficient mice from lethal fulminant liver failure induced by toxin and can provide engraftment up to 5% of the recipient liver [28,29]. MSCs can also reduce oxidative stress in an injured liver and accelerate the proliferation of endogenous hepatocytes, thus suggesting possible roles of paracrine effects.

Adipose tissue-derived stem cells

MSCs can be derived from various adult tissues and can differentiate into many cell types in vitro. Human adipose tissue-derived MSCs (AT-MSCs) can differentiate into hepatocyte-like cells with similar gene expression, morphology, and metabolic activity as hepatocytes [30]. A xenogeneic transplantation model of liver regeneration involving mice transplanted with pre-differentiated AT-MSCs revealed long-term engraftment of AT-MSCs-derived hepatocyte-like cells with a high repopulation rate (more than 10%), accompanied by improved serum albumin levels [27]. The use of AT-MSCs as regenerative cells would be advantageous based on ethical and safety issues. These autologous cells are immuno-compatible and exhibit controlled differentiation and multi-functional abilities. In addition, they do not undergo post-transplantation rejection or unwanted differentiation that may lead to in teratoma formation.

Embryonic stem cells and induced pluripotent stem cells

ESCs can be generated from the inner cell mass of human embryos and offer the advantage of being expandable and totipotent [31]. Therefore, they are more versatile than any other population of stem cells utilized for liver regeneration. Multiple protocols for ESC differentiation into cells of hepatic lineage have been studied. Growth factors used for differentiation include activin-A, FGF-2, bone morphogenetic protein-2, HGF, dexamethasone, and oncostatin M [32,33]. However, their use in therapy is limited by ethical issues, immunosuppression, and low repopulation rate [34].

Therefore, the development of ESC-like induced pluripotent stem cells (iPSCs) may prove useful in solving the above mentioned limitations. iPSCs are adult cells reprogrammed into an ESC-like state that express stem cell markers and are capable of generating cells characteristic of all three germ layers. In 2006, Takahashi and coworkers were the first to report on the induction of pluripotent stem cells, which had similar morphology, proliferation, and teratoma formation as ESCs, from mouse embryonic fibroblast cultures by introduction of Oct4, Sox2, c-Myc, and Klf4 [35]. One year later, the first iPSCs from adult human cells were created by retroviral infection with Oct4, Sox2, c-Myc, and Klf4 [36] or Oct4, Sox2, Nanog, and Lin28 [37]. Since then, several groups had tried to generate iPSCs from various types of cells, e.g., mouse and human (embryonic) fibroblasts [38], adult dermal fibroblasts [39,40], neural stem cells [41], and human keratinocytes [42], using retroviral gene-transfer of up to four different factors. Meanwhile, the use of chemical inhibitors to support genetic reprogramming was proposed by Li and coworkers [43]. Having similar characteristics as ESCs, iPSCs might have a promising future application in cell therapy. Although iPSCs raise less ethical issues than ESCs, there are still many other issues that have to be investigated before these cells can be used for medicine, e.g. the risk of teratoma formation and the safety of retroviral gene transfer.

Mechanisms of stem cells in liver regeneration

Critical questions about the process of stem cell transformation into hepatocytes remain unanswered. At present, two possible mechanisms for exploring the functions of bone marrow stem cells have been proposed: via sole transdifferentiation of stem cells and via cell fusion of stem cells and hepatocytes. In addition, the potential for paracrine effects, and promoting angiogenesis promotion, liver progenitors, and hepatocyte proliferation should also be taken into consideration in the process of liver regeneration.

Transdifferentiation

Great interest in the possibility of the so-called adult stem cell plasticity was sparked when Y-chromosome-positive hepatocytes were identified in female patients who had received a male bone marrow (BM) graft [44,45]. It was suggested that extrahepatic cells may generate hepatocytes in vivo via transdifferentiation. Further investigation found that transplanted human MSCs into rat liver by intrasplenical route differentiated into human hepatocytes. Cell fusion was not likely involved since both human and rat chromosomes were independently identified by fluorescence in situ hybridization [46]. Subsequent analysis demonstrated that hematopoietic stem cells convert into liver cells without cell fusion [47].

Cell fusion

Other studies showed that transdifferentiation is not the mechanism of bone marrow stem cell (BMSC) transformation into hepatocytes. In the mouse model of hereditary type I tryosinaemia, the fumarylacetoacetate hydrolase (FAH) is knocked out. This potentially fatal enzyme deficiency could be rescued through repopulation by BM cells derived from wild-type donors, and the restoration to normal phenotype of the FAH-deficient liver was explained by monocyte-hepatocyte fusion [48]. Findings of other studies, such as the detection of multiploid hepatocytes in the recipient liver, support the conclusion that cell fusion is the principal source of bone marrow-derived hepatocytes [49-51]. In vivo fate mapping of transplanted myeloid lineage cells showed that HSC-derived hepatocytes are derived from mature myelomonocytic cells and that myeloid cells spontaneously fuse with host hepatocytes [52]. Cell fusion might be a novel mechanism for liver function restoration.

Methods for stem cell delivery

For therapeutic purposes, stem cells can be administered via different routes; e.g. systemic infusion, intrahepatic injection, portal vein injection, intrasplenic injection, and extracorporeal liver support devices. Every route has its own advantages and disadvantages. The ideal strategy of stem cell delivery should be easy to perform, minimally invasive and traumatic, has very little side effects, has high stem cell survival, and be based on clinical settings.

Systemic infusion

Peripheral intravenous infusion is an easy and convenient method of stem cell delivery. Most implantations are performed using this method because it is minimally invasive and traumatic, and does not require advanced medical equipment. However, the systemic administration is often accompanied by complicated side effects, such as fever, immunoreaction, and the risk of donor cell entrapment in the lungs.

Intrahepatic injection

Intrahepatic injection of stem cells is another route of local administration. Direct hepatic infusion of stem cells would be favorable for the cells repopulating the liver, but this procedure is highly complicated and invasive, and severe trauma may occur. In addition, the microenvironment of a diseased liver may not be conducive for stem cell repopulation and function.

Portal vein injection

Stem cells infused through the portal vein may reside in the periportal areas and repopulate more quickly compared with peripheral systemic infusion. However, this procedure has several disadvantages: (i) the procedure is highly complicated and invasive; (ii) it may cause portal hypertension and further liver damage; and (iii) transplanted cells may migrate to systemic veins and cause embolism of other organs, such as the lung and brain.

Intrasplenic injection

Intrasplenic injection has long been used as a route of cell delivery in liver diseases. This is advantageous when the diseased liver is not suitable for cell transplantation because the spleen has anatomic affinity with the liver. Some of the transplanted cells may be retained in the spleen and implement their functions there instead of in the diseased liver. Since the splenic vein drains directly into the portal vein, a majority of the cells will migrate to the liver via the portal vein system; this may cause portal hypertension and portal vein embolism.

Extracorporeal liver support devices

The applications of stem cell-derived hepatocytes in extracorporeal hepatic support device are rare compared with other implantation approaches. Stem cells are potential cell sources used in the bioartificial liver and hybrid liver assist device.

Modalities of liver stem cell therapy

The numerous types of hepatic progenitor cells offer a cellular basis for stem cell therapy for the treatment of liver disease. Stem cell-derived hepatocytes can be potentially used for drug screening and disease modeling, human bio-artificial liver construction, and transplantation therapy potentially. Major constraints remain to be difficulties in finding sources and maintaining viable hepatocytes. The development of ESC and iPSC technologies provides a research tool in the investigation of human liver disease. Their use could also prove invaluable in constructing drug metabolism models and in providing functional humanized extracorporeal support. Moreover, it is important to recognize the role of the hepatocellular microenvironment during the process of stem cell therapy. There is accumulating evidence suggesting that cocultures of hepatocytes and non-parenchymal cells, including hepatic stellate cells [53-55] and endothelial cells [56,57], greatly improve hepatocyte functionality in vitro. Manipulation of the in vitro culture substrate using different extracellular matrix components or their synthetic chemical mimics also leads to improved and prolonged biological function [54,58,59].

Although stem cell therapy has produced surprising outcomes in the treatment of liver diseases, cell therapy alone cannot suffice to regenerate large tissue defects or even replace whole organs. In these settings, “tissue engineering” seems to be a promising strategy in future. Tissue engineering involves a scaffold imitating the architecture of tissue-specific extracellular matrix that is seeded with tissue-specific cells. This technique has already been successfully used to synthesize experimental replacement for simple tissues, such as skin, bone, and fat [60]. At present, tissue engineering is still far from successful in generating tissues for therapeutic replacement of highly complicated structures, such as kidney, liver, or intestine.

Risks or problems with future stem cell therapy

Despite the overall positive potential of stem cell therapy, some important theoretical and practical issues have to be addressed before stem cell transplantation becomes a routine procedure for the treatment of liver disease.

ESCs have full developmental potential to become all types of cells or tissues in the body, and they have been reported to become integrated into organs such as the liver. However, their therapeutic use is limited by the recipient’s immune responses and the potential development of teratomas. Moreover, the use of human embryos for the production of these cells has raised many ethical issues that remain unsolved. Therefore, ESC technology is quite limited to several countries and has not been used for clinical application.

Difficulties in finding sources and maintaining viable hepatocytes in stem cell therapeutic application, raising additional questions about availability, cryopreservation, or storage of the stem cells, remain to be the main limitations for application. Moreover, the route of cell application, engraftment, or “homing” of cells to the diseased tissue or at the least, cell survival in another organ where they may partially fulfill their initial role are issues that have not been solved yet. The role of immunosuppression also remains to be studied.

Finally, fibrogenic cells in the liver, such as hepatic stellate cells and myofibroblasts, can originate from stem cells. It may make the situation worse for patients with cirrhosis if unwanted or profibrotic cells are transplanted [61,62]. Further studies may investigate the inhibition of profibrotic cell derivation during stem cell therapeutic application.

Conclusion

Although recent advances in the production of stem cell-derived hepatocytes hold great promise for the treatment of liver disease, a registered clinical application based on stem cell technology will take at least an additional 5 to 10 years. Based on recent findings, the use of adult stem cells seems to be a “more” realistic option that can be applied in clinical settings because of its autologous source. The so-called iPS cells may overcome the dilemma of ethical controversy and source shortage raised by ESCs but may raise others, such as the risk of teratoma formation and safety of retroviral gene transfer. Therefore, the next essential step forward will be to define the milestones of safety and efficacy that need to be achieved before clinical application of liver stem cell therapies and standardize progenitor cell definition, preclinical animal models, and endpoint parameters.

References

[1] O’Leary J G, Lepe R, Davis G L. Indications for liver transplantation. Gastroenterology, 2008, 134(6): 1764-1776

[2] Kung J W, Forbes S J. Stem cells and liver repair. Curr Opin Biotechnol, 2009, 20(5): 568-574

[3] Ogawa S, Miyagawa S. Potentials of regenerative medicine for liver disease. Surg Today, 2009, 39(12): 1019-1025

[4] Ehnert S, Glanemann M, Schmitt A, Vogt S, Shanny N, Nussler N C, Stöckle U, Nussler A. The possible use of stem cells in regenerative medicine: dream or reality? Langenbecks Arch Surg, 2009, 394(6): 985-997

[5] Alison M R, Islam S, Lim S. Stem cells in liver regeneration, fibrosis and cancer: the good, the bad and the ugly. J Pathol, 2009, 217(2): 282-298

[6] Fausto N, Campbell J S, Riehle K J. Liver regeneration. Hepatology, 2006, 43 (Suppl 1): S45-S53

[7] Roskams T A, Theise N D, Balabaud C, Bhagat G, Bhathal P S, Bioulac-Sage P, Brunt E M, Crawford J M, Crosby H A, Desmet V, Finegold M J, Geller S A, Gouw A S, Hytiroglou P, Knisely A S, Kojiro M, Lefkowitch J H, Nakanuma Y, Olynyk J K, Park Y N, Portmann B, Saxena R, Scheuer P J, Strain A J, Thung S N, Wanless I R, West A B. Nomenclature of the finer branches of the biliary tree: canals, ductules, and ductular reactions in human livers. Hepatology, 2004, 39(6): 1739-1745

[8] Dorrell C, Grompe M. Liver repair by intra- and extrahepatic progenitors. Stem Cell Rev, 2005, 1(1): 61-64

[9] Lee J S, Heo J, Libbrecht L, Chu I S, Kaposi-Novak P, Calvisi D F, Mikaelyan A, Roberts L R, Demetris A J, Sun Z, Nevens F, Roskams T, Thorgeirsson S S. A novel prognostic subtype of human hepatocellular carcinoma derived from hepatic progenitor cells. Nat Med, 2006, 12(4): 410-416

[10] Tanaka M, Okabe M, Suzuki K, Kamiya Y, Tsukahara Y, Saito S, Miyajima A. Mouse hepatoblasts at distinct developmental stages are characterized by expression of EpCAM and DLK1: drastic change of EpCAM expression during liver development. Mech Dev, 2009, 126(8-9): 665-676

[11] Okabe M, Tsukahara Y, Tanaka M, Suzuki K, Saito S, Kamiya Y, Tsujimura T, Nakamura K, Miyajima A. Potential hepatic stem cells reside in EpCAM+ cells of normal and injured mouse liver. Development, 2009, 136(11): 1951-1960

[12] Schmelzer E, Zhang L, Bruce A, Wauthier E, Ludlow J, Yao H L, Moss N, Melhem A, McClelland R, Turner W, Kulik M, Sherwood S, Tallheden T, Cheng N, Furth M E, Reid L M. Human hepatic stem cells from fetal and postnatal donors. J Exp Med, 2007, 204(8): 1973-1987

[13] Sangan C B, Tosh D. Hepatic progenitor cells. Cell Tissue Res, 2010, 342(2): 131-137

[14] Oertel M, Menthena A, Dabeva M D, Shafritz D A. Cell competition leads to a high level of normal liver reconstitution by transplanted fetal liver stem/progenitor cells. Gastroenterology, 2006, 130(2): 507-520

[15] Oertel M, Menthena A, Chen Y Q, Teisner B, Jensen C H, Shafritz D A. Purification of fetal liver stem/progenitor cells containing all the repopulation potential for normal adult rat liver. Gastroenterology, 2008, 134(3): 823-832

[16] Mahieu-Caputo D, Allain J E, Branger J, Coulomb A, Delgado J P, Andreoletti M, Mainot S, Frydman R, Leboulch P, Di Santo J P, Capron F, Weber A. Repopulation of athymic mouse liver by cryopreserved early human fetal hepatoblasts. Hum Gene Ther, 2004, 15(12): 1219-1228

[17] Kallis Y N, Alison M R, Forbes S J. Bone marrow stem cells and liver disease. Gut, 2007, 56(5): 716-724

[18] Petersen B E, Bowen W C, Patrene K D, Mars W M, Sullivan A K, Murase N, Boggs S S, Greenberger J S, Goff J P. Bone marrow as a potential source of hepatic oval cells. Science, 1999, 284(5417): 1168-1170

[19] Gilchrist E S, Plevris J N. Bone marrow-derived stem cells in liver repair: 10 years down the line. Liver Transpl, 2010, 16(2): 118-129

[20] Houlihan D D, Newsome P N. Critical review of clinical trials of bone marrow stem cells in liver disease. Gastroenterology, 2008, 135(2): 438-450

[21] Levicar N, Pai M, Habib N A, Tait P, Jiao L R, Marley S B, Davis J, Dazzi F, Smadja C, Jensen S L, Nicholls J P, Apperley J F, Gordon M Y. Long-term clinical results of autologous infusion of mobilized adult bone marrow derived CD34+ cells in patients with chronic liver disease. Cell Prolif, 2008, 41 (Suppl 1): 115-125

[22] Tajima F, Tsuchiya H, Nishikawa K, Kataoka M, Hisatome I, Shiota G. Hepatocyte growth factor mobilizes and recruits hematopoietic progenitor cells into liver through a stem cell factor-mediated mechanism. Hepatol Res, 2010, 40(7): 711-719

[23] Yamaguchi K, Itoh K, Masuda T, Umemura A, Baum C, Itoh Y, Okanoue T, Fujita J. In vivo selection of transduced hematopoietic stem cells and little evidence of their conversion into hepatocytes in vivo. J Hepatol, 2006, 45(5): 681-687

[24] Friedenstein A J, Chailakhjan R K, Lalykina K S. The development of fibroblast colonies in monolayer cultures of guinea-pig bone marrow and spleen cells. Cell Tissue Kinet, 1970, 3(4): 393-403

[25] Lee K D, Kuo T K, Whang-Peng J, Chung Y F, Lin C T, Chou S H, Chen J R, Chen Y P, Lee O K. In vitro hepatic differentiation of human mesenchymal stem cells. Hepatology, 2004, 40(6): 1275-1284

[26] Liu Z J, Zhuge Y, Velazquez O C. Trafficking and differentiation of mesenchymal stem cells. J Cell Biochem, 2009, 106(6): 984-991

[27] Aurich H, Sgodda M, Kaltwasser P, Vetter M, Weise A, Liehr T, Brulport M, Hengstler J G, Dollinger M M, Fleig W E, Christ B. Hepatocyte differentiation of mesenchymal stem cells from human adipose tissue in vitro promotes hepatic integration in vivo. Gut, 2009, 58(4): 570-581

[28] Cho K A, Ju S Y, Cho S J, Jung Y J, Woo S Y, Seoh J Y, Han H S, Ryu K H. Mesenchymal stem cells showed the highest potential for the regeneration of injured liver tissue compared with other subpopulations of the bone marrow. Cell Biol Int, 2009, 33(7): 772-777

[29] Kuo T K, Hung S P, Chuang C H, Chen C T, Shih Y R, Fang S C, Yang V W, Lee O K. Stem cell therapy for liver disease: parameters governing the success of using bone marrow mesenchymal stem cells. Gastroenterology, 2008; 134(7):2111-2121, 2121.e1-3

[30] Banas A, Teratani T, Yamamoto Y, Tokuhara M, Takeshita F, Quinn G, Okochi H, Ochiya T. Adipose tissue-derived mesenchymal stem cells as a source of human hepatocytes. Hepatology, 2007, 46(1): 219-228

[31] Wang Z, Lu H, Wang Y C, Cong X Q. Human embryonic stem cells and liver diseases: from basic research to future clinical application. J Dig Dis, 2008, 9(1): 14-19

[32] Cai J, Zhao Y, Liu Y, Ye F, Song Z, Qin H, Meng S, Chen Y, Zhou R, Song X, Guo Y, Ding M, Deng H. Directed differentiation of human embryonic stem cells into functional hepatic cells. Hepatology, 2007, 45(5): 1229-1239

[33] Dan Y Y, Yeoh G C. Liver stem cells: a scientific and clinical perspective. J Gastroenterol Hepatol, 2008, 23(5): 687-698

[34] Dalgetty D M, Medine C N, Iredale J P, Hay D C. Progress and future challenges in stem cell-derived liver technologies. Am J Physiol Gastrointest Liver Physiol, 2009, 297(2): G241-G248

[35] Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell, 2006, 126(4): 663-676

[36] Okita K, Ichisaka T, Yamanaka S. Generation of germline-competent induced pluripotent stem cells. Nature, 2007, 448(7151): 313-317

[37] Yu J, Vodyanik M A, Smuga-Otto K, Antosiewicz-Bourget J, Frane J L, Tian S, Nie J, Jonsdottir G A, Ruotti V, Stewart R, Slukvin I I, Thomson J A. Induced pluripotent stem cell lines derived from human somatic cells. Science, 2007, 318(5858): 1917-1920

[38] Wernig M, Meissner A, Foreman R, Brambrink T, Ku M, Hochedlinger K, Bernstein B E, Jaenisch R. In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state. Nature, 2007, 448(7151): 318-324

[39] Lowry W E, Richter L, Yachechko R, Pyle A D, Tchieu J, Sridharan R, Clark A T, Plath K. Generation of human induced pluripotent stem cells from dermal fibroblasts. Proc Natl Acad Sci USA, 2008, 105(8): 2883-2888

[40] Park I H, Zhao R, West J A, Yabuuchi A, Huo H, Ince T A, Lerou P H, Lensch M W, Daley G Q. Reprogramming of human somatic cells to pluripotency with defined factors. Nature, 2008, 451(7175): 141-146

[41] Kim J B, Zaehres H, Wu G, Gentile L, Ko K, Sebastiano V, Araúzo-Bravo M J, Ruau D, Han D W, Zenke M, Schöler H R. Pluripotent stem cells induced from adult neural stem cells by reprogramming with two factors. Nature, 2008, 454(7204): 646-650

[42] Carey B W, Markoulaki S, Hanna J, Saha K, Gao Q, Mitalipova M, Jaenisch R. Reprogramming of murine and human somatic cells using a single polycistronic vector. Proc Natl Acad Sci USA, 2009, 106(1): 157-162

[43] Li W, Wei W, Zhu S, Zhu J, Shi Y, Lin T, Hao E, Hayek A, Deng H, Ding S. Generation of rat and human induced pluripotent stem cells by combining genetic reprogramming and chemical inhibitors. Cell Stem Cell, 2009, 4(1): 16-19

[44] Theise N D, Nimmakayalu M, Gardner R, Illei P B, Morgan G, Teperman L, Henegariu O, Krause D S. Liver from bone marrow in humans. Hepatology, 2000, 32(1): 11-16

[45] Alison M R, Poulsom R, Jeffery R, Dhillon A P, Quaglia A, Jacob J, Novelli M, Prentice G, Williamson J, Wright N A. Hepatocytes from non-hepatic adult stem cells. Nature, 2000, 406(6793): 257

[46] Sato Y, Araki H, Kato J, Nakamura K, Kawano Y, Kobune M, Sato T, Miyanishi K, Takayama T, Takahashi M, Takimoto R, Iyama S, Matsunaga T, Ohtani S, Matsuura A, Hamada H, Niitsu Y. Human mesenchymal stem cells xenografted directly to rat liver are differentiated into human hepatocytes without fusion. Blood, 2005, 106(2): 756-763

[47] Jang Y Y, Collector M I, Baylin S B, Diehl A M, Sharkis S J. Hematopoietic stem cells convert into liver cells within days without fusion. Nat Cell Biol, 2004, 6(6): 532-539

[48] Willenbring H, Bailey A S, Foster M, Akkari Y, Dorrell C, Olson S, Finegold M, Fleming W H, Grompe M. Myelomonocytic cells are sufficient for therapeutic cell fusion in liver. Nat Med, 2004, 10(7): 744-748

[49] Quintana-Bustamante O, Alvarez-Barrientos A, Kofman A V, Fabregat I, Bueren J A, Theise N D, Segovia J C. Hematopoietic mobilization in mice increases the presence of bone marrow-derived hepatocytes via in vivo cell fusion. Hepatology, 2006, 43(1): 108-116

[50] Rodić N, Rutenberg M S, Terada N. Cell fusion and reprogramming: resolving our transdifferences. Trends Mol Med, 2004, 10(3): 93-96

[51] Dahlke M H, Popp F C, Larsen S, Schlitt H J, Rasko J E. Stem cell therapy of the liver—fusion or fiction? Liver Transpl, 2004, 10(4): 471-479

[52] Camargo F D, Finegold M, Goodell M A. Hematopoietic myelomonocytic cells are the major source of hepatocyte fusion partners. J Clin Invest, 2004, 113(9): 1266-1270

[53] Krause P, Saghatolislam F, Koenig S, Unthan-Fechner K, Probst I. Maintaining hepatocyte differentiation in vitro through co-culture with hepatic stellate cells. In Vitro Cell Dev Biol Anim, 2009, 45(5-6): 205-212

[54] Woodrow K A, Wood M J, Saucier-Sawyer J K, Solbrig C, Saltzman W M. Biodegradable meshes printed with extracellular matrix proteins support micropatterned hepatocyte cultures. Tissue Eng Part A, 2009, 15(5): 1169-1179

[55] Wen F, Chang S, Toh Y C, Arooz T, Zhuo L, Teoh S H, Yu H. Development of dual-compartment perfusion bioreactor for serial coculture of hepatocytes and stellate cells in poly(lactic-co-glycolic acid)-collagen scaffolds. J Biomed Mater Res B Appl Biomater, 2008, 87(1): 154-162

[56] Jindal R, Nahmias Y, Tilles A W, Berthiaume F, Yarmush M L. Amino acid-mediated heterotypic interaction governs performance of a hepatic tissue model. FASEB J, 2009, 23(7): 2288-2298

[57] Xiong A, Austin T W, Lagasse E, Uchida N, Tamaki S, Bordier B B, Weissman I L, Glenn J S, Millan M T. Isolation of human fetal liver progenitors and their enhanced proliferation by three-dimensional coculture with endothelial cells. Tissue Eng Part A, 2008, 14(6): 995-1006

[58] Hoshiba T, Cho C S, Murakawa A, Okahata Y, Akaike T. The effect of natural extracellular matrix deposited on synthetic polymers on cultured primary hepatocytes. Biomaterials, 2006, 27(26): 4519-4528

[59] Hoshiba T, Wakejima M, Cho C S, Shiota G, Akaike T. Different regulation of hepatocyte behaviors between natural extracellular matrices and synthetic extracellular matrices by hepatocyte growth factor. J Biomed Mater Res A, 2008, 85(1): 228-235

[60] Hidaka M, Su G N, Chen J K, Mukaisho K, Hattori T, Yamamoto G. Transplantation of engineered bone tissue using a rotary three-dimensional culture system. In Vitro Cell Dev Biol Anim, 2007, 43(2): 49-58

[61] Chen Z, Qi L Z, Zeng R, Li H Y, Dai L J. Stem cells and hepatic cirrhosis. Panminerva Med, 2010, 52(2): 149-165

[62] Russo F P, Alison M R, Bigger B W, Amofah E, Florou A, Amin F, Bou-Gharios G, Jeffery R, Iredale J P, Forbes S J. The bone marrow functionally contributes to liver fibrosis. Gastroenterology, 2006, 130(6): 1807-1821

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