International Journal of Stem Cells Vol. 2, No. 2, 2009
SPECIAL ISSUE
Liver Stem Cells Derived from the Bone Marrow and Umbilical Cord Blood Kyung Ha Ryu Department of Pediatrics, College of Medicine, Ewha womans University, Seoul, Korea
stem cells possible. In other words, somatic stem cells generate differentiated cells beyond their own tissue boundaries, a process referred to as "developmental plasticity".
A new concept in stem cell therapy Current medical investigations of diseases that damage organs now focus on replacement of the damaged cells with healthy cells in addition to pharmacological treatments. Moreover, regeneration of an organ using stem cells has now become an important focus of study (Fig. 1). Stem cells are used for organ regeneration due to their well-known unique characteristics. First, multipotent stem cells are long-term sources of cells due to their capacity for self renewal; second, stem cells can mobilize to injured organs. Stem cells can be divided into two different types: the embryonic stem cells and the somatic stem cells (1). Both types of stem cells have merits and limitations with regard t to the treatment of disease. For example, the embryonic stem cells are excellent with regard to proliferation; however, there are problems with directing them to differentiate to the cell type needed for the damaged organ. On the other hand, somatic stem cells are likely to be capable of site-specific differentiation; however, can they proliferate to the degree needed to provide functional recovery of the damaged organ (2). A single, fertilized egg develops into embryonic stem cells, multipotent stem cells and tissue specific stem cells. These cells differentiate into blood, liver, nerve and muscle stem cells that finally develop into an individual human being. However, multipotent stem cells can be obtained from tissues, making cell therapy using somatic
The use of stem cells for liver regeneration Allogeneic liver transplantation remains the only effective treatment available for patients with liver failure. However, due to the serious shortage of liver donors, other alternative therapeutic approaches are urgently needed. Transplantation of hepatocytes derived from adult or fetal livers are not a good candidate for alternative treatment because the source of such cells is presently limited to the human liver. Therefore, extrahepatic sources of cells with a hepatocyte lineage have been explored for use in cell therapy (3-5). Recently, stem cell-based cytotherapy has shown promising results in animal models and in some patients (6). This review highlights the recent progress in this field, emphasizing the therapeutic potential of bone marrow (BM) or umbilical cord blood (UCB)-derived mesenchymal stem cells (MSCs) used for the treatment of liver diseases. UCB transplantation for a variety of hematopoietic diseases has resulted in successful hematopoietic reconstitution and a lower incidence of graft-versus-host disease than was generally obtained with BM transplantation. Furthermore, the use of UCB creates no ethical problems for laboratory based studies or clinical applications. UCB cells can be collected without any harm to the newborn infant. In addition, UCB hematopoietic stem cell grafts can be cryopreserved and transplanted to a host, after thawing without losing their ability to reproduce. For these reasons, UCB might be a reliable source of cells for transplantation in a variety of diseases. BM derived cells have several advantages. BM samples
Accepted for publication October 25, 2009 Correspondence to Kyung Ha Ryu Department of Pediatrics, School of Medicine, Ewha Womans University, 911-1, Mok-dong, Yangchun-gu, Seoul 158-710, Korea Tel: +82-2-2650-2678, Fax: +82-2-2653-3718 E-mail: [email protected]
97
98 International Journal of Stem Cells 2009;2:97-101
Fig. 1. The direction of cell therapy in medical progress.
can be obtained from living donors or from the recipients themselves with a moderately invasive procedure. There is no problem with limited donors, which a problem that has greatly restricted liver and hepatocyte transplantations (7). Based on their ability for self-renewal, the amplification of BM stem cells can be performed. Utilizing the patient’s own BM for repopulating both the BM and the hepatic system might be a useful approach that can avoid or reduce the immunological reactions that usually affect recipients for their entire life. However, the utilization of human BM as the graft source is limited by a shortage of healthy stem cell donors.
The presence of liver stem cells in BM and UCB The first demonstration of the existence of putative liver stem cells in the BM was reported by Petersen et al in 1999 (8). They showed that bone marrow cells transplanted into lethally irradiated mice was engrafted in the recipient's liver and differentiated into liver stem cells (oval cells) or mature hepatocytes. Hepatocytes were originally described as differentiating from the endoderm during the process of embryonic development. Recent studies have shown, however, that hepatocytes can also be derived from a mesodermal origin such as the BM and UCB. Hepatocytes derived from BM have also been found in the liver after BM transplantation was performed in humans (9). In addition, it has been reported that UCB contains mesenchymal progenitor cells that are capable of differentiating into marrow stroma, bone, cartilage, muscle, connective tissues and hepatocytes. MSCs show different characteristics compared to other components when differentiating into hepatocytes. In a previous study, we investigated, in vitro, whether
UCB and BM contain hepatoblasts that could supply hepatocytes and cholangiocytes as potential sources of transplantation cells for liver injury (10). Mononuclear cells from UCB and BM were cultured with fibroblast growth factor (FGF)-1, FGF-2, stem cell factor (SCF) and hepatocyte growth factor (HGF). The morphology of the cultured cells was monitored by inverted light microscopy. The cultured cells were analyzed by reverse transcriptionpolymerase chain reaction (RT-PCR) and immunofluorescent (IF) staining analysis to detect hepatocyte lineage markers. In addition, flow cytometry was used to determine whether hepatocyte lineage differentiation occurred rather than hematopoietic differentiation. When the hepatocyte lineage markers were examined, ALB mRNA was detected in the UCB and BM cells in the 7 day culture that contained exogenous growth/differentiation factors. RT-PCR analysis revealed that transcripts of CK-18 and AFP were also expressed in the 7 day cultured cells. When comparing the UCB and BM, the BM derived cells produced more dense RT-PCR bands throughout the culture period with the same dose of cells compared to the UCB (10). The results showed that human UCB as well as BM cells generated cells of hepatic and cholangiocytic lineage in the primary culture system supplemented with a combination of growth/differentiation factors. These findings indicate that UCB and BM-derived hepatocytes may play a useful role in the treatment of liver failure. Therefore, human UCB and BM might provide potential alternative sources of cells that could be used for the treatment of liver failure.
Kyung Ha Ryu: Liver Stem Cells Derived from the Bone Marrow and Umbilical Cord Blood 99
Mechanism of BM cells homing to the liver The potential therapeutic benefit of MSCs can only be realized through their homing efficiency to the required site. The true identity of the circulating stem cell population and the factors responsible for recruitment/chemotaxis are poorly understood (11). The following questions have been frequently encountered while pursuing cell-based therapeutic investigations: what is the best method for the delivery of cells, how do the cells get to the sites of injury and by what mechanisms are they targeted? The methods of MSC administration can be classified into three categories, directional or site-specific delivery, semi-directional delivery, and systemic delivery (12). For examples, MSCs have been administered to the injured liver by intrahepatic injection, which is considered directional delivery (13). Intrasplenic injection (14) and intravenous infusion (15) are examples of semi-directional and systemic delivery. Under certain circumstances, a combination of more than one of above mentioned method and repeated administration may also be considered. The migration of MSC from the circulation into the damaged tissues is the most crucial step for effective MSC therapy. Biological signals released from the injured area and corresponding receptors expressed on the cell surface of MSCs are critical determinants of this step (16). Stem cell migration with tissue damage is a complicated and highly regulated process coordinated by a large number of growth factors, cytokines, and chemokines, all of which are derived from a variety of tissues that are usually not found under normal conditions; for example, SDF-1α increases after tissue injury and increases further after BM cell infusion. They were stained within hepatocytes, thus indicating their production origin, and BM cell recruitment. In our previous study, we used MHC-identical mice as a pair. CCl4 was administered to induce liver cirrhosis. Then, MHC-identical BM cells from the uninjured mouse were infused into the liver injured recipient. The CCl4 infusion group showed marked hepatosplenomegaly and hepatic fibrosis; however, these findings resolved and the liver recovered to a normal condition after BM cell infusion (17). In addition, hematopoietic stem cell markers of the CCl4 injured liver were increased after BM transplantation. Intravenously injected CD34 and CD45 positive BM cells homed to the injured liver rather than BM and spleen by seven days after the transplantation (6). The
CCl4 only treated mouse liver also showed a high fraction of hematopoietic stem cell marker positive cells compared to the normal liver. Therefore, BM cells appear to have the potential to respond to stress signals from an injured liver, which is important for tissue targeting and repair (17). In an animal study, we observed the recovery of hepatic function and histological recovery of hepatic fibrosis of a mouse with liver cirrhosis after receiving BM cell transplantation from a healthy mouse. Externally injected BM cells, as well as endogenous stem cells, have a pivotal role in this process and the chemokine, SDF-1, secreted from injured liver tissue has a role in the homing process (16).
Clinical application and perspectives The translation of preclinical research on MSCs to clinical use on cirrhotic patients has generated great interest, due to the growing population of patients with advanced liver disease and the critical shortage of available liver donors. In cardiology, large-scale controlled and double-blinded clinical trials were performed in a number of centers (18). Some studies have demonstrated clinical benefits, whereas in others the differences have been less significant (Table 1) (9, 19-25). Especially G-CSF-based BM cell mobilization, induced improvement of liver function in chronic liver failure in one case (Case No 3 Table) (21); this procedure was safe and effective and can be exploited as a simple therapeutic strategy in patients with end stage liver disease. In addition, Yannaki E et. al (20). reported two cases treated with booster infusions of autologous mobilized hematopoietic stem cells to regenerate a cirrhotic liver (Case No 4 Table). In this report the clinical course improved over the 30 months of follow up. Based on the knowledge from preclinical studies and previous clinical trials, the following considerations should be addressed in clinical trials of patients with liver disease: 1) Utilization of purified MSC population, 2) MSC passage, 3) MSC delivery route and 4) Evaluation standards. Because bone marrow derived fibrogenic cells play a role in the progression of liver fibrogenesis, the use of a purified MSC population could avoid this complication. MSCs isolated from patients share the same surface markers and similar biological characteristics as those isolated from healthy humans (26, 27). About one billion or at least one million/kg body weight of MSCs are required for each transplantation. Longer in vitro culture time and unnecessary manipulation may introduce more unexpected effects in the ex vivo expanded MSCs. Three to five-passage cultures are known to be safe and practical. During this culture period
100 International Journal of Stem Cells 2009;2:97-101
Table 1. Clinical trials using somatic stem cells for liver regeneration No of patients
Type of stem cell infused
Infusion route
Results
Autologous bone marrow
3
CD133＋ cells
PV
Mobilized autologous PB Mobilization only Mobilized autologous PB Autologous bone marrow Autologous bone marrow
5
CD34＋ cells
PV
−
−
Liver volume increase LFT improve CP, MELD improve CP, MELD improve CP improve LFT improve
No
Liver condition
Source of stem cells
1
Waiting extensive hepatectomy Chronic liver failure Liver cirrhosis
2 3 4 5
Alcoholic cirrhosis Liver cirrhosis
6
Chronic liver failure
7
Waiting extensive hepatectomy Liver cirrhosis
8 2 9
MNCs Booster (3) MNCs
PB
MNCs
HA
CD133＋ cells
PV
MNCs
HA
PB
10
13
8
Autologous bone marrow 9 Autologous bone marrow
MSC retain their cytogenetic stability; sufficient numbers of cells can be obtained for transplantation starting with 10 to 15 ml of BM aspirate. Intravenous infusion is the first choice in most circumstances. Although portal vein or hepatic artery delivery may enhance MSC homing efficacy, precautions must be taken when catheterization is used in patients with liver disease (13-15). In order to objectively assess the therapeutic effects of MSC transplantation, standards must be established prior to MSC administration. The following should be included: I) patient enrollment requirements, e.g., age, gender and type of hepatic disease; II) hepatic function assessment; III) liver parenchyma assessment, e.g., ultrasonography, computed tomography and MRI; and IV) hepatic fibrosis assessment (22).
Conclusions Although the mechanism of action of stem cells in the human liver remains elusive, there are new insights into the varied effects of different subclasses of stem cells in the injured liver based on the results of animal studies. Even though stem cell clinical trials do not show an unequivocal benefit to date, they do provide renewed hope for the future, which is timely in the current climate of increasing morbidity and mortality associated with trans-
Liver volume increase MELD not improve
References Stem Cells 2005;23:463 Stem Cells 2006;24:1822 J Hepatol 2006;45:13 Exp Hematol 2006;34:1583 Stem Cells 2006;24:2292 World J Gastroenterol 2007;13:1067 Radiology 2007;243:171 World J Gastroenterol 2007;13:3359
plant waiting lists around the world. Whether stem cell therapy can provide a bridge to definitive treatment or is the definitive treatment requires further research.
Acknowledgements This study was supported by a grant of the Korean Health Technology R&D Project, Ministry for Health, Welfare & Family Affairs, Republic of Korea (A084067). Potential Conflict of Interest The authors have no conflicting financial interest.
References 1. Andersson ER, Lendahl U. Regenerative medicine: a 2009 overview. J Intern Med 2009;266:303-310 2. Colman A, Dreesen O. Induced pluripotent stem cells and the stability of the differentiated state. EMBO Rep 2009;10: 714-721 3. Cantz T, Manns MP, Ott M. Stem cells in liver regeneration and therapy. Cell Tissue Res 2008;331:271-282 4. Kuo TK, Hung SP, Chuang CH, Chen CT, Shih YR, Fang SC, Yang VW, Lee OK. Stem cell therapy for liver disease: parameters governing the success of using bone marrow mesenchymal stem cells. Gastroenterology 2008;134:2111-2121 5. Fujii H, Hirose T, Oe S, Yasuchika K, Azuma H, Fujikawa T, Nagao M, Yamaoka Y. Contribution of bone marrow cells to liver regeneration after partial hepatectomy in mice. J Hepatol 2002;36:653-659
Kyung Ha Ryu: Liver Stem Cells Derived from the Bone Marrow and Umbilical Cord Blood 101
6. Jung YJ, Ryu KH, Cho SJ, Woo SY, Seoh JY, Chun CH, Yoo K, Moon IH, Han HS. Syngenic bone marrow cells restore hepatic function in carbon tetrachloride-induced mouse liver injury. Stem Cells Dev 2006;15:687-695 7. Dai LJ, Li HY, Guan LX, Ritchie G, Zhou JX. The therapeutic potential of bone marrow-derived mesenchymal stem cells on hepatic cirrhosis. Stem Cell Res 2009;2:16-25 8. Petersen BE, Bowen WC, Patrene KD, Mars WM, Sullivan AK, Murase N, Boggs SS, Greenberger JS, Goff JP. Bone marrow as a potential source of hepatic oval cells. Science 1999;284:1168-1170 9. Gordon MY, Levicar N, Pai M, Bachellier P, Dimarakis I, Al-Allaf F, M'Hamdi H, Thalji T, Welsh JP, Marley SB, Davies J, Dazzi F, Marelli-Berg F, Tait P, Playford R, Jiao L, Jensen S, Nicholls JP, Ayav A, Nohandani M, Farzaneh F, Gaken J, Dodge R, Alison M, Apperley JF, Lechler R, Habib NA. Characterization and clinical application of human CD34＋ stem/progenitor cell populations mobilized into the blood by granulocyte colony-stimulating factor. Stem Cells 2006;24:1822-1830 10. Jung YJ, Ryu KH, Cho KA, Woo SY, Seoh JY, Cho SJ, Joo SY, Yoo K, Ho-Seoung H. In vitro hepatic differentiation of human umbilical cord blood and bone marrow cells. Pediatr Hematol Oncol Sep 2008;25:481-491 11. Jung YJ, Ju SY, Yoo ES, Cho SJ, Cho KA, Woo SY, Seoh JY, Park JW, Han HS, Ryu KH. MSC-DC interactions: MSC inhibit maturation and migration of BM-derived DC. Cytotherapy 2007;9:451-458 12. Zhang ZX, Guan LX, Zhang K, Zhang Q, Dai LJ. A combined procedure to deliver autologous mesenchymal stromal cells to patients with traumatic brain injury. Cytotherapy 2008;10:134-139 13. 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;106:756-763 14. Aurich I, Mueller LP, Aurich H, Luetzkendorf J, Tisljar K, Dollinger MM, Schormann W, Walldorf J, Hengstler JG, Fleig WE, Christ B. Functional integration of hepatocytes derived from human mesenchymal stem cells into mouse livers. Gut 2007;56:405-415 15. Fang B, Shi M, Liao L, Yang S, Liu Y, Zhao RC. Systemic infusion of FLK1(＋) mesenchymal stem cells ameliorate carbon tetrachloride-induced liver fibrosis in mice. Transplantation 2004;78:83-88 16. Cho KA, Ju SY, Cho SJ, Jung YJ, Woo SY, Seoh JY, Han HS, Ryu KH. 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;33:772-777 17. Ju SY, Cho KA, Cho SJ, Jung YJ, Woo SY, Seoh JY, Han HS, Ryu KH. Effect of hypoxic treatment on bone marrow cells that are able to migrate to the injured liver. Cell Biol
Int 2009;33:31-35 18. Kang HJ, Kim HS, Zhang SY, Park KW, Cho HJ, Koo BK, Kim YJ, Soo Lee D, Sohn DW, Han KS, Oh BH, Lee MM, Park YB. Effects of intracoronary infusion of peripheral blood stem-cells mobilised with granulocyte-colony stimulating factor on left ventricular systolic function and restenosis after coronary stenting in myocardial infarction: the MAGIC cell randomised clinical trial. Lancet 2004;363:751-756 19. Am Esch JS 2nd, Knoefel WT, Klein M, Ghodsizad A, Fuerst G, Poll LW, Piechaczek C, Burchardt ER, Feifel N, Stoldt V, Stockschläder M, Stoecklein N, Tustas RY, Eisenberger CF, Peiper M, Häussinger D, Hosch SB. Portal application of autologous CD133＋ bone marrow cells to the liver: a novel concept to support hepatic regeneration. Stem Cells 2005;23:463-470 20. Yannaki E, Anagnostopoulos A, Kapetanos D, Xagorari A, Iordanidis F, Batsis I, Kaloyannidis P, Athanasiou E, Dourvas G, Kitis G, Fassas A. Lasting amelioration in the clinical course of decompensated alcoholic cirrhosis with boost infusions of mobilized peripheral blood stem cells. Exp Hematol 2006;34:1583-1587 21. Gaia S, Smedile A, Omedè P, Olivero A, Sanavio F, Balzola F, Ottobrelli A, Abate ML, Marzano A, Rizzetto M, Tarella C. Feasibility and safety of G-CSF administration to induce bone marrow-derived cells mobilization in patients with end stage liver disease. J Hepatol 2006;45:13-19 22. Terai S, Ishikawa T, Omori K, Aoyama K, Marumoto Y, Urata Y, Yokoyama Y, Uchida K, Yamasaki T, Fujii Y, Okita K, Sakaida I. Improved liver function in patients with liver cirrhosis after autologous bone marrow cell infusion therapy. Stem Cells 2006;24:2292-2298 23. Lyra AC, Soares MB, da Silva LF, Fortes MF, Silva AG, Mota AC, Oliveira SA, Braga EL, de Carvalho WA, Genser B, dos Santos RR, Lyra LG. Feasibility and safety of autologous bone marrow mononuclear cell transplantation in patients with advanced chronic liver disease. World J Gastroenterol 2007;13:1067-1073 24. Mohamadnejad M, Namiri M, Bagheri M, Hashemi SM, Ghanaati H, Zare Mehrjardi N, Kazemi Ashtiani S, Malekzadeh R, Baharvand H. Phase 1 human trial of autologous bone marrow-hematopoietic stem cell transplantation in patients with decompensated cirrhosis. World J Gastroenterol 2007;13:3359-3363 25. Fürst G, Schulte am Esch J, Poll LW, Hosch SB, Fritz LB, Klein M, Godehardt E, Krieg A, Wecker B, Stoldt V, Stockschläder M, Eisenberger CF, Mödder U, Knoefel WT. Portal vein embolization and autologous CD133＋ bone marrow stem cells for liver regeneration: initial experience. Radiology 2007;243:171-179 26. Houlihan DD, Newsome PN. Critical review of clinical trials of bone marrow stem cells in liver disease. Gastroenterology 2008;135:438-450 27. Friedman SL. Mechanisms of hepatic fibrogenesis. Gastroenterology 2008;134:1655-1669
SPECIAL ISSUE
Liver Stem Cells Derived from the Bone Marrow and Umbilical Cord Blood Kyung Ha Ryu Department of Pediatrics, College of Medicine, Ewha womans University, Seoul, Korea
stem cells possible. In other words, somatic stem cells generate differentiated cells beyond their own tissue boundaries, a process referred to as "developmental plasticity".
A new concept in stem cell therapy Current medical investigations of diseases that damage organs now focus on replacement of the damaged cells with healthy cells in addition to pharmacological treatments. Moreover, regeneration of an organ using stem cells has now become an important focus of study (Fig. 1). Stem cells are used for organ regeneration due to their well-known unique characteristics. First, multipotent stem cells are long-term sources of cells due to their capacity for self renewal; second, stem cells can mobilize to injured organs. Stem cells can be divided into two different types: the embryonic stem cells and the somatic stem cells (1). Both types of stem cells have merits and limitations with regard t to the treatment of disease. For example, the embryonic stem cells are excellent with regard to proliferation; however, there are problems with directing them to differentiate to the cell type needed for the damaged organ. On the other hand, somatic stem cells are likely to be capable of site-specific differentiation; however, can they proliferate to the degree needed to provide functional recovery of the damaged organ (2). A single, fertilized egg develops into embryonic stem cells, multipotent stem cells and tissue specific stem cells. These cells differentiate into blood, liver, nerve and muscle stem cells that finally develop into an individual human being. However, multipotent stem cells can be obtained from tissues, making cell therapy using somatic
The use of stem cells for liver regeneration Allogeneic liver transplantation remains the only effective treatment available for patients with liver failure. However, due to the serious shortage of liver donors, other alternative therapeutic approaches are urgently needed. Transplantation of hepatocytes derived from adult or fetal livers are not a good candidate for alternative treatment because the source of such cells is presently limited to the human liver. Therefore, extrahepatic sources of cells with a hepatocyte lineage have been explored for use in cell therapy (3-5). Recently, stem cell-based cytotherapy has shown promising results in animal models and in some patients (6). This review highlights the recent progress in this field, emphasizing the therapeutic potential of bone marrow (BM) or umbilical cord blood (UCB)-derived mesenchymal stem cells (MSCs) used for the treatment of liver diseases. UCB transplantation for a variety of hematopoietic diseases has resulted in successful hematopoietic reconstitution and a lower incidence of graft-versus-host disease than was generally obtained with BM transplantation. Furthermore, the use of UCB creates no ethical problems for laboratory based studies or clinical applications. UCB cells can be collected without any harm to the newborn infant. In addition, UCB hematopoietic stem cell grafts can be cryopreserved and transplanted to a host, after thawing without losing their ability to reproduce. For these reasons, UCB might be a reliable source of cells for transplantation in a variety of diseases. BM derived cells have several advantages. BM samples
Accepted for publication October 25, 2009 Correspondence to Kyung Ha Ryu Department of Pediatrics, School of Medicine, Ewha Womans University, 911-1, Mok-dong, Yangchun-gu, Seoul 158-710, Korea Tel: +82-2-2650-2678, Fax: +82-2-2653-3718 E-mail: [email protected]
97
98 International Journal of Stem Cells 2009;2:97-101
Fig. 1. The direction of cell therapy in medical progress.
can be obtained from living donors or from the recipients themselves with a moderately invasive procedure. There is no problem with limited donors, which a problem that has greatly restricted liver and hepatocyte transplantations (7). Based on their ability for self-renewal, the amplification of BM stem cells can be performed. Utilizing the patient’s own BM for repopulating both the BM and the hepatic system might be a useful approach that can avoid or reduce the immunological reactions that usually affect recipients for their entire life. However, the utilization of human BM as the graft source is limited by a shortage of healthy stem cell donors.
The presence of liver stem cells in BM and UCB The first demonstration of the existence of putative liver stem cells in the BM was reported by Petersen et al in 1999 (8). They showed that bone marrow cells transplanted into lethally irradiated mice was engrafted in the recipient's liver and differentiated into liver stem cells (oval cells) or mature hepatocytes. Hepatocytes were originally described as differentiating from the endoderm during the process of embryonic development. Recent studies have shown, however, that hepatocytes can also be derived from a mesodermal origin such as the BM and UCB. Hepatocytes derived from BM have also been found in the liver after BM transplantation was performed in humans (9). In addition, it has been reported that UCB contains mesenchymal progenitor cells that are capable of differentiating into marrow stroma, bone, cartilage, muscle, connective tissues and hepatocytes. MSCs show different characteristics compared to other components when differentiating into hepatocytes. In a previous study, we investigated, in vitro, whether
UCB and BM contain hepatoblasts that could supply hepatocytes and cholangiocytes as potential sources of transplantation cells for liver injury (10). Mononuclear cells from UCB and BM were cultured with fibroblast growth factor (FGF)-1, FGF-2, stem cell factor (SCF) and hepatocyte growth factor (HGF). The morphology of the cultured cells was monitored by inverted light microscopy. The cultured cells were analyzed by reverse transcriptionpolymerase chain reaction (RT-PCR) and immunofluorescent (IF) staining analysis to detect hepatocyte lineage markers. In addition, flow cytometry was used to determine whether hepatocyte lineage differentiation occurred rather than hematopoietic differentiation. When the hepatocyte lineage markers were examined, ALB mRNA was detected in the UCB and BM cells in the 7 day culture that contained exogenous growth/differentiation factors. RT-PCR analysis revealed that transcripts of CK-18 and AFP were also expressed in the 7 day cultured cells. When comparing the UCB and BM, the BM derived cells produced more dense RT-PCR bands throughout the culture period with the same dose of cells compared to the UCB (10). The results showed that human UCB as well as BM cells generated cells of hepatic and cholangiocytic lineage in the primary culture system supplemented with a combination of growth/differentiation factors. These findings indicate that UCB and BM-derived hepatocytes may play a useful role in the treatment of liver failure. Therefore, human UCB and BM might provide potential alternative sources of cells that could be used for the treatment of liver failure.
Kyung Ha Ryu: Liver Stem Cells Derived from the Bone Marrow and Umbilical Cord Blood 99
Mechanism of BM cells homing to the liver The potential therapeutic benefit of MSCs can only be realized through their homing efficiency to the required site. The true identity of the circulating stem cell population and the factors responsible for recruitment/chemotaxis are poorly understood (11). The following questions have been frequently encountered while pursuing cell-based therapeutic investigations: what is the best method for the delivery of cells, how do the cells get to the sites of injury and by what mechanisms are they targeted? The methods of MSC administration can be classified into three categories, directional or site-specific delivery, semi-directional delivery, and systemic delivery (12). For examples, MSCs have been administered to the injured liver by intrahepatic injection, which is considered directional delivery (13). Intrasplenic injection (14) and intravenous infusion (15) are examples of semi-directional and systemic delivery. Under certain circumstances, a combination of more than one of above mentioned method and repeated administration may also be considered. The migration of MSC from the circulation into the damaged tissues is the most crucial step for effective MSC therapy. Biological signals released from the injured area and corresponding receptors expressed on the cell surface of MSCs are critical determinants of this step (16). Stem cell migration with tissue damage is a complicated and highly regulated process coordinated by a large number of growth factors, cytokines, and chemokines, all of which are derived from a variety of tissues that are usually not found under normal conditions; for example, SDF-1α increases after tissue injury and increases further after BM cell infusion. They were stained within hepatocytes, thus indicating their production origin, and BM cell recruitment. In our previous study, we used MHC-identical mice as a pair. CCl4 was administered to induce liver cirrhosis. Then, MHC-identical BM cells from the uninjured mouse were infused into the liver injured recipient. The CCl4 infusion group showed marked hepatosplenomegaly and hepatic fibrosis; however, these findings resolved and the liver recovered to a normal condition after BM cell infusion (17). In addition, hematopoietic stem cell markers of the CCl4 injured liver were increased after BM transplantation. Intravenously injected CD34 and CD45 positive BM cells homed to the injured liver rather than BM and spleen by seven days after the transplantation (6). The
CCl4 only treated mouse liver also showed a high fraction of hematopoietic stem cell marker positive cells compared to the normal liver. Therefore, BM cells appear to have the potential to respond to stress signals from an injured liver, which is important for tissue targeting and repair (17). In an animal study, we observed the recovery of hepatic function and histological recovery of hepatic fibrosis of a mouse with liver cirrhosis after receiving BM cell transplantation from a healthy mouse. Externally injected BM cells, as well as endogenous stem cells, have a pivotal role in this process and the chemokine, SDF-1, secreted from injured liver tissue has a role in the homing process (16).
Clinical application and perspectives The translation of preclinical research on MSCs to clinical use on cirrhotic patients has generated great interest, due to the growing population of patients with advanced liver disease and the critical shortage of available liver donors. In cardiology, large-scale controlled and double-blinded clinical trials were performed in a number of centers (18). Some studies have demonstrated clinical benefits, whereas in others the differences have been less significant (Table 1) (9, 19-25). Especially G-CSF-based BM cell mobilization, induced improvement of liver function in chronic liver failure in one case (Case No 3 Table) (21); this procedure was safe and effective and can be exploited as a simple therapeutic strategy in patients with end stage liver disease. In addition, Yannaki E et. al (20). reported two cases treated with booster infusions of autologous mobilized hematopoietic stem cells to regenerate a cirrhotic liver (Case No 4 Table). In this report the clinical course improved over the 30 months of follow up. Based on the knowledge from preclinical studies and previous clinical trials, the following considerations should be addressed in clinical trials of patients with liver disease: 1) Utilization of purified MSC population, 2) MSC passage, 3) MSC delivery route and 4) Evaluation standards. Because bone marrow derived fibrogenic cells play a role in the progression of liver fibrogenesis, the use of a purified MSC population could avoid this complication. MSCs isolated from patients share the same surface markers and similar biological characteristics as those isolated from healthy humans (26, 27). About one billion or at least one million/kg body weight of MSCs are required for each transplantation. Longer in vitro culture time and unnecessary manipulation may introduce more unexpected effects in the ex vivo expanded MSCs. Three to five-passage cultures are known to be safe and practical. During this culture period
100 International Journal of Stem Cells 2009;2:97-101
Table 1. Clinical trials using somatic stem cells for liver regeneration No of patients
Type of stem cell infused
Infusion route
Results
Autologous bone marrow
3
CD133＋ cells
PV
Mobilized autologous PB Mobilization only Mobilized autologous PB Autologous bone marrow Autologous bone marrow
5
CD34＋ cells
PV
−
−
Liver volume increase LFT improve CP, MELD improve CP, MELD improve CP improve LFT improve
No
Liver condition
Source of stem cells
1
Waiting extensive hepatectomy Chronic liver failure Liver cirrhosis
2 3 4 5
Alcoholic cirrhosis Liver cirrhosis
6
Chronic liver failure
7
Waiting extensive hepatectomy Liver cirrhosis
8 2 9
MNCs Booster (3) MNCs
PB
MNCs
HA
CD133＋ cells
PV
MNCs
HA
PB
10
13
8
Autologous bone marrow 9 Autologous bone marrow
MSC retain their cytogenetic stability; sufficient numbers of cells can be obtained for transplantation starting with 10 to 15 ml of BM aspirate. Intravenous infusion is the first choice in most circumstances. Although portal vein or hepatic artery delivery may enhance MSC homing efficacy, precautions must be taken when catheterization is used in patients with liver disease (13-15). In order to objectively assess the therapeutic effects of MSC transplantation, standards must be established prior to MSC administration. The following should be included: I) patient enrollment requirements, e.g., age, gender and type of hepatic disease; II) hepatic function assessment; III) liver parenchyma assessment, e.g., ultrasonography, computed tomography and MRI; and IV) hepatic fibrosis assessment (22).
Conclusions Although the mechanism of action of stem cells in the human liver remains elusive, there are new insights into the varied effects of different subclasses of stem cells in the injured liver based on the results of animal studies. Even though stem cell clinical trials do not show an unequivocal benefit to date, they do provide renewed hope for the future, which is timely in the current climate of increasing morbidity and mortality associated with trans-
Liver volume increase MELD not improve
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plant waiting lists around the world. Whether stem cell therapy can provide a bridge to definitive treatment or is the definitive treatment requires further research.
Acknowledgements This study was supported by a grant of the Korean Health Technology R&D Project, Ministry for Health, Welfare & Family Affairs, Republic of Korea (A084067). Potential Conflict of Interest The authors have no conflicting financial interest.
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