- Email: [email protected]
Hog Heaven on the Road to Liver Cell Therapy
450
EDITORIALS
GASTROENTEROLOGY Vol. 132, No. 1
Hog Heaven on the Road to Liver Cell Therapy
See “Prolonged survival of porcine hepatocytes in cynomolgus monkeys” by Nagata H, Nishitai R, Shirota C, Zhang J-L, Koch CA, Cai J, Awwad M, Schuurman H-J, Christians U, Abe M, Baranowska–Kortylewicz J, Platt JL, Fox IJ, on page 321.
O
ver the past 25 years, travel on the road to liver cell therapy has not always been easy. Initially, when hepatocyte transplantation improved outcomes in animals with liver failure, metabolic disease, and so on, it seemed that clinical applications would follow shortly.1 Early studies of ex vivo gene therapy in familial hypercholesterolemia with autologous hepatocytes,2 and in Crigler-Najjar syndrome with healthy allogeneic hepatocytes,3 highlighted inefficient gene transfer, cell loss during in vitro manipulations, limited cell engraftment, and absence of transplanted cell proliferation as major problems. Since then, much has been accomplished in applying liver cell therapy to people, although more needs to be done.4 Numerous animal studies advanced insights into liver repopulation, including genetic modification of cells with superior vectors and correction of a variety of disorders characterized by healthy and/or diseased livers.5–11 Significant bodies of work have accumulated over the past several years with respect to mechanisms concerning the potential of mature hepatocytes, fetal liver-derived stem/progenitor cells, embryonic stem cells, as well as extrahepatic stem cells from the bone marrow or cord blood.12 Suffice it to say, interest in stem cells capable of repopulating the liver seems appropriate, although fundamental issues remain in respect with generating mature hepatocytes from pluripotential stem cells and whether the progeny of stem cells could be expanded in vitro and in vivo without oncogenicity in case of embryonic stem cells and of the proclivity for cell fusion in case of hematopoietic stem cells. Use of embryonic stem cells, in particular, needs further resolution of social and political issues. However, fetal human liver stem/progenitor cells show extensive replication and differentiation potential, including the capacity to generate mature hepatocytes after transplantation in immunodeficient animals.13,14 Indeed, fetal rodent cells showed superior replication potential compared with adult hepatocytes.15 Significant numbers of epithelial cells can be isolated from a midgestation fetal human liver and these cells can be expanded enormously in vitro, by up to 5 ⫻ 107-fold, that is, trillions of cells could be generated from a single
fetal liver. However, the supply of fetal human tissues is also not unlimited. The replication potential of fetal liver cells has been extended through reconstitution of telomerase activity,16,17 which maintains chromosomal integrity during cell division, although the possibility of oncogenic perturbations needs further study. The choice of donor cells is particularly relevant because mature hepatocytes themselves possess extensive replication capacity, as shown, for example, in FAH mutant mice, which model hereditary tyrosinemia type 1.18 Suitable mechanisms have been identified to alter the condition of healthy rat and mouse livers before cell transplantation. Under these circumstances, transplanted hepatocytes can replace virtually the entire liver over several weeks.7,11,15 Although animal studies often use toxic chemicals, more recently, genotoxicity with radiation plus ischemia–reperfusion proved effective in liver repopulation.11 After suitable types of liver injury, transplanted cells require ⬍10 population doublings to repopulate the healthy rat liver. Therefore, clinical protocols may require transplantation of relatively few cells to repopulate the liver. To ensure that transplanted cells have the best opportunities for engrafting and proliferating in the liver, critical mechanisms have been examined, extending from the hepatic vasomotor tone, sinusoidal endothelial barrier, receptor-mediated cell targeting, and cell– cell interactions, which regulate transplanted cell engraftment, the role of the hepatic microenvironment in regulating the function of transplanted cells, and the underlying state of the hepatic parenchyma and modifying factors that determine whether transplanted cells will proliferate.19 –23 On the other hand, the shortage of donor human organs represents a major road-bump in clinical applications of cell therapy. To overcome this barrier, Nagata et al24 propose in this issue of GASTROENTEROLOGY that xenogeneic pig hepatocytes will be suitable for transplantation. Through careful studies in cynomolgus monkeys— by no means an easy task—they offer convincing evidence that porcine cells engrafted and functioned under immunosuppression conditions that can be used in humans. This represents a significant advance in considering xenogeneic donors for liver cell therapy. Looking back at xenotransplantation, you might be surprised to learn from an excellent recent review that animal products had already been transplanted into people in the 16th century!25 In the ancient Hindu tradition, Lord Ganesha is shown to bear an elephant head on a human torso. The myth goes that upon returning from a trip, Ganesha’s father, the omnipotent God, Shiva, found him lying next to his consort, the Goddess Parvati, and in
January 2007
a jealous rage beheaded Ganesha. Shiva did not realize that Ganesha, who was born large, was his own recentborn son. Parvati went into great agitation and threatened to destroy the entire universe unless Shiva restored Ganesha to her. Therefore, in mad haste, Shiva found a head that could fit the torso of Ganesha, and that turned out to be from an elephant! Over the past centuries, tissue and cell xenotransplants in people have been documented from diverse animals, including rabbit, guinea pig, lamb, sheep, dog, calf, pig, ape, and baboon. Remarkably, from 1969 to 1993, chimpanzee, baboon, and pig livers were transplanted into 11 people, including by Starzl and Makowka in the United States; the longest survival, however, was 70 days. Survival of xenogeneic tissues, cells, or organs was limited by a robust host immune response. Therefore, it is with this background that one needs to consider xenotransplantation, particularly pig tissues and organs, as these are most readily procured for human applications. Recent studies identified fundamental differences in the immune response to allografts versus xenografts. For instance, host immune responses after xenografting of porcine solid organs is characterized by hyperacute rejection (HAR), with irreversible graft injury within minutes to hours due to pre-existing host xenoreactive natural antibodies (XNA).26 This is followed by acute humoral xenograft rejection (AHXR), largely from activation of macrophages, neutrophils, CD8⫹ T cells and natural killer cells, within 24 hours after xenografting. The processes of HAR and AHXR constitute critical host immune responses with rapid and severe endothelial damage leading to xenograft loss. Fortunately, progress in understanding the pathophysiology of these processes is beginning to yield ways to modulate HAR and AHXR. For instance, the most important XNA is against the porcine galactosyltransferase epitope, Gal␣1-3 Gal14GlcNAc-R. Recently, genetically modified pigs lacking this epitope were generated,27 which in combination with strategies to avoid activation of the complement cascade will help in solid organ xenografts.26 Similarly, strategies are being developed to eliminate AHXR. The initial process of HAR and AHXR may be followed by an acute cellular xenograft response, characterized by cellular reactions without vascular thrombosis, and finally, chronic xenorejection, which is less well characterized. Transplantation of nonvascularized tissues (eg, pancreatic islets) elicits a different xenograft response, due to low or minimal expression of the ␣-Gal epitope on cells compared with solid organs, as well as the absence of XNA response.28,29 In recent studies, transplanted porcine islets were shown to restore insulin secretion in monkeys over the long term (⬎180 days). However, porcine islets in monkeys still generated inflammatory reaction, including activation of complement and inflammatory cells, with damage to transplanted islets.
EDITORIALS
451
As indicated by Nagata et al,24 in previous studies, porcine hepatocytes ameliorated serum cholesterol levels in Watanabe rabbits,30 and in their own studies improved outcomes in rats with advanced liver cirrhosis.9 The latter studies used an extrahepatic reservoir of liver cells in the spleen, such that transplanted cells would supplement the metabolic capacity of the cirrhotic liver. Similarly, an extrahepatic reservoir of cells was created with porcine hepatocytes in monkeys,24 by mixing sodium alginate to promote cell entrapment in the spleen. Recipient monkeys were immunosuppressed, with induction using thymoglobulin, methylprednisolone, and an anti–interleukin-2 receptor antibody, followed by maintenance with cyclosporine, everolimus—an inhibitor of the mammalian target of rapamycin (mTOR)—the investigational drug FTY720, and methylprednisolone. Transplanted porcine hepatocytes secreted albumin, expressed the asialoglycoprotein receptor, which was identified by scintigraphic studies using a radioligand, and survived in monkeys for 25– 86 days. Cell recipients did not express significant XNA responses and antibodies against the ␣-Gal epitope were mostly absent. Moreover, porcine hepatocytes were successfully retransplanted and in one monkey, transplanted cells were shown to function for a total of 253 days. These studies established that xenogeneic porcine hepatocytes will be less immunogenic than the whole organ and perhaps other types of xenografts, such as pancreatic islets.28,29 Although immunosuppression was associated with some infectious complications in monkeys, drug dosing can obviously be adjusted to avoid excessive immunosuppression. In choosing the most effective immunosuppression regime, it should be noteworthy that drugs can affect transplanted cells. For instance, the mTOR inhibitory drug, rapamycin, impaired transplanted cell proliferation.22 Nagata et al24 indicated that in monkeys with an intact liver, it was difficult to assess the metabolic contribution of transplanted cells, and that due to feedback regulation, serum albumin levels would not have been effective for such analysis. Nonetheless, creating an extrahepatic reservoir of liver cells in the proposed manner should be helpful in tiding over patients with liver failure. In the setting of acute liver failure, temporary metabolic support should facilitate recovery of the native organ, or at least offer a bridge to orthotopic liver transplantation. Similarly, during decompensation in chronic liver disease, an additional hepatocyte reservoir should help to treat complications such as hepatic encephalopathy, although in debilitated individuals, immunosuppression would increase the risk of infectious complications. Cell transplantation in the setting of chronic liver failure could potentially help prolong life, as shown by Nagata et al in studies of porcine cell transplantation in cirrhotic rats.9 This new road of porcine hepatocyte xenografting will be of much interest for addressing the potential of liver
452
EDITORIALS
repopulation in suitable experimental systems. Perhaps it is appropriate to consider additional issues regarding porcine xenografts in this context. For instance, concerns have been raised regarding molecular differences in complement, coagulation systems, and soluble human and porcine molecules.31 However, such concerns must be considered separately from solid organ xenotransplants, because cell transplants need not replace an organ entirely, and native proteins should still be available. The possibility of zoonotic disease transmission, for example, porcine endogenous retrovirus, which is embedded in the pig genome, and could possibly be reactivated through genetic recombination,32 has been under debate. So far, emergence of active porcine endogenous retrovirus infection in recipients of porcine material has, reassuringly, not been documented.
SANJEEV GUPTA Marion Bessin Liver Research Center Diabetes Center, Cancer Research Center, and Center for hESC Research Departments of Medicine and Pathology Albert Einstein College of Medicine Bronx, New York References 1. Gupta S, Roy Chowdhury J. Hepatocyte transplantation: back to the future! Hepatology 1992;15:156 –162. 2. Fisher RA, Strom SC. Human hepatocyte transplantation: worldwide results. Transplantation 2006;82:441– 449. 3. Grossman M, Rader DJ, Muller DWM, Kolansky DM, Kozarsky K, Clark, BJ III, Stein EA, Lupien PJ, Brewer, BH Jr, Raper SE, Wilson JM. A pilot study of ex vivo gene therapy for homozygous familiar hypercholesterolaemia. Nat Med 1995;1:1148 –1154. 4. Fox IJ, Chowdhury JR, Kaufman SS, Goertzen TC, Chowdhury NR, Warkentin PI, Dorko K, Sauter BV, Strom SC. Treatment of the Crigler-Najjar syndrome type I with hepatocyte transplantation. N Engl J Med 1998;338:1422–1426. 5. De Vree JM, Ottenhoff R, Bosma PJ, Smith AJ, Aten J, Oude Elferink RP. Correction of liver disease by hepatocyte transplantation in a mouse model of progressive familial intrahepatic cholestasis. Gastroenterology 2000;119:1720 –1730. 6. Wang X, Al-Dhalimy M, Lagasse E, Finegold M, Grompe M. Liver repopulation and correction of metabolic liver disease by transplanted adult mouse pancreatic cells. Am J Pathol 2001;158: 571–579. 7. Guha C, Parashar B, Deb NJ, Garg M, Gorla GR, Singh A, RoyChowdhury N, Vikram B, Roy-Chowdhury J. Normal hepatocytes correct serum bilirubin after repopulation of Gunn rat liver subjected to irradiation/partial resection. Hepatology 2002;36:354 – 362. 8. Malhi H, Irani AN, Volenberg I, Schilsky ML, Gupta S. Early cell transplantation in LEC rats modeling Wilson’s disease eliminates hepatic copper with reversal of liver disease. Gastroenterology 2002;122:438 – 447. 9. Nagata H, Ito M, Cai J, Edge AS, Platt JL, Fox IJ. Treatment of cirrhosis and liver failure in rats by hepatocyte xenotransplantation. Gastroenterology 2003;124:422– 431. 10. Merle U, Encke J, Tuma S, Volkmann M, Naldini L, Stremmel W. Lentiviral gene transfer ameliorates disease progression in LongEvans cinnamon rats: an animal model for Wilson disease. Scand J Gastroenterol 2006;41:974 –982.
GASTROENTEROLOGY Vol. 132, No. 1
11. Malhi H, Gorla GR, Irani AN, Annamaneni P, Gupta S. Cell transplantation after oxidative hepatic preconditioning with radiation and ischemia-reperfusion leads to extensive liver repopulation. Proc Natl Acad Sci U S A 2002;99:13114 –13119. 12. Grompe M. The origin of hepatocytes. Gastroenterology 2005; 128:2158 –2160. 13. Malhi H, Irani AN, Gagandeep S, Gupta S. Isolation of human progenitor liver epithelial cells with extensive replication capacity and differentiation into mature hepatocytes. J Cell Sci 2002;115: 2679 –2688. 14. Dan YY, Riehle KJ, Lazaro C, Teoh N, Haque J, Campbell JS, Fausto N. Isolation of multipotent progenitor cells from human fetal liver capable of differentiating into liver and mesenchymal lineages. Proc Natl Acad Sci U S A 2006;103:9912–9917. 15. Oertel M, Menthena A, Dabeva MD, Shafritz DA. Cell competition leads to a high level of normal liver reconstitution by transplanted fetal liver stem/progenitor cells. Gastroenterology 2006;130: 507–520. 16. Wege H, Le HT, Chui MS, Liu L, Wu J, Giri RK, Malhi H, Sappal BS, Kumaran V, Gupta S, Zern MA. Telomerase reconstitution immortalizes human fetal hepatocytes without disrupting their differentiation potential. Gastroenterology 2003;124:432– 444. 17. Zalzman M, Gupta S, Giri RK, Berkovich I, Sappal BS, Karnieli O, Zern MA, Fleischer N, Efrat S. Reversal of hyperglycemia in mice using human expandable insulin-producing cells differentiated from fetal liver progenitor cells. Proc Natl Acad Sci U S A 2003; 100:7253–7258. 18. Overturf K, Al-Dhalimy M, Ou C-N, Finegold M, Grompe M. Serial transplantation reveals the stem-cell-like regenerative potential of adult mouse hepatocytes. Am J Pathol 1997;151:1273–1280. 19. Gupta S, Rajvanshi P, Sokhi R, Vaidya S, Irani AN, Gorla GR. Position-specific gene expression in the liver lobule is directed by the microenvironment and not by the previous cell differentiation state. J Biol Chem 1999;274:2157–2165. 20. Kumaran V, Joseph B, Benten D, Gupta S. Integrin and extracellular matrix interactions regulate engraftment of transplanted hepatocytes in the rat liver. Gastroenterology 2005;129:1643–1653. 21. Benten D, Kumaran V, Joseph B, Gupta S. Hepatocyte transplantation activates hepatic stellate cells with beneficial modulation of cell engraftment. Hepatology 2005;42:1072–1081. 22. Wu YM, Joseph B, Gupta S. Immunosuppression using the mTOR inhibition mechanism affects replacement of the rat liver with transplanted cells. Hepatology 2006;44:410 – 419. 23. Joseph B, Kumaran V, Berishvili E, Bhargava KK, Palestro CJ, Gupta S. Monocrotaline promotes transplanted cell engraftment and advances liver repopulation in rats via liver conditioning. Hepatology 2006;44:1411–1420. 24. Nagata H, Nishitai R, Shirota C, Zhang J-L, Koch CA, Cai J, Awwad M, Schuurman HJ, Christians U, Abe M, Baranowska–Kortylewicz J, Platt JL, Fox IJ. Prolonged survival of porcine hepatocytes in cynomolgus monkeys. Gastroenterology 2007;132:321–329. 25. Deschamps JY, Roux FA, Sai P, Gouin E. History of xenotransplantation. Xenotransplantation 2005;12:91–109. 26. Cozzi E, Bosio E, Seveso M, Vadori M, Ancona E. Xenotransplantation— current status and future perspectives. Br Med Bull 2006;75–76:99 –114. 27. Tseng YL, Moran K, Dor FJ, Sanderson TM, Li W, Lancos CJ, Schuurman HJ, Sachs DH, Cooper DK. Elicited antibodies in baboons exposed to tissues from alpha1,3-galactosyltransferase geneknockout pigs. Transplantation 2006;81:1058 –1062. 28. Hering BJ, Wijkstrom M, Graham ML, et al. Prolonged diabetes reversal after intraportal xenotransplantation of wild-type porcine islets in immunosuppressed nonhuman primates. Nat Med 2006;12:301–303. 29. Cardona K, Korbutt GS, Milas Z, Lyon J, Cano J, Jiang W, BelloLaborn H, Hacquoil B, Strobert E, Gangappa S, Weber CJ, Pearson TC, Rajotte RV, Larsen CP. Long-term survival of neonatal
January 2007
porcine islets in nonhuman primates by targeting costimulation pathways. Nat Med 2006;12:304 –306. 30. Gunsalus JR, Brady DA, Coulter SM, Gray BM, Edge AS. Reduction of serum cholesterol in Watanabe rabbits by xenogeneic hepatocellular transplantation. Nat Med 1997;3:48 –53. 31. Ibrahim Z, Busch J, Awwad M, Wagner R, Wells K, Cooper DK. Selected physiologic compatibilities and incompatibilities between human and porcine organ systems. Xenotransplantation 2006;13:488–499. 32. Martina Y, Marcucci KT, Cherqui S, Szabo A, Drysdale T, Srinivisan U, Wilson CA, Patience C, Salomon DR. Mice transgenic for a human porcine endogenous retrovirus receptor are susceptible to productive viral infection. J Virol 2006;80:3135–3146.
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Address requests for reprints to: Sanjeev Gupta, MD, Albert Einstein College of Medicine, Ullmann Building, Room 625, 1300 Morris Park Avenue, Bronx, New York 10461. e-mail: [email protected]; fax: (718) 430-8975. Supported in part by NIH grants R01-DK46952, R01-DK071111, P01-DK52956, P20-GM0750375, P30-DK41296, P30-CA13330, and M01-RR12248-9. © 2007 by the AGA Institute
0016-5085/07/$32.00 doi:10.1053/j.gastro.2006.11.047
EDITORIALS
GASTROENTEROLOGY Vol. 132, No. 1
Hog Heaven on the Road to Liver Cell Therapy
See “Prolonged survival of porcine hepatocytes in cynomolgus monkeys” by Nagata H, Nishitai R, Shirota C, Zhang J-L, Koch CA, Cai J, Awwad M, Schuurman H-J, Christians U, Abe M, Baranowska–Kortylewicz J, Platt JL, Fox IJ, on page 321.
O
ver the past 25 years, travel on the road to liver cell therapy has not always been easy. Initially, when hepatocyte transplantation improved outcomes in animals with liver failure, metabolic disease, and so on, it seemed that clinical applications would follow shortly.1 Early studies of ex vivo gene therapy in familial hypercholesterolemia with autologous hepatocytes,2 and in Crigler-Najjar syndrome with healthy allogeneic hepatocytes,3 highlighted inefficient gene transfer, cell loss during in vitro manipulations, limited cell engraftment, and absence of transplanted cell proliferation as major problems. Since then, much has been accomplished in applying liver cell therapy to people, although more needs to be done.4 Numerous animal studies advanced insights into liver repopulation, including genetic modification of cells with superior vectors and correction of a variety of disorders characterized by healthy and/or diseased livers.5–11 Significant bodies of work have accumulated over the past several years with respect to mechanisms concerning the potential of mature hepatocytes, fetal liver-derived stem/progenitor cells, embryonic stem cells, as well as extrahepatic stem cells from the bone marrow or cord blood.12 Suffice it to say, interest in stem cells capable of repopulating the liver seems appropriate, although fundamental issues remain in respect with generating mature hepatocytes from pluripotential stem cells and whether the progeny of stem cells could be expanded in vitro and in vivo without oncogenicity in case of embryonic stem cells and of the proclivity for cell fusion in case of hematopoietic stem cells. Use of embryonic stem cells, in particular, needs further resolution of social and political issues. However, fetal human liver stem/progenitor cells show extensive replication and differentiation potential, including the capacity to generate mature hepatocytes after transplantation in immunodeficient animals.13,14 Indeed, fetal rodent cells showed superior replication potential compared with adult hepatocytes.15 Significant numbers of epithelial cells can be isolated from a midgestation fetal human liver and these cells can be expanded enormously in vitro, by up to 5 ⫻ 107-fold, that is, trillions of cells could be generated from a single
fetal liver. However, the supply of fetal human tissues is also not unlimited. The replication potential of fetal liver cells has been extended through reconstitution of telomerase activity,16,17 which maintains chromosomal integrity during cell division, although the possibility of oncogenic perturbations needs further study. The choice of donor cells is particularly relevant because mature hepatocytes themselves possess extensive replication capacity, as shown, for example, in FAH mutant mice, which model hereditary tyrosinemia type 1.18 Suitable mechanisms have been identified to alter the condition of healthy rat and mouse livers before cell transplantation. Under these circumstances, transplanted hepatocytes can replace virtually the entire liver over several weeks.7,11,15 Although animal studies often use toxic chemicals, more recently, genotoxicity with radiation plus ischemia–reperfusion proved effective in liver repopulation.11 After suitable types of liver injury, transplanted cells require ⬍10 population doublings to repopulate the healthy rat liver. Therefore, clinical protocols may require transplantation of relatively few cells to repopulate the liver. To ensure that transplanted cells have the best opportunities for engrafting and proliferating in the liver, critical mechanisms have been examined, extending from the hepatic vasomotor tone, sinusoidal endothelial barrier, receptor-mediated cell targeting, and cell– cell interactions, which regulate transplanted cell engraftment, the role of the hepatic microenvironment in regulating the function of transplanted cells, and the underlying state of the hepatic parenchyma and modifying factors that determine whether transplanted cells will proliferate.19 –23 On the other hand, the shortage of donor human organs represents a major road-bump in clinical applications of cell therapy. To overcome this barrier, Nagata et al24 propose in this issue of GASTROENTEROLOGY that xenogeneic pig hepatocytes will be suitable for transplantation. Through careful studies in cynomolgus monkeys— by no means an easy task—they offer convincing evidence that porcine cells engrafted and functioned under immunosuppression conditions that can be used in humans. This represents a significant advance in considering xenogeneic donors for liver cell therapy. Looking back at xenotransplantation, you might be surprised to learn from an excellent recent review that animal products had already been transplanted into people in the 16th century!25 In the ancient Hindu tradition, Lord Ganesha is shown to bear an elephant head on a human torso. The myth goes that upon returning from a trip, Ganesha’s father, the omnipotent God, Shiva, found him lying next to his consort, the Goddess Parvati, and in
January 2007
a jealous rage beheaded Ganesha. Shiva did not realize that Ganesha, who was born large, was his own recentborn son. Parvati went into great agitation and threatened to destroy the entire universe unless Shiva restored Ganesha to her. Therefore, in mad haste, Shiva found a head that could fit the torso of Ganesha, and that turned out to be from an elephant! Over the past centuries, tissue and cell xenotransplants in people have been documented from diverse animals, including rabbit, guinea pig, lamb, sheep, dog, calf, pig, ape, and baboon. Remarkably, from 1969 to 1993, chimpanzee, baboon, and pig livers were transplanted into 11 people, including by Starzl and Makowka in the United States; the longest survival, however, was 70 days. Survival of xenogeneic tissues, cells, or organs was limited by a robust host immune response. Therefore, it is with this background that one needs to consider xenotransplantation, particularly pig tissues and organs, as these are most readily procured for human applications. Recent studies identified fundamental differences in the immune response to allografts versus xenografts. For instance, host immune responses after xenografting of porcine solid organs is characterized by hyperacute rejection (HAR), with irreversible graft injury within minutes to hours due to pre-existing host xenoreactive natural antibodies (XNA).26 This is followed by acute humoral xenograft rejection (AHXR), largely from activation of macrophages, neutrophils, CD8⫹ T cells and natural killer cells, within 24 hours after xenografting. The processes of HAR and AHXR constitute critical host immune responses with rapid and severe endothelial damage leading to xenograft loss. Fortunately, progress in understanding the pathophysiology of these processes is beginning to yield ways to modulate HAR and AHXR. For instance, the most important XNA is against the porcine galactosyltransferase epitope, Gal␣1-3 Gal14GlcNAc-R. Recently, genetically modified pigs lacking this epitope were generated,27 which in combination with strategies to avoid activation of the complement cascade will help in solid organ xenografts.26 Similarly, strategies are being developed to eliminate AHXR. The initial process of HAR and AHXR may be followed by an acute cellular xenograft response, characterized by cellular reactions without vascular thrombosis, and finally, chronic xenorejection, which is less well characterized. Transplantation of nonvascularized tissues (eg, pancreatic islets) elicits a different xenograft response, due to low or minimal expression of the ␣-Gal epitope on cells compared with solid organs, as well as the absence of XNA response.28,29 In recent studies, transplanted porcine islets were shown to restore insulin secretion in monkeys over the long term (⬎180 days). However, porcine islets in monkeys still generated inflammatory reaction, including activation of complement and inflammatory cells, with damage to transplanted islets.
EDITORIALS
451
As indicated by Nagata et al,24 in previous studies, porcine hepatocytes ameliorated serum cholesterol levels in Watanabe rabbits,30 and in their own studies improved outcomes in rats with advanced liver cirrhosis.9 The latter studies used an extrahepatic reservoir of liver cells in the spleen, such that transplanted cells would supplement the metabolic capacity of the cirrhotic liver. Similarly, an extrahepatic reservoir of cells was created with porcine hepatocytes in monkeys,24 by mixing sodium alginate to promote cell entrapment in the spleen. Recipient monkeys were immunosuppressed, with induction using thymoglobulin, methylprednisolone, and an anti–interleukin-2 receptor antibody, followed by maintenance with cyclosporine, everolimus—an inhibitor of the mammalian target of rapamycin (mTOR)—the investigational drug FTY720, and methylprednisolone. Transplanted porcine hepatocytes secreted albumin, expressed the asialoglycoprotein receptor, which was identified by scintigraphic studies using a radioligand, and survived in monkeys for 25– 86 days. Cell recipients did not express significant XNA responses and antibodies against the ␣-Gal epitope were mostly absent. Moreover, porcine hepatocytes were successfully retransplanted and in one monkey, transplanted cells were shown to function for a total of 253 days. These studies established that xenogeneic porcine hepatocytes will be less immunogenic than the whole organ and perhaps other types of xenografts, such as pancreatic islets.28,29 Although immunosuppression was associated with some infectious complications in monkeys, drug dosing can obviously be adjusted to avoid excessive immunosuppression. In choosing the most effective immunosuppression regime, it should be noteworthy that drugs can affect transplanted cells. For instance, the mTOR inhibitory drug, rapamycin, impaired transplanted cell proliferation.22 Nagata et al24 indicated that in monkeys with an intact liver, it was difficult to assess the metabolic contribution of transplanted cells, and that due to feedback regulation, serum albumin levels would not have been effective for such analysis. Nonetheless, creating an extrahepatic reservoir of liver cells in the proposed manner should be helpful in tiding over patients with liver failure. In the setting of acute liver failure, temporary metabolic support should facilitate recovery of the native organ, or at least offer a bridge to orthotopic liver transplantation. Similarly, during decompensation in chronic liver disease, an additional hepatocyte reservoir should help to treat complications such as hepatic encephalopathy, although in debilitated individuals, immunosuppression would increase the risk of infectious complications. Cell transplantation in the setting of chronic liver failure could potentially help prolong life, as shown by Nagata et al in studies of porcine cell transplantation in cirrhotic rats.9 This new road of porcine hepatocyte xenografting will be of much interest for addressing the potential of liver
452
EDITORIALS
repopulation in suitable experimental systems. Perhaps it is appropriate to consider additional issues regarding porcine xenografts in this context. For instance, concerns have been raised regarding molecular differences in complement, coagulation systems, and soluble human and porcine molecules.31 However, such concerns must be considered separately from solid organ xenotransplants, because cell transplants need not replace an organ entirely, and native proteins should still be available. The possibility of zoonotic disease transmission, for example, porcine endogenous retrovirus, which is embedded in the pig genome, and could possibly be reactivated through genetic recombination,32 has been under debate. So far, emergence of active porcine endogenous retrovirus infection in recipients of porcine material has, reassuringly, not been documented.
SANJEEV GUPTA Marion Bessin Liver Research Center Diabetes Center, Cancer Research Center, and Center for hESC Research Departments of Medicine and Pathology Albert Einstein College of Medicine Bronx, New York References 1. Gupta S, Roy Chowdhury J. Hepatocyte transplantation: back to the future! Hepatology 1992;15:156 –162. 2. Fisher RA, Strom SC. Human hepatocyte transplantation: worldwide results. Transplantation 2006;82:441– 449. 3. Grossman M, Rader DJ, Muller DWM, Kolansky DM, Kozarsky K, Clark, BJ III, Stein EA, Lupien PJ, Brewer, BH Jr, Raper SE, Wilson JM. A pilot study of ex vivo gene therapy for homozygous familiar hypercholesterolaemia. Nat Med 1995;1:1148 –1154. 4. Fox IJ, Chowdhury JR, Kaufman SS, Goertzen TC, Chowdhury NR, Warkentin PI, Dorko K, Sauter BV, Strom SC. Treatment of the Crigler-Najjar syndrome type I with hepatocyte transplantation. N Engl J Med 1998;338:1422–1426. 5. De Vree JM, Ottenhoff R, Bosma PJ, Smith AJ, Aten J, Oude Elferink RP. Correction of liver disease by hepatocyte transplantation in a mouse model of progressive familial intrahepatic cholestasis. Gastroenterology 2000;119:1720 –1730. 6. Wang X, Al-Dhalimy M, Lagasse E, Finegold M, Grompe M. Liver repopulation and correction of metabolic liver disease by transplanted adult mouse pancreatic cells. Am J Pathol 2001;158: 571–579. 7. Guha C, Parashar B, Deb NJ, Garg M, Gorla GR, Singh A, RoyChowdhury N, Vikram B, Roy-Chowdhury J. Normal hepatocytes correct serum bilirubin after repopulation of Gunn rat liver subjected to irradiation/partial resection. Hepatology 2002;36:354 – 362. 8. Malhi H, Irani AN, Volenberg I, Schilsky ML, Gupta S. Early cell transplantation in LEC rats modeling Wilson’s disease eliminates hepatic copper with reversal of liver disease. Gastroenterology 2002;122:438 – 447. 9. Nagata H, Ito M, Cai J, Edge AS, Platt JL, Fox IJ. Treatment of cirrhosis and liver failure in rats by hepatocyte xenotransplantation. Gastroenterology 2003;124:422– 431. 10. Merle U, Encke J, Tuma S, Volkmann M, Naldini L, Stremmel W. Lentiviral gene transfer ameliorates disease progression in LongEvans cinnamon rats: an animal model for Wilson disease. Scand J Gastroenterol 2006;41:974 –982.
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Address requests for reprints to: Sanjeev Gupta, MD, Albert Einstein College of Medicine, Ullmann Building, Room 625, 1300 Morris Park Avenue, Bronx, New York 10461. e-mail: [email protected]; fax: (718) 430-8975. Supported in part by NIH grants R01-DK46952, R01-DK071111, P01-DK52956, P20-GM0750375, P30-DK41296, P30-CA13330, and M01-RR12248-9. © 2007 by the AGA Institute
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