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Therapeutic potential of hepatocyte transplantation
seminars in CELL & DEVELOPMENTAL BIOLOGY, Vol. 13, 2002: pp. 439–446 doi:10.1016/S1084–9521(02)00132-5, available online at http://www.idealibrary.com on
Therapeutic potential of hepatocyte transplantation Sanjeev Gupta∗ and Jayanta Roy Chowdhury
donor livers become available in the United States annually, whereas the number of people on waiting lists for OLT is several times greater. Therefore, it is imperative that alternative strategies, such as cell therapy, are developed. Percutaneous catheters can be utilized to transplant cells simply and the procedure is ‘reversible’ because the host liver remains intact. Due to the successes of hepatocyte transplantation in animals, several groups are preparing to evaluate cell therapy in a variety of diseases (Table 1). Among potentially significant applications of cell transplantation are included provision of metabolic support in acute or chronic liver failure and definitive treatment of inherited metabolic disorders. Moreover, hepatocytes are an excellent vehicle for ex vivo gene therapy. While hepatocytes are the most abundant liver cell type (∼60%), other cell types include biliary epithelial cells, hepatic stellate cells (found in the space of Disse, juxtaposed between hepatocytes and liver sinusoids), endothelial cells lining sinusoids, fibroblasts, pit cells, and littoral cells, such as Kupffer cells. Separation of these liver cell types and analysis of the biology of individual cell types is beginning to provide myriad insights into how cell–cell interactions could benefit hepatocyte transplantation. Recent insights in stem/progenitor cells isolated from the liver itself, from extrahepatic organs, as well as from pluripotential embryonic stem cells offer exciting new opportunities for cell therapy.
Liver repopulation with transplanted cells offers unique opportunities for treating a variety of diseases and for studies of fundamental mechanisms in cell biology. Our understanding of the basis of liver repopulation has come from studies of transplanted cells in animal models. A variety of studies established that transplanted hepatocytes as well as stem/progenitor cells survive, engraft, and function in the liver. Transplanted cells survive life-long, although cells do not proliferate in the normal liver. On the other hand, the liver is repopulated extensively when diseases or other injuries afflict native hepatocytes but spare transplanted cells. The identification of ways to repopulate the liver with transplanted cells has greatly reinvigorated the field of liver cell therapy. The confluence of insights in stem/progenitor cells, transplantation immunology, cryobiology, and liver repopulation in specific models of human diseases indicates that the field of liver cell therapy will begin to reap the promised fruit in the near future. Key words: liver repopulation / hepatocytes / transplanted cells © 2002 Elsevier Science Ltd. All rights reserved.
Introduction The prognosis for patients with liver failure, or potentially fatal inherited metabolic disorders is greatly improved by orthotopic liver transplantation (OLT), which refers to the replacement of the native liver with a donor liver. However, OLT is technically quite complex and has been greatly limited by shortages of donor organs. For instance, only approximately 5000
Mechanisms of transplanted cell engraftment, function, and proliferation Liver cells display major histocompatibility complex (MHC) antigens and mismatched cells are promptly rejected.1, 2 Therefore, liver cells must be transplanted into MHC-compatible or immunodeficient animals. In early studies, cells were transplanted into ectopic sites because methods to distinguish between transplanted and native cells in the liver were not then available.3 These studies showed that transplanted cells survived in the spleen, interscapular fat pad, peritoneal cavity,
From the Marion Bessin Liver Research Center, Departments of Medicine, Pathology and Molecular Genetics, Albert Einstein College of Medicine, Bronx, NY 10461, USA. * Corresponding author. Albert Einstein College of Medicine, Room 625, Ullmann Building, 1300 Morris Park Avenue, Bronx, NY 10461, USA. E-mail: [email protected] © 2002 Elsevier Science Ltd. All rights reserved. 1084–9521 / 02 / $– see front matter
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Table 1. Conditions potentially suitable for liver cell therapy Liver is diseased
Extrahepatic organs manifest disease
Genetic disorders Wilson’s disease α-1 antitrypsin deficiency Erythropoietic protoporphyria Lipidoses, e.g. Niemann–Pick disease Tyrosinemia type 1
Metabolic deficiency states Congenital hyperbilirubinemia, e.g. Crigler–Najjar syndrome Familial hypercholesterolemia Hyperammonemia syndromes Defects of carbohydrate metabolism Oxalosis
Acquired disorders Acute liver failure Chronic viral hepatitis Cirrhosis and liver failure
Coagulation disorders Hemophilia A Factor IX deficiency Immune disorders Hereditary angioedema
antigen (HBsAg), human α-1 antitrypsin (hAAT), and Escherichia coli β-galactosidase (LacZ).8, 9 Other reporters include green fluorescence protein (GFP), sex chromosome markers, or unique proteins that are deficient in recipient animals.10, 11 Use of the dipeptidyl peptidase IV deficient (DPPIV)-rat has been particularly helpful in elucidating the biology of transplanted cells.12 DPPIV is abundantly expressed in bile canaliculi, which provides methods to demonstrate whether transplanted cells are integrated in the liver parenchyma.13, 14 Studies in these animal models established that transplanted hepatocytes enter the liver plate after deposition in
and less effectively in a variety of other extrahepatic sites.4–6 The capacity of the spleen to accommodate cells is limited, although an enormous mass of transplanted hepatocytes could potentially be created in the peritoneal cavity. However, cell survival in the peritoneal cavity requires biomaterials for extracellular matrix support and transplanted cells show inferior gene expression in the peritoneal cavity.4 Numerous animal models are now available for studying transplanted cell survival in the liver (Table 2).7 Inbred transgenice mice constituted the earliest models, where donor hepatocytes expressed unique reporters, such as hepatitis B virus surface
Table 2. Useful animal models for studying liver cell therapy Nature of animal model
Animal designation
Animal origin or manipulation
Disease reproduced
Genetically determined
DPPIV-rat
Natural mutation (rat)
None
DPPIV-mouse FAH mouse Gunn rat LEC rat P-glycoprotein (mdr 2) mouse
Gene knockout Gene knockout Spontaneous mutation Spontaneous mutation Gene knockout
Watanabe heritable hyperlipidemic rabbit (WHHL) Nagase analbuminemic rat (NAR)
Spontaneous mutation
Tyrosinemia type 1 Crigler–Najjar syndrome Wilson’s disease Biliary phospholipid transport defect Familial hypercholesterolemia
Spontaneous mutation
Analbuminemia
Acetaminophen, carbon tetrachloride, d-galactosamine, other chemicals, subtotal partial hepatectomy Repeated carbon tetrachloride
Acute liver failure
Iatrogenically produced
Mice, rats, rabbits, pigs, dogs
Rats
440
Liver cirrhosis
Cell therapy
Figure 1. Syngeneic F344 rat hepatocytes localized by histochemistry in the DPPIV-rat liver following intrasplenic transplantation. (A) Transplanted cells are located immediately adjacent to a portal area (Pa) (arrows). (B) Extensive liver repopulation in a rat subjected to liver radiation and ischemia-reperfusion injury before intrasplenic cell transplantation. Transplanted cells exhibit DPPIV activity (red color) in the bile canalicular domain and have replaced most of the liver. Tissues were stained lightly with hematoxylin to visualize cell nuclei.
liver sinusoids. Transplanted hepatocytes develop conjoint bile canaliculi and gap junctions with adjacent native hepatocytes, express liver genes in a physiologically regulated manner, and respond to mitogenic stimuli.13–17 However, transplanted cells do not proliferate in the normal rat or mouse liver,9, 18, 19 with the exceptions being the liver of either very young or old F344 rats, where transplanted cells exhibit spontaneous proliferative activity.19 This translates into the repopulation of only 0.5–1% of the liver following transplantation of 20 million cells in the rat or 2 million cells in the mouse liver, which can be increased to approximately 5% by transplanting cells repeatedly.20 However, correction of specific diseases might be incomplete with this magnitude of liver reconstitution and means to induce significant liver repopulation have been investigated. Induction of transplanted cell proliferation in the liver requires selective ablation of native hepatocytes with chemicals, e.g. carbon tetrachloride (CCl4 ), etc., or hepatotoxic trangenes (Reference 17 and see the following description). For instance, extensive liver repopulation occurs in animals with hepatic expression of urokinase-type plasminogen activator (alb-uPA mouse),21 the FAH mouse, which models hereditary tyrosinemia type 1,22 Fas ligand-induced apoptosis,23 prodrug activation of herpes simplex virus thymidine kinase (HSV-TK),24 expression of the cell cycle regulator Mad1,25 use of toxic bile salts in mice deficient in mdr 2 gene, which impairs biliary phopholipid excretion with hepatobiliary injury,26 etc. In the rat liver, genotoxic hepatic damage has been helpful for liver repopulation. The pyrollizidine
alkaloid, retrorsine, which alkylates DNA and inhibits hepatocellular proliferation has been highly effective for liver repopulation in rat.27 Retrorsine induces extensive hepatic polyploidy, which is also induced by two-thirds partial hepatectomy and the thyroid hormone, tri-iodothyronine (T3),28, 29 and the combination of these manipulations plays synergistic roles in liver repopulation.30 However, retrorsine is potentially oncogenic and is unsuitable for clinical use. Additional mechanisms for inducing genotoxic liver injury for liver repopulation include ionizing radiation and this has been as effective as retrorsine in repopulating the rat liver31 (Figure 1). It is noteworthy that radiation is ineffective by itself and requires either partial hepatectomy or ischemia-reperfusion injury, which cause oxidative DNA damage in the liver and promote radiation injury in the liver.32, 33 As radiation can be used clinically, this has raised the possibility of achieving extensive liver repopulation in the clinical setting.
Treatment of metabolic diseases Several metabolic diseases have been corrected in laboratory animals. The Gunn rat model of Crigler–Najjar syndrome type-1, has been highly characterized.6 In this hepatic metabolic disorder, bilirubin-UDP-glucuronosyltransferase (UGT1A1) activity is deficient and unconjugated bilirubin accumulates in toxic levels. Nagase analbuminemic rats (NAR) exhibit extremely low levels of serum albumin due to defective hepatic expression of the albumin 441
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gene.34 Transplantation of normal hepatocytes ameliorates the metabolic abnormality in Gunn rats as well as NAR.6 Additional animal models of metabolic disorders that are amenable to liver cell therapy include the Watanabe heritable hyperlipidemic rabbit (WHHL), which lack cell surface receptors for low density lipoproteins and models familial hypercholesterolemia,35 the Long–Evans Cinnamon (LEC) rat, an animal model for Wilson’s disease,36 the FAH mouse, which models hereditary tyrosinemia type-1,22 and mdr 2 knockout mice, which model progressive familial intrahepatic cholestasis.26 Studies in these animals have established a number of critical insights. For instance, transplantation of relatively small numbers of liver cells is insufficient for correcting metabolic deficiencies in the animals and significant liver repopulation has been necessary. These paradigms are critical for clinical applications. Early studies in patients have reproduced the experience in laboratory animals, which further validates the value of cell therapy studies in animal models. This early clinical experience extends to the transplantation of autologous genetically modified cells in patients with familial hypercholesterolemia37 and of allogeneic hepatocytes in patients with ornithine transcarbamylase (OTC) deficiency, α-1 antitrypsin deficiency, and Crigler–Najjar syndrome type-1.38–40 Treatment of one child with OTC deficiency led to limited reconstitution of hepatic enzyme activity but this was insufficient and the patient succumbed shortly thereafter to severe hyperammonemia.38 After cell transplantation in one patient with Crigler–Najjar syndrome type-1, serum bilirubin decreased significantly, hepatic bilirubin glucuronidating activity rose to approximately 5% of normal, and bilirubin glucuronides appeared in bile.40 However, the patient still required phototherapy and ultimately needed OLT. Therefore, further studies will be necessary with suitable strategies to amplify the transplanted hepatocyte mass for achieving cures.
liver. The potential of cell transplantation has been tested in a variety of animal models of acute liver failure.41 Multiple early studies showed that transplantation of hepatocytes in the peritoneal cavity, spleen, or liver improved mortality. However, it was unclear whether improved outcomes could be directly ascribed to transplanted cells or were due to unknown mechanisms, including mitogenic stimulation, cytoprotection, etc., because mortality improved following injection of conditioned medium from cultured cells, of fragmented cells, and even of xenogeneic cells that were promptly rejected in animals.42–44 More recently, transplanted hepatocytes were shown to engraft in the liver of animals with acute liver failure.45 Further studies in novel genetic models, where acute liver failure was induced by activation of ganciclovir by HSV-TK or Mad1 expression, showed that cell transplantation improved mortality.24, 25 Also, cell transplantation has been shown to prevent the development of intracranial hypertension in pigs following acute liver failure.46 Early studies of hepatocyte transplantation in patients with acute liver failure have generally been too limited to make firm conclusions regarding its efficacy. For example, seven patients with acute liver failure were treated with intraperitoneal transplantation of fetal human hepatocytes, which possibly improved survival in some patients, although it was unknown whether this was directly due to transplanted cell survival, as isolated hepatocytes do not survive for long following injection in the peritoneal cavity.47 In another clinical study, after hepatocyte transplantation through the splenic artery, five of seven patients with acute liver failure survived long enough to undergo OLT.48 In additional patients with acute liver failure, infusion of hepatocytes into the portal vein was shown to result in cell engraftment in the liver, along with some possible improvement in hepatic encephalopathy, cerebral perfusion pressure, etc.38 Perhaps the most important message from these early studies is that cells can be transplanted safely in patients with acute liver failure. Obviously, cell therapy for acute liver failure requires further study. Pertinent issues concern utilization of highly viable cells, identification of patients with comparable disease for demonstrating therapeutic outcomes, and correlations between the transplanted cell mass and outcomes. It is also noteworthy that currently available animal models may not reproduce the clinical syndrome of acute liver failure adequately and superior animal models are required. Also, mechanisms concerning transplanted cell engraftment
Hepatocyte transplantation for acute liver failure Cell therapy for acute liver failure is quite attractive for two reasons. First, prolongation of survival and amelioration of specific complications, such as raised intracranial pressure, could bridge patients to OLT. Second, OLT could be avoided altogether if cell transplantation would permit regeneration of the native 442
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and proliferation in acute liver failure need further analysis because the process of cell transplantation may aggravate liver injury to begin with by inducing ischemia-reperfusion-type changes, engraftment of transplanted cells might vary in different parts of the liver, and transplanted cell proliferation is delayed until transplanted cells are fully integrated in the liver parenchyma.24, 45, 49
cirrhotic animals. However, transplanted cell proliferation in the liver was limited and animals did not show any differences in mortality over a 12-month period. On the other hand, other investigators found that intrasplenic cell transplantation in extremely sick cirrhotic rats was associated with improvement in liver tests, coagulation abnormality, and outcomes.52 These findings have been interpreted to suggest that creation of an additional reservoir of cells in the liver will prolong survival in end-stage liver disease, although it is unclear as to why transplanted hepatocytes demonstrate superior function compared with the native liver in these animals. Studies in the LEC rat again established that transplanted cells engraft and survive in the diseased liver.36, 54, 55 However, transplanted cell proliferation in LEC rats requires selection pressure or development of extensive disease. In otherwise unmanipulated LEC rats, transplanted cell proliferation requires several months.54, 55 On the other hand, when copper accumulation and/or toxicity are exacerbated by retrorsine and partial hepatectomy, liver repopulation was greatly accelerated.54 It is noteworthy that cell transplantation can actually reverse established liver disease in LEC rats,55 which is in agreement with the potential of curing people with chronic viral hepatitis, were it possible to transplant cells that would resist viral infection. Our clinical experience of cell transplantation in patients with chronic liver disease is rather fledgling. In an early study, 10 Japanese patients with cirrhosis were transplanted into various sites, including spleen, with hepatocytes isolated from a piece of their own liver.56 In one of these patients, nuclear medicine studies showed transplanted hepatocytes in the spleen 11 months following transplantation, although whether any patients improved directly due to cell transplantation was unclear. Several patients with chronic liver disease have been transplanted with allogeneic hepatocytes through the splenic artery in the United States.38 However, it was unclear whether hepatocyte transplantation was responsible for improved liver function in these patients. The initial experience of cell transplantation suggests that further insights are necessary for optimizing various clinical procedures. For example, whether different results in animals and people might be related to injection of cells into the splenic artery in people requires study, because unlike cell engraftment in the red pulp of the spleen, injection of hepatocytes into arterial beds leads to rapid loss of transplanted cells, presumably due to shear injury and absence of cell anchorage. Similarly, whether immunosuppressive drugs might alter the fate or
Treatment of chronic liver failure Patients with end-stage liver disease and hepatic encephalopathy or other liver complications require OLT. However, many patients are unfit for receiving OLT and in many parts of the world OLT is not available. The general consideration is that cell transplantation could offer an additional mass of functioning liver, either in the liver itself or in the spleen, to supplement metabolic or synthetic function of the liver. Animals with established cirrhosis constitute an excellent model to establish these principles of cell therapy. On the other hand, if ongoing disease, e.g. chronic hepatitis, were to be ameliorated or cured, one would envision that transplantation of cells that are disease resistant would be needed. LEC rats modeling Wilson’s disease offer such a model because these animals exhibit genetically determined copper toxicosis, where excessive amounts of copper circulate in the body and exist within hepatocytes, which leads to extensive fibrosis and liver disease.36 The therapeutic potential of hepatocyte transplantation in decompensated liver disease has begun to be explored only recently. In rats with hepatic encephalopathy induced by end-to-side portacaval shunt, intrasplenic transplantation of hepatocytes improved encephalopathy scores and partially corrected changes in serum amino acid levels.50 Similarly, hepatocyte transplantation ameliorated hepatic encephalopathy in portacaval-shunted rats burdened with a load of ammonia.51, 52 Transplanted cells did not enter the liver in these animals due to portacaval shunting. Transplanted cells were present in the spleen, although the overall mass of transplanted cells created in the spleen was unknown. Animals with cirrhosis induced by repeated CCl4 administration develop significant liver fibrosis, portal hypertension, and ascites.53 Studies have been conducted in these animals by transplanting cells after discontinuing CCl4 to avoid clearance of transplanted cells. Transplanted cells could integrate in the liver parenchyma despite extensive fibrosis in 443
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function of transplanted cells in people is unknown. Finally, whether the concept of creating therapeutically useful additional mass of transplanted hepatocytes in patients with end-stage liver disease is indeed appropriate to begin with, requires consideration.
demonstrated that fetal human hepatocytes exhibit unique properties, including capacity for extensive proliferation and excellent recovery following cryopreservation. Moreover, following transplantation in immunodeficient mice, fetal human liver cells engraft in the liver parenchyma and generate mature hepatocytes. Use of hematopoeitic stem cells (HSC) derived from the bone marrow, cord blood, or peripheral blood has excited considerable interest. Petersen et al. initially showed that bone marrow-derived HSC generated hepatocytes in the DPPIV-rat.61 These observations were verified in the mouse as well as humans.62–66 Finally, isolation of pluripotential human embryonic stem cells or mesenchymal stem cells and early successes in differentiating these cells along specific lineages without tumorigenesis suggests that an unlimited supply of self-renewable donor cells will eventually become available. In the meantime, efforts continue to enhance the replication capacity of mature hepatocytes by genetic perturbations, such as introduction of the simian virus 40 T antigen, which drives cell proliferation or reconstitution of telomerase, in the absence of which cells enter replicative senescence.67, 68
Ongoing issues Further insights into mechanisms regulating transplanted cell engraftment and proliferation are needed to improve results. For example, only 20–30% of the transplanted hepatocytes survive and engraft in the liver.15 Recent studies indicate that perturbation of initial cell distributions in the liver sinusoids, interference with the phagocytotic responses activated by cell transplantation, and manipulation of the sinusoidal endothelial integrity prior to cell transplantation could have salutary effects on transplanted cell engraftment and subsequent liver repopulation.57–59 Similarly, it is possible to begin applying insights concerning the central role of genotoxic liver injury for inducing proliferation in transplanted cells. Use of ischemia-reperfusion injury, in combination with radiation, offers a potential mechanism for preconditioning the liver for inducing liver repopulation with transplanted cells. Abrogation of rejection following cell transplantation constitutes another important area, where much progress is being made, but space limitations preclude discussion of this area here. Similarly, advances in cryopreservation of hepatocytes will greatly facilitate clinical applications. In general, hepatocytes are highly susceptible to freeze–thaw damage and significant fractions of cells are lost following cryopreservation. If the initial cell isolate is of relatively marginal viability to begin with, it becomes difficult to obtain adequate numbers of cells for transplantation. Therefore, development of methods to procure highly viable cell populations and of ways to cryopreserve cells, such that frozen cells can be banked for use at short notice will greatly facilitate cell therapy. As the number of donor human livers will remain limited in the foreseeable future, use of stem cells is of great interest for expanding donor availability. Potential sources of such cells could concern hepatocytes themselves, which can replicate extensively, as shown by transplantation studies in FAH mice, where hepatocytes showed >80 cell divisions without loss of the replication potential.60 Isolation of progenitor liver cells, especially from the fetal liver, offers a practical way of expanding donor livers. The fetal liver contains an abundant supply of hepatoblasts.11 Recent studies
Acknowledgements This work was supported in part by NIH Grants R01 DK46952, R01 DK46057, P33DK41296, and P30 CA13330, and MO1 RR12248. The author gratefully acknowledges the contributions of his colleagues in many studies cited here.
References 1. Bumgardner GL, Gao D, Li J, Baskin JH, Heininger M, Orosz CG (2000) Rejection responses to allogeneic hepatocytes by reconstituted SCID mice, CD4, KO, and CD8 KO mice. Transplantation 70:1771–1780 2. Reddy B, Gupta S, Chuzhin Y, Kalergis A, Budhai L, Zhang M, Droguett G, Horwitz M, Roy Chowdhury J, Nathenson SG, Davidson A (2001) Differential effect of CD28/B7 blockade with CTLA4Ig on alloreactive CD4+, CD8+ and B cells in murine isolated hepatocyte transplantation. Transplantation 71:801– 811 3. Gupta S, Rajvanshi P, Bhargava KK, Kerr A (1996) Hepatocyte transplantation: progress toward liver repopulation. Prog Liver Dis 14:199–222 4. Gupta S, Vemuru RP, Lee C-D, Yerneni P, Aragona E, Burk RD (1994) Hepatocytes exhibit superior transgene expression after transplantation into liver and spleen compared with peritoneal cavity or dorsal fat pad: implications for hepatic gene therapy. Hum Gene Ther 5:959–967
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5. Jirtle RL, Biles C, Michalopoulos G (1980) Morphologic and histochemical analysis of hepatocytes transplanted into syngeneic hosts. Am J Pathol 101:115–126 6. Demetriou AA, Levenson SM, Novikoff PM, Novikoff AB, Roy Chowdhury N, Whiting J, Reisner A, Roy Chowdhury J (1986) Survival, organization and function of microcarrier-attached hepatocytes transplanted in rats. Proc Natl Acad Sci USA 83:7475–7479 7. Gupta S, Rogler CE (1999) Liver repopulation systems and study of pathophysiological mechanisms in animals. Am J Physiol 277:G1097–G1102 8. Gupta S, Roy Chowdhury R, Jagtiani R, Gustin K, Shafritz DA, Roy Chowdhury J, Burk RD (1990) A novel system for transplantation of isolated hepatocytes utilizing HBsAg producing transgenic donor cells. Transplantation 50:472–475 9. Ponder KP, Gupta S, Leland F, Darlington G, Finegold M, DeMayo J, Ledley FD, Roy Chowdhury J, Woo SLC (1991) Mouse hepatocytes migrate to liver parenchyma and function indefinitely after intrasplenic transplantation. Proc Natl Acad Sci USA 88:1217–1221 10. Zahler MH, Irani A, Malhi H, Reutens AT, Albanese C, Bouzahzah B, Joyce D, Gupta S, Pestell RG (2000) The application of a lentiviral vector for gene transfer in fetal human hepatocytes. J Gene Med 2:186–193 11. Malhi H, Irani AN, Gagandeep S, Gupta S (2002) Isolation of human progenitor liver epithelial cells with extensive replication capacity and differentiation into mature hepatocytes. J Cell Sci 115:2679–2688 12. Thompson NL, Hixson DC, Callanan H, Panzica M, Flanagan D, Faris RA, Hong WJ, Hartel-Schenk S, Doyle D (1991) A Fischer rat substrain deficient in dipeptidyl peptidase IV activity makes normal steady-state RNA levels and an altered protein. Use as a liver-cell transplantation model. Biochem J 273:497–502 13. Gupta S, Rajvanshi P, Lee C-D (1995) Integration of transplanted hepatocytes in host liver plates demonstrated with dipeptidyl peptidase IV deficient rats. Proc Natl Acad Sci USA 92:5860–5864 14. Rajvanshi P, Kerr A, Bhargava KK, Burk RD, Gupta S (1996) Studies of liver repopulation using the dipeptidyl peptidase IV deficient rat and other rodent recipients: cell size and structure relationships regulate capacity for increased transplanted hepatocyte mass in the liver lobule. Hepatology 23:482–496 15. Gupta S, Rajvanshi P, Sokhi RP, Slehria S, Yam A, Kerr A, Novikoff PM (1999) Entry and integration of transplanted hepatocytes in liver plates occur by disruption of hepatic sinusoidal endothelium. Hepatology 29:509–519 16. Gupta S, Rajvanshi P, Sokhi R, Vaidya S, Irani AN, Gorla GR (1999) Position-specific gene expression in the liver lobule is directed by the microenvironment and not by the previous cell differentiation state. J Biol Chem 274:2157–2165 17. Gupta S, Rajvanshi P, Aragona E, Yerneni PR, Lee C-D, Burk RD (1996) Transplanted hepatocytes proliferate differently after CCl4 treatment and hepatocyte growth factor infusion. Am J Physiol 276:G629–G638 18. Gupta S, Aragona E, Vemuru RP, Bhargava K, Burk RD, Roy Chowdhury J (1991) Permanent engraftment and function of hepatocytes delivered to the liver: implications for gene therapy and liver repopulation. Hepatology 14:144–149 19. Sokhi RP, Rajvanshi P, Gupta S (2000) Transplanted reporter cells help in defining onset of hepatocyte proliferation during the life of F344 rats. Am J Physiol Gastrointest Liver Physiol 279:631–640 20. Rajvanshi P, Kerr A, Bhargava KK, Burk RD, Gupta S (1996) Efficacy and safety of repeated hepatocyte transplantation for
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52. Kobayashi N, Ito M, Nakamura J, Cai J, Gao C, Hammel JM, Fox IJ (2000) Hepatocyte transplantation in rats with decompensated cirrhosis. Hepatology 31:851–857 53. Gagandeep S, Rajvanshi P, Sokhi R, Slehria S, Palestro CJ, Bhargava KK, Gupta S (2000) Transplanted hepatocytes engraft, survive and proliferate in the liver of rats with carbon tetrachloride-induced cirrhosis. J Pathol 191:78–85 54. Irani AN, Malhi H, Slehria S, Gorla GR, Volenberg I, Schilsky ML, Gupta S (2001) Correction of liver disease following transplantation of normal hepatocytes in LEC rats modeling Wilson’s disease. Mol Ther 3:302–309 55. Malhi H, Irani AN, Volenberg I, Schilsky ML, Gupta S (2002) Early cell transplantation in LEC rats modeling Wilson’s disease eliminates hepatic copper with reversal of liver disease. Gastroenterology 122:438–447 56. Mito M, Kusano M, Kauwara Y (1992) Hepatocyte transplantation in man. Transplant Proc 24:3052–3053 57. Slehria S, Rajvanshi P, Ito Y, Sokhi R, Bhargava KK, Palestro CJ, McCuskey RS, Gupta S (2002) Hepatic sinusoidal vasodilators improve transplanted cell engraftment and ameliorate microcirculatory perturbations in the liver. Hepatology 35: 1320–1328 58. Malhi H, Annamaneni P, Slehria S, Joseph B, Bhargava KK, Palestro CJ, Novikoff PM, Gupta S (2002) Cyclophosphamide disrupts hepatic sinusoidal endothelium and improves transplanted cell engraftment in rat liver. Hepatology 36:112–121 59. Joseph B, Malhi H, Bhargava KK, Palestro CJ, McCuskey RS, Gupta S (2002) Kupffer cells participate in early clearance of syngeneic hepatocytes transplanted in the rat liver. Gastroenterology, in press 60. Overturf K, Al-Dhalimy M, Ou CN, Finegold M, Grompe M (1997) Serial transplantation reveals the stem-cell-like regenerative potential of adult mouse hepatocytes. Am J Pathol 151:1273–1280 61. Petersen BE, Bowen WC, Patrene KD, Mars WM, Sullivan AK, Murase N, Boggs SS, Greenberger JS, Goff JP (1999) Bone marrow as a potential source of hepatic oval cells. Science 284:1168– 1170 62. Theise ND, Badve S, Saxena R, Henegariu O, Sell S, Crawford JM, Krause DS (2000) Derivation of hepatocytes from bone marrow cells in mice after radiation-induced myeloablation. Hepatology 31:235–240 63. Theise ND, Nimmakayalu M, Gardner R, Illei PB, Morgan G, Teperman L, Henegariu O, Kause DS (2000) Liver from bone marrow in humans. Hepatology 32:11–16 64. Alison MR, Poulson R, Jeffrey R, Dhillon AP, Quaglia A, Jacob J, Novelli M, Prentice G, Williamson J, Wright NA (2000) Hepatocytes from non-hepatic adult stem cells. Nature 406:257 65. Lagasse E, Connors H, Al Dhalimy M, Reitsma M, Dohse M, Osborne L, Wang X, Finegold M, Weissman IL, Grompe M (2000) Purified hematopoietic stem cells can differentiate into hepatocytes in vivo. Nat Med 6:1229–1234 66. Korbling M, Katz RL, Khanna A, Ruifrok AC, Rondon G, Albitar M, Champlin RE, Estrov Z (2002) Hepatocytes and epithelial cells of donor origin in recipients of peripheral-blood stem cells. New Engl J Med 346:738–746 67. Kobayashi N, Fujiwara T, Westerman KA, Inoue Y, Sakaguchi M, Noguchi H, Miyazaki M, Cai J, Tanaka N, Fox IJ, Leboulch P (2000) Prevention of acute liver failure in rats with reversibly immortalized human hepatocytes. Science 287:1258–1262 68. Wege H, Le H, Wu J, Giri R, Malhi H, Gupta S, Zern MA (2001) Telomerase reconstitution immortalizes human hepatocytes. Hepatology 34:249A (abstract)
in Long–Evans cinnamon rats. Gastroenterology 111:1654– 1660 Grossman M, Rader DJ, Muller DWM, Kolansky DM, Kozarsky K, ClarkBJ III , Stein EA, Lupien PJ, Bryan BrewerH Jr , Raper SE, Wilson JM (1995) A pilot study of ex vivo gene therapy for homozygous familiar hypercholesterolaemia. Nat Med 1:1148– 1154 Strom SC, Fisher RA, Rubinstein WS, Barranger JA, Towbin RB, Charron M, Mieles L, Pisarov LA, Dorko K, Thompson MT, Reyes J (1997) Transplantation of human hepatocytes. Transplant Proc 29:2103–2106 Strom SC, Fisher RA, Thompson MT, Sanyal AJ, Cole PE, Ham JM, Posner MP (1997) Hepatocyte transplantation as a bridge to orthotopic liver transplantation in terminal liver failure. Transplantation 63:559–569 Fox IJ, Chowdhury JR, Kaufman SS, Goertzen TC, Chowdhury NR, Warkentin PI, Dorko K, Sauter BV, Strom SC (1998) Treatment of the Crigler–Najjar syndrome type I with hepatocyte transplantation. New Engl J Med 338:1422–1426 Chamuleau RAFM (1999) Hepatocyte transplantation for acute hepatic failure, in Hepatocyte Transplantation (Mito M, Sawa M, eds) pp. 159–167. Karger Landes Systems, Basel Makowka L, Rotstein LE, Falk RE, Falk JA, Langer B, Nossal NA, Blendis LM, Phillips JM (1980) Reversal of toxic and anoxic induced hepatic failure by syngeneic, allogeneic and xenogeneic hepatocyte transplantation, allogeneic and xenogeneic hepatocyte transplantation. Surgery 88:2243–2253 Miyazaki M, Makowka L, Falk RE, Falk W, Ventura D (1983) Reversal of lethal, chemotherapeutically induced acute hepatic necrosis in rats by regenerating liver cytosol. Surgery 94:142– 150 Baumgartner D, LaPlante-O’Neill PM, Sutherland DE, Najarian JS (1983) Effects of intrasplenic injection of hepatocytes, hepatocyte fragments and hepatocyte culture supernatants on d-galactosamine-induced liver failure in rats. Eur Surg Res 15:129–135 Gupta S, Rajvanshi P, Irani AN, Palestro CJ, Bhargava KK (2000) Integration and proliferation of transplanted cells in hepatic parenchyma following d-galactosamine-induced acute injury in F344 rats. J Pathol 190:203–210 Arkadopoulos N, Chen SC, Khalili TM, Detry O, Hewitt WR, Lilja H, Kamachi H, Petrovic L, Mullon CJ, Demetriou AA, Rozga J (1998) Transplantation of hepatocytes for prevention of intracranial hypertension in pigs with ischemic liver failure. Cell Transplant 7:357–363 Habibullah CM (1992) Hepatocyte transplantation: need for liver cell bank. Trop Gastroenterol 13:129–131 Bilir BM, Guinette D, Karrer F, Kumpe DA, Krysl J, Stephens J, McGavran L, Ostrowska A, Durham J (2000) Hepatocyte transplantation in acute liver failure. Liver Transplant 6:32–40 Gupta S, Rajvanshi P, Malhi H, Sokhi RP, Slehria S, Vasa SRG, Dabeva M, Shafritz DA, Kerr A (2000) Cell transplantation causes loss of gap junctions and activates GGT expression permanently in host liver. Am J Physiol Gastrointest Liver Physiol 279:G815–G826 Ribiero J, Nordlinger B, Ballet F, Cynober L, Coudray-Lucas C, Baudromont M, Legendre C, Delelo R, Panis Y (1992) Intrasplenic hepatocellular transplantation corrects hepatic encephalopathy in portacaval shunted rats. Hepatology 15:12–18 Schumacher IK, Okamoto T, Kim BH, Chowdhury NR, Chowdhury JR, Fox IJ (1996) Transplantation of conditionally immortalized hepatocytes to treat hepatic encephalopathy. Hepatology 24:337–343
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donor livers become available in the United States annually, whereas the number of people on waiting lists for OLT is several times greater. Therefore, it is imperative that alternative strategies, such as cell therapy, are developed. Percutaneous catheters can be utilized to transplant cells simply and the procedure is ‘reversible’ because the host liver remains intact. Due to the successes of hepatocyte transplantation in animals, several groups are preparing to evaluate cell therapy in a variety of diseases (Table 1). Among potentially significant applications of cell transplantation are included provision of metabolic support in acute or chronic liver failure and definitive treatment of inherited metabolic disorders. Moreover, hepatocytes are an excellent vehicle for ex vivo gene therapy. While hepatocytes are the most abundant liver cell type (∼60%), other cell types include biliary epithelial cells, hepatic stellate cells (found in the space of Disse, juxtaposed between hepatocytes and liver sinusoids), endothelial cells lining sinusoids, fibroblasts, pit cells, and littoral cells, such as Kupffer cells. Separation of these liver cell types and analysis of the biology of individual cell types is beginning to provide myriad insights into how cell–cell interactions could benefit hepatocyte transplantation. Recent insights in stem/progenitor cells isolated from the liver itself, from extrahepatic organs, as well as from pluripotential embryonic stem cells offer exciting new opportunities for cell therapy.
Liver repopulation with transplanted cells offers unique opportunities for treating a variety of diseases and for studies of fundamental mechanisms in cell biology. Our understanding of the basis of liver repopulation has come from studies of transplanted cells in animal models. A variety of studies established that transplanted hepatocytes as well as stem/progenitor cells survive, engraft, and function in the liver. Transplanted cells survive life-long, although cells do not proliferate in the normal liver. On the other hand, the liver is repopulated extensively when diseases or other injuries afflict native hepatocytes but spare transplanted cells. The identification of ways to repopulate the liver with transplanted cells has greatly reinvigorated the field of liver cell therapy. The confluence of insights in stem/progenitor cells, transplantation immunology, cryobiology, and liver repopulation in specific models of human diseases indicates that the field of liver cell therapy will begin to reap the promised fruit in the near future. Key words: liver repopulation / hepatocytes / transplanted cells © 2002 Elsevier Science Ltd. All rights reserved.
Introduction The prognosis for patients with liver failure, or potentially fatal inherited metabolic disorders is greatly improved by orthotopic liver transplantation (OLT), which refers to the replacement of the native liver with a donor liver. However, OLT is technically quite complex and has been greatly limited by shortages of donor organs. For instance, only approximately 5000
Mechanisms of transplanted cell engraftment, function, and proliferation Liver cells display major histocompatibility complex (MHC) antigens and mismatched cells are promptly rejected.1, 2 Therefore, liver cells must be transplanted into MHC-compatible or immunodeficient animals. In early studies, cells were transplanted into ectopic sites because methods to distinguish between transplanted and native cells in the liver were not then available.3 These studies showed that transplanted cells survived in the spleen, interscapular fat pad, peritoneal cavity,
From the Marion Bessin Liver Research Center, Departments of Medicine, Pathology and Molecular Genetics, Albert Einstein College of Medicine, Bronx, NY 10461, USA. * Corresponding author. Albert Einstein College of Medicine, Room 625, Ullmann Building, 1300 Morris Park Avenue, Bronx, NY 10461, USA. E-mail: [email protected] © 2002 Elsevier Science Ltd. All rights reserved. 1084–9521 / 02 / $– see front matter
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Table 1. Conditions potentially suitable for liver cell therapy Liver is diseased
Extrahepatic organs manifest disease
Genetic disorders Wilson’s disease α-1 antitrypsin deficiency Erythropoietic protoporphyria Lipidoses, e.g. Niemann–Pick disease Tyrosinemia type 1
Metabolic deficiency states Congenital hyperbilirubinemia, e.g. Crigler–Najjar syndrome Familial hypercholesterolemia Hyperammonemia syndromes Defects of carbohydrate metabolism Oxalosis
Acquired disorders Acute liver failure Chronic viral hepatitis Cirrhosis and liver failure
Coagulation disorders Hemophilia A Factor IX deficiency Immune disorders Hereditary angioedema
antigen (HBsAg), human α-1 antitrypsin (hAAT), and Escherichia coli β-galactosidase (LacZ).8, 9 Other reporters include green fluorescence protein (GFP), sex chromosome markers, or unique proteins that are deficient in recipient animals.10, 11 Use of the dipeptidyl peptidase IV deficient (DPPIV)-rat has been particularly helpful in elucidating the biology of transplanted cells.12 DPPIV is abundantly expressed in bile canaliculi, which provides methods to demonstrate whether transplanted cells are integrated in the liver parenchyma.13, 14 Studies in these animal models established that transplanted hepatocytes enter the liver plate after deposition in
and less effectively in a variety of other extrahepatic sites.4–6 The capacity of the spleen to accommodate cells is limited, although an enormous mass of transplanted hepatocytes could potentially be created in the peritoneal cavity. However, cell survival in the peritoneal cavity requires biomaterials for extracellular matrix support and transplanted cells show inferior gene expression in the peritoneal cavity.4 Numerous animal models are now available for studying transplanted cell survival in the liver (Table 2).7 Inbred transgenice mice constituted the earliest models, where donor hepatocytes expressed unique reporters, such as hepatitis B virus surface
Table 2. Useful animal models for studying liver cell therapy Nature of animal model
Animal designation
Animal origin or manipulation
Disease reproduced
Genetically determined
DPPIV-rat
Natural mutation (rat)
None
DPPIV-mouse FAH mouse Gunn rat LEC rat P-glycoprotein (mdr 2) mouse
Gene knockout Gene knockout Spontaneous mutation Spontaneous mutation Gene knockout
Watanabe heritable hyperlipidemic rabbit (WHHL) Nagase analbuminemic rat (NAR)
Spontaneous mutation
Tyrosinemia type 1 Crigler–Najjar syndrome Wilson’s disease Biliary phospholipid transport defect Familial hypercholesterolemia
Spontaneous mutation
Analbuminemia
Acetaminophen, carbon tetrachloride, d-galactosamine, other chemicals, subtotal partial hepatectomy Repeated carbon tetrachloride
Acute liver failure
Iatrogenically produced
Mice, rats, rabbits, pigs, dogs
Rats
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Liver cirrhosis
Cell therapy
Figure 1. Syngeneic F344 rat hepatocytes localized by histochemistry in the DPPIV-rat liver following intrasplenic transplantation. (A) Transplanted cells are located immediately adjacent to a portal area (Pa) (arrows). (B) Extensive liver repopulation in a rat subjected to liver radiation and ischemia-reperfusion injury before intrasplenic cell transplantation. Transplanted cells exhibit DPPIV activity (red color) in the bile canalicular domain and have replaced most of the liver. Tissues were stained lightly with hematoxylin to visualize cell nuclei.
liver sinusoids. Transplanted hepatocytes develop conjoint bile canaliculi and gap junctions with adjacent native hepatocytes, express liver genes in a physiologically regulated manner, and respond to mitogenic stimuli.13–17 However, transplanted cells do not proliferate in the normal rat or mouse liver,9, 18, 19 with the exceptions being the liver of either very young or old F344 rats, where transplanted cells exhibit spontaneous proliferative activity.19 This translates into the repopulation of only 0.5–1% of the liver following transplantation of 20 million cells in the rat or 2 million cells in the mouse liver, which can be increased to approximately 5% by transplanting cells repeatedly.20 However, correction of specific diseases might be incomplete with this magnitude of liver reconstitution and means to induce significant liver repopulation have been investigated. Induction of transplanted cell proliferation in the liver requires selective ablation of native hepatocytes with chemicals, e.g. carbon tetrachloride (CCl4 ), etc., or hepatotoxic trangenes (Reference 17 and see the following description). For instance, extensive liver repopulation occurs in animals with hepatic expression of urokinase-type plasminogen activator (alb-uPA mouse),21 the FAH mouse, which models hereditary tyrosinemia type 1,22 Fas ligand-induced apoptosis,23 prodrug activation of herpes simplex virus thymidine kinase (HSV-TK),24 expression of the cell cycle regulator Mad1,25 use of toxic bile salts in mice deficient in mdr 2 gene, which impairs biliary phopholipid excretion with hepatobiliary injury,26 etc. In the rat liver, genotoxic hepatic damage has been helpful for liver repopulation. The pyrollizidine
alkaloid, retrorsine, which alkylates DNA and inhibits hepatocellular proliferation has been highly effective for liver repopulation in rat.27 Retrorsine induces extensive hepatic polyploidy, which is also induced by two-thirds partial hepatectomy and the thyroid hormone, tri-iodothyronine (T3),28, 29 and the combination of these manipulations plays synergistic roles in liver repopulation.30 However, retrorsine is potentially oncogenic and is unsuitable for clinical use. Additional mechanisms for inducing genotoxic liver injury for liver repopulation include ionizing radiation and this has been as effective as retrorsine in repopulating the rat liver31 (Figure 1). It is noteworthy that radiation is ineffective by itself and requires either partial hepatectomy or ischemia-reperfusion injury, which cause oxidative DNA damage in the liver and promote radiation injury in the liver.32, 33 As radiation can be used clinically, this has raised the possibility of achieving extensive liver repopulation in the clinical setting.
Treatment of metabolic diseases Several metabolic diseases have been corrected in laboratory animals. The Gunn rat model of Crigler–Najjar syndrome type-1, has been highly characterized.6 In this hepatic metabolic disorder, bilirubin-UDP-glucuronosyltransferase (UGT1A1) activity is deficient and unconjugated bilirubin accumulates in toxic levels. Nagase analbuminemic rats (NAR) exhibit extremely low levels of serum albumin due to defective hepatic expression of the albumin 441
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gene.34 Transplantation of normal hepatocytes ameliorates the metabolic abnormality in Gunn rats as well as NAR.6 Additional animal models of metabolic disorders that are amenable to liver cell therapy include the Watanabe heritable hyperlipidemic rabbit (WHHL), which lack cell surface receptors for low density lipoproteins and models familial hypercholesterolemia,35 the Long–Evans Cinnamon (LEC) rat, an animal model for Wilson’s disease,36 the FAH mouse, which models hereditary tyrosinemia type-1,22 and mdr 2 knockout mice, which model progressive familial intrahepatic cholestasis.26 Studies in these animals have established a number of critical insights. For instance, transplantation of relatively small numbers of liver cells is insufficient for correcting metabolic deficiencies in the animals and significant liver repopulation has been necessary. These paradigms are critical for clinical applications. Early studies in patients have reproduced the experience in laboratory animals, which further validates the value of cell therapy studies in animal models. This early clinical experience extends to the transplantation of autologous genetically modified cells in patients with familial hypercholesterolemia37 and of allogeneic hepatocytes in patients with ornithine transcarbamylase (OTC) deficiency, α-1 antitrypsin deficiency, and Crigler–Najjar syndrome type-1.38–40 Treatment of one child with OTC deficiency led to limited reconstitution of hepatic enzyme activity but this was insufficient and the patient succumbed shortly thereafter to severe hyperammonemia.38 After cell transplantation in one patient with Crigler–Najjar syndrome type-1, serum bilirubin decreased significantly, hepatic bilirubin glucuronidating activity rose to approximately 5% of normal, and bilirubin glucuronides appeared in bile.40 However, the patient still required phototherapy and ultimately needed OLT. Therefore, further studies will be necessary with suitable strategies to amplify the transplanted hepatocyte mass for achieving cures.
liver. The potential of cell transplantation has been tested in a variety of animal models of acute liver failure.41 Multiple early studies showed that transplantation of hepatocytes in the peritoneal cavity, spleen, or liver improved mortality. However, it was unclear whether improved outcomes could be directly ascribed to transplanted cells or were due to unknown mechanisms, including mitogenic stimulation, cytoprotection, etc., because mortality improved following injection of conditioned medium from cultured cells, of fragmented cells, and even of xenogeneic cells that were promptly rejected in animals.42–44 More recently, transplanted hepatocytes were shown to engraft in the liver of animals with acute liver failure.45 Further studies in novel genetic models, where acute liver failure was induced by activation of ganciclovir by HSV-TK or Mad1 expression, showed that cell transplantation improved mortality.24, 25 Also, cell transplantation has been shown to prevent the development of intracranial hypertension in pigs following acute liver failure.46 Early studies of hepatocyte transplantation in patients with acute liver failure have generally been too limited to make firm conclusions regarding its efficacy. For example, seven patients with acute liver failure were treated with intraperitoneal transplantation of fetal human hepatocytes, which possibly improved survival in some patients, although it was unknown whether this was directly due to transplanted cell survival, as isolated hepatocytes do not survive for long following injection in the peritoneal cavity.47 In another clinical study, after hepatocyte transplantation through the splenic artery, five of seven patients with acute liver failure survived long enough to undergo OLT.48 In additional patients with acute liver failure, infusion of hepatocytes into the portal vein was shown to result in cell engraftment in the liver, along with some possible improvement in hepatic encephalopathy, cerebral perfusion pressure, etc.38 Perhaps the most important message from these early studies is that cells can be transplanted safely in patients with acute liver failure. Obviously, cell therapy for acute liver failure requires further study. Pertinent issues concern utilization of highly viable cells, identification of patients with comparable disease for demonstrating therapeutic outcomes, and correlations between the transplanted cell mass and outcomes. It is also noteworthy that currently available animal models may not reproduce the clinical syndrome of acute liver failure adequately and superior animal models are required. Also, mechanisms concerning transplanted cell engraftment
Hepatocyte transplantation for acute liver failure Cell therapy for acute liver failure is quite attractive for two reasons. First, prolongation of survival and amelioration of specific complications, such as raised intracranial pressure, could bridge patients to OLT. Second, OLT could be avoided altogether if cell transplantation would permit regeneration of the native 442
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and proliferation in acute liver failure need further analysis because the process of cell transplantation may aggravate liver injury to begin with by inducing ischemia-reperfusion-type changes, engraftment of transplanted cells might vary in different parts of the liver, and transplanted cell proliferation is delayed until transplanted cells are fully integrated in the liver parenchyma.24, 45, 49
cirrhotic animals. However, transplanted cell proliferation in the liver was limited and animals did not show any differences in mortality over a 12-month period. On the other hand, other investigators found that intrasplenic cell transplantation in extremely sick cirrhotic rats was associated with improvement in liver tests, coagulation abnormality, and outcomes.52 These findings have been interpreted to suggest that creation of an additional reservoir of cells in the liver will prolong survival in end-stage liver disease, although it is unclear as to why transplanted hepatocytes demonstrate superior function compared with the native liver in these animals. Studies in the LEC rat again established that transplanted cells engraft and survive in the diseased liver.36, 54, 55 However, transplanted cell proliferation in LEC rats requires selection pressure or development of extensive disease. In otherwise unmanipulated LEC rats, transplanted cell proliferation requires several months.54, 55 On the other hand, when copper accumulation and/or toxicity are exacerbated by retrorsine and partial hepatectomy, liver repopulation was greatly accelerated.54 It is noteworthy that cell transplantation can actually reverse established liver disease in LEC rats,55 which is in agreement with the potential of curing people with chronic viral hepatitis, were it possible to transplant cells that would resist viral infection. Our clinical experience of cell transplantation in patients with chronic liver disease is rather fledgling. In an early study, 10 Japanese patients with cirrhosis were transplanted into various sites, including spleen, with hepatocytes isolated from a piece of their own liver.56 In one of these patients, nuclear medicine studies showed transplanted hepatocytes in the spleen 11 months following transplantation, although whether any patients improved directly due to cell transplantation was unclear. Several patients with chronic liver disease have been transplanted with allogeneic hepatocytes through the splenic artery in the United States.38 However, it was unclear whether hepatocyte transplantation was responsible for improved liver function in these patients. The initial experience of cell transplantation suggests that further insights are necessary for optimizing various clinical procedures. For example, whether different results in animals and people might be related to injection of cells into the splenic artery in people requires study, because unlike cell engraftment in the red pulp of the spleen, injection of hepatocytes into arterial beds leads to rapid loss of transplanted cells, presumably due to shear injury and absence of cell anchorage. Similarly, whether immunosuppressive drugs might alter the fate or
Treatment of chronic liver failure Patients with end-stage liver disease and hepatic encephalopathy or other liver complications require OLT. However, many patients are unfit for receiving OLT and in many parts of the world OLT is not available. The general consideration is that cell transplantation could offer an additional mass of functioning liver, either in the liver itself or in the spleen, to supplement metabolic or synthetic function of the liver. Animals with established cirrhosis constitute an excellent model to establish these principles of cell therapy. On the other hand, if ongoing disease, e.g. chronic hepatitis, were to be ameliorated or cured, one would envision that transplantation of cells that are disease resistant would be needed. LEC rats modeling Wilson’s disease offer such a model because these animals exhibit genetically determined copper toxicosis, where excessive amounts of copper circulate in the body and exist within hepatocytes, which leads to extensive fibrosis and liver disease.36 The therapeutic potential of hepatocyte transplantation in decompensated liver disease has begun to be explored only recently. In rats with hepatic encephalopathy induced by end-to-side portacaval shunt, intrasplenic transplantation of hepatocytes improved encephalopathy scores and partially corrected changes in serum amino acid levels.50 Similarly, hepatocyte transplantation ameliorated hepatic encephalopathy in portacaval-shunted rats burdened with a load of ammonia.51, 52 Transplanted cells did not enter the liver in these animals due to portacaval shunting. Transplanted cells were present in the spleen, although the overall mass of transplanted cells created in the spleen was unknown. Animals with cirrhosis induced by repeated CCl4 administration develop significant liver fibrosis, portal hypertension, and ascites.53 Studies have been conducted in these animals by transplanting cells after discontinuing CCl4 to avoid clearance of transplanted cells. Transplanted cells could integrate in the liver parenchyma despite extensive fibrosis in 443
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function of transplanted cells in people is unknown. Finally, whether the concept of creating therapeutically useful additional mass of transplanted hepatocytes in patients with end-stage liver disease is indeed appropriate to begin with, requires consideration.
demonstrated that fetal human hepatocytes exhibit unique properties, including capacity for extensive proliferation and excellent recovery following cryopreservation. Moreover, following transplantation in immunodeficient mice, fetal human liver cells engraft in the liver parenchyma and generate mature hepatocytes. Use of hematopoeitic stem cells (HSC) derived from the bone marrow, cord blood, or peripheral blood has excited considerable interest. Petersen et al. initially showed that bone marrow-derived HSC generated hepatocytes in the DPPIV-rat.61 These observations were verified in the mouse as well as humans.62–66 Finally, isolation of pluripotential human embryonic stem cells or mesenchymal stem cells and early successes in differentiating these cells along specific lineages without tumorigenesis suggests that an unlimited supply of self-renewable donor cells will eventually become available. In the meantime, efforts continue to enhance the replication capacity of mature hepatocytes by genetic perturbations, such as introduction of the simian virus 40 T antigen, which drives cell proliferation or reconstitution of telomerase, in the absence of which cells enter replicative senescence.67, 68
Ongoing issues Further insights into mechanisms regulating transplanted cell engraftment and proliferation are needed to improve results. For example, only 20–30% of the transplanted hepatocytes survive and engraft in the liver.15 Recent studies indicate that perturbation of initial cell distributions in the liver sinusoids, interference with the phagocytotic responses activated by cell transplantation, and manipulation of the sinusoidal endothelial integrity prior to cell transplantation could have salutary effects on transplanted cell engraftment and subsequent liver repopulation.57–59 Similarly, it is possible to begin applying insights concerning the central role of genotoxic liver injury for inducing proliferation in transplanted cells. Use of ischemia-reperfusion injury, in combination with radiation, offers a potential mechanism for preconditioning the liver for inducing liver repopulation with transplanted cells. Abrogation of rejection following cell transplantation constitutes another important area, where much progress is being made, but space limitations preclude discussion of this area here. Similarly, advances in cryopreservation of hepatocytes will greatly facilitate clinical applications. In general, hepatocytes are highly susceptible to freeze–thaw damage and significant fractions of cells are lost following cryopreservation. If the initial cell isolate is of relatively marginal viability to begin with, it becomes difficult to obtain adequate numbers of cells for transplantation. Therefore, development of methods to procure highly viable cell populations and of ways to cryopreserve cells, such that frozen cells can be banked for use at short notice will greatly facilitate cell therapy. As the number of donor human livers will remain limited in the foreseeable future, use of stem cells is of great interest for expanding donor availability. Potential sources of such cells could concern hepatocytes themselves, which can replicate extensively, as shown by transplantation studies in FAH mice, where hepatocytes showed >80 cell divisions without loss of the replication potential.60 Isolation of progenitor liver cells, especially from the fetal liver, offers a practical way of expanding donor livers. The fetal liver contains an abundant supply of hepatoblasts.11 Recent studies
Acknowledgements This work was supported in part by NIH Grants R01 DK46952, R01 DK46057, P33DK41296, and P30 CA13330, and MO1 RR12248. The author gratefully acknowledges the contributions of his colleagues in many studies cited here.
References 1. Bumgardner GL, Gao D, Li J, Baskin JH, Heininger M, Orosz CG (2000) Rejection responses to allogeneic hepatocytes by reconstituted SCID mice, CD4, KO, and CD8 KO mice. Transplantation 70:1771–1780 2. Reddy B, Gupta S, Chuzhin Y, Kalergis A, Budhai L, Zhang M, Droguett G, Horwitz M, Roy Chowdhury J, Nathenson SG, Davidson A (2001) Differential effect of CD28/B7 blockade with CTLA4Ig on alloreactive CD4+, CD8+ and B cells in murine isolated hepatocyte transplantation. Transplantation 71:801– 811 3. Gupta S, Rajvanshi P, Bhargava KK, Kerr A (1996) Hepatocyte transplantation: progress toward liver repopulation. Prog Liver Dis 14:199–222 4. Gupta S, Vemuru RP, Lee C-D, Yerneni P, Aragona E, Burk RD (1994) Hepatocytes exhibit superior transgene expression after transplantation into liver and spleen compared with peritoneal cavity or dorsal fat pad: implications for hepatic gene therapy. Hum Gene Ther 5:959–967
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