Ira J. Fox

A Whole-Organ Regenerative Medicine Approach for Liver Replacement

BACKGROUND & AIMS The therapy of choice for end-stage liver disease is whole-organ liver transplantation, but this option is limited by a shortage of donor organs. Cell-based therapies and hepatic tissue engineering have been considered as alternatives to liver transplantation, but neither has proven effective to date. A regenerative medicine approach for liver replacement has recently been described that includes the use of a three-dimensional organ scaffold prepared by decellularization of xenogeneic liver. The present study investigates a new, minimally disruptive method for whole-organ liver decellularization and three different cell reseeding strategies to engineer functional liver tissue. METHODS A combination of enzymatic, detergent, and mechanical methods are used to remove all cells from isolated rat livers. Whole-organ perfusion is used in a customized organ chamber and the decellularized livers are examined by morphologic, biochemical, and immunolabeling techniques for preservation of the native matrix architecture and composition. Three different methods for hepatocyte seeding of the resultant three-dimensional liver scaffolds are evaluated to maximize cell survival and function: (1) direct parenchymal injection, (2) multistep infusion, or (3) continuous perfusion. RESULTS The decellularization process preserves the three-dimensional macrostructure, the ultrastructure, the composition of the extracellular matrix components, the native microvascular network of the liver, and the bile drainage system, and up to 50% of growth factor content. The three-dimensional liver matrix reseeded with the multistep infusion of hepatocytes generated ∼90% of cell engraftment and supported liver-specific functional capacities of the engrafted cells, including albumin production, urea metabolism, and cytochrome P450 induction. CONCLUSIONS Whole-organ liver decellularization is possible with maintenance of structure and composition suitable to support functional hepatocytes.

New ISSCR Guidelines Underscore Major Principles for Responsible Translational Stem Cell Research

The International Society for Stem Cell Research (ISSCR) task force that developed new Guidelines for the Clinical Translation of Stem Cells discusses core principles that should guide the responsible transition of basic stem cell research into appropriate clinical applications.

Use of differentiated pluripotent stem cells in replacement therapy for treating disease

BACKGROUND Decades of laboratory and clinical investigation have led to successful therapies using hematopoietic stem cells (HSCs), but few other cell therapies have transitioned from experimental to standard clinical care. Providing patients with autologous rather than allogeneic HSCs reduces morbidity and mortality, and in some circumstances broader use could expand the range of conditions amenable to HSC transplantation. The availability of a homogeneous supply of mature blood cells would also be advantageous. An unlimited supply of pluripotent stem cells (PSCs) directed to various cell fates holds great promise as source material for cell transplantation and minimally invasive therapies to treat a variety of disorders. In this Review, we discuss past experience and challenges ahead and examine the extent to which hematopoietic stem cell transplantation and cell therapy for diabetes, liver disease, muscular dystrophies, neurodegenerative disorders, and heart disease would be affected by the availability of precisely differentiated PSCs. Unlimited populations of differentiated PSCs should facilitate blood therapies and hematopoietic stem cell transplantation, as well as the treatment of heart, pancreas, liver, muscle, and neurologic disorders. However, successful cell transplantation will require optimizing the best cell type and site for engraftment, overcoming limitations to cell migration and tissue integration, and possibly needing to control immunologic reactivity (challenges indicated in red). iPSC, induced PSC; ES cells, embryonic stem cells. Unlimited populations of differentiated PSCs should facilitate blood therapies and hematopoietic stem cell transplantation, as well as the treatment of heart, pancreas, liver, muscle, and neurologic disorders. However, successful cell transplantation will require optimizing the best cell type and site for engraftment, overcoming limitations to cell migration and tissue integration, and possibly needing to control immunologic reactivity (challenges indicated in red). iPSC, induced PSC; ES cells, embryonic stem cells. ADVANCES Although it is not yet possible to differentiate PSCs to cells with characteristics identical to those in the many organs that need replacement, it is likely a matter of time before these “engineering” problems can be overcome. Experience with cell therapies, both in the laboratory and the clinic, however, indicate that many challenges remain for treatment of diseases other than those involving the hematopoietic system. There are issues of immunity, separate from controlling graft rejection, and identifying the optimal cell type for treatment in the case of muscular dystrophies and heart disease. Optimization is also needed for the transplant site, as in diabetes, or when dealing with disruption of the extracellular matrix in treating degenerative diseases, such as chronic liver and heart disease. Finally, when the pathologic process is diffuse and migration of transplanted cells is limited, as is the case with Alzheimer’s disease, amyotrophic lateral sclerosis, and the muscular dystrophies, identifying the best means and location for cell delivery will require further study. OUTLOOK Considering the pace of progress in generating transplantable cells with a mature phenotype, and the availability of PSC-derived lineages in sufficient mass to treat some patients already, the challenges to scaling up production and eliminating cells with tumor-forming potential are probably within reach. However, generation of enough cells to treat an individual patient requires time for expansion, differentiation, selection, and testing to exclude contamination by tumorigenic precursors. Current methods are far too long and costly to address the treatment of acute organ injury or decompensated function. Immune rejection of engrafted cells, however, is likely to be overcome through transplantation of autologous cells from patient-derived PSCs. Availability of PSC-derived cell populations will have a dramatic effect on blood cell transfusion and the use of hematopoietic stem cell transplantation, and it will likely facilitate treatment of diabetes, some forms of liver disease and neurologic disorders, retinal diseases, and possibly heart disease. Close collaboration between scientists and clinicians—including surgeons and interventional radiologists—and between academia and industry will be critical to overcoming challenges and to bringing new therapies to patients in need. Challenges for stem cell–based therapies Patient-derived pluripotent stem cells (PSCs) hold promise in the treatment of injury and disease. An ever-increasing number of specific cell types can be generated from PSCs, but technical challenges remain in applying these cells in the clinic. Fox et al. review the challenges in attaining this goal. These include gene modification, cell rejection, and delivery and localization issues involved in transplantation of cells for the treatment of diabetes and disorders of the blood, liver, heart, and brain. Science, this issue 10.1126/science.1247391 Pluripotent stem cells (PSCs) directed to various cell fates holds promise as source material for treating numerous disorders. The availability of precisely differentiated PSC-derived cells will dramatically affect blood component and hematopoietic stem cell therapies and should facilitate treatment of diabetes, some forms of liver disease and neurologic disorders, retinal diseases, and possibly heart disease. Although an unlimited supply of specific cell types is needed, other barriers must be overcome. This review of the state of cell therapies highlights important challenges. Successful cell transplantation will require optimizing the best cell type and site for engraftment, overcoming limitations to cell migration and tissue integration, and occasionally needing to control immunologic reactivity, as well as a number of other challenges. Collaboration among scientists, clinicians, and industry is critical for generating new stem cell–based therapies.

Improving the techniques for human hepatocyte transplantation: Report from a consensus meeting in London

On September 6 and 7, 2009 a meeting was held in London to identify and discuss what are perceived to be current roadblocks to effective hepatocyte transplantation as it is currently practiced in the clinics and, where possible, to offer suggestions to overcome the blocks and improve the outcomes for this cellular therapy. Present were representatives of most of the active clinical hepatocyte transplant programs along with other scientists who have contributed substantial basic research to this field. Over the 2-day sessions based on the experience of the participants, numerous roadblocks or challenges were identified, including the source of cells for the transplants and problems with tracking cells following transplantation. Much of the discussion was focused on methods to improve engraftment and proliferation of donor cells posttransplantation. The group concluded that, for now, parenchymal hepatocytes isolated from donor livers remain the best cell source for transplantation. It was reported that investigations with other cell sources, including stem cells, were at the preclinical and early clinical stages. Numerous methods to modulate the immune reaction and vascular changes that accompany hepatocyte transplantation were proposed. It was agreed that, to obtain sufficient levels of repopulation of liver with donor cells in patients with metabolic liver disease, some form of liver preconditioning would likely be required to enhance the engraftment and/or proliferation of donor cells. It was reported that clinical protocols for preconditioning by hepatic irradiation, portal vein embolization, and surgical resection had been developed and that clinical studies using these protocols would be initiated in the near future. Participants concluded that sharing information between the groups, including standard information concerning the quality and function of the transplanted cells prior to transplantation, clinical information on outcomes, and standard preconditioning protocols, would help move the field forward and was encouraged.

Barriers to the successful treatment of liver disease by hepatocyte transplantation

Management of patients with hepatic failure and liver-based metabolic disorders is complex and expensive. Hepatic failure results in impaired coagulation, altered consciousness and cerebral function, a heightened risk of multiple organ system failure, and sepsis [1]. Such manifold problems are only treatable today and for the foreseeable future by transplantation. In fact, whole or auxiliary partial liver transplantation is often the only available treatment option for severe, even if transient, hepatic failure. Patients with life-threatening liver-based metabolic disorders similarly require organ transplantation even though their metabolic diseases are typically the result of a single enzyme deficiency, and the liver otherwise functions normally. For all of the benefits it may confer, liver transplantation is not an ideal therapy, even for severe hepatic failure. More than 17,000 patients currently await liver transplantation in the United States, a number that seriously underestimates the number of patients that need treatment [2], as it has been estimated that more than a million patients could benefit from transplantation [3]. Unfortunately, use of whole liver transplantation to treat these disorders is limited by a severe shortage of donors and by the risks to the recipient associated with major surgery [4].

Cell and Tissue Engineering for Liver Disease

Advances in cell and tissue engineering are moving cell-based therapies for treating liver disease and liver failure closer to the clinic. Despite the tremendous hurdles presented by the complexity of the liver’s structure and function, advances in liver physiology, stem cell biology and reprogramming, and the engineering of tissues and devices are accelerating the development of cell-based therapies for treating liver disease and liver failure. This State of the Art Review discusses both the near- and long-term prospects for such cell-based therapies and the unique challenges for clinical translation.

Route of hepatocyte delivery affects hepatocyte engraftment in the spleen1

In laboratory animals, intrasplenic hepatocyte transplantation corrects the physiologic abnormalities associated with decompensated liver disease. The clinical experience with hepatocyte transplantation for cirrhosis has been disappointing when compared with laboratory experience. The route of hepatocyte delivery may influence hepatocyte engraftment and function. Outbred pigs were recipients of allogeneic pig hepatocytes. Donor hepatocytes were isolated by collagenase perfusion and labeled using 5(6)-carboxyfluorescein diacetate succinimidyl-ester (CMFSE). Cells were introduced into pig spleens by infusion through the splenic artery or by direct splenic puncture. Direct intrasplenic injection produced engraftment that was far superior to that obtained using splenic artery infusion. Splenic artery infusion produced a gastric erosion and large areas of splenic necrosis secondary to vascular occlusion with hepatocytes, whereas direct splenic injection was associated with clinically insignificant intraabdominal hemorrhage. The route of hepatocyte delivery may influence hepatocyte engraftment and explain the disparity in efficacy of hepatocyte transplantation between the laboratory and clinic.

Spontaneous hepatic repopulation in transgenic mice expressing mutant human α1-antitrypsin by wild-type donor hepatocytes

α1-Antitrypsin deficiency is an inherited condition that causes liver disease and emphysema. The normal function of this protein, which is synthesized by the liver, is to inhibit neutrophil elastase, a protease that degrades connective tissue of the lung. In the classical form of the disease, inefficient secretion of a mutant α1-antitrypsin protein (AAT-Z) results in its accumulation within hepatocytes and reduced protease inhibitor activity, resulting in liver injury and pulmonary emphysema. Because mutant protein accumulation increases hepatocyte cell stress, we investigated whether transplanted hepatocytes expressing wild-type AAT might have a competitive advantage relative to AAT-Z-expressing hepatocytes, using transgenic mice expressing human AAT-Z. Wild-type donor hepatocytes replaced 20%-98% of mutant host hepatocytes, and repopulation was accelerated by injection of an adenovector expressing hepatocyte growth factor. Spontaneous hepatic repopulation with engrafted hepatocytes occurred in the AAT-Z-expressing mice even in the absence of severe liver injury. Donor cells replaced both globule-containing and globule-devoid cells, indicating that both types of host hepatocytes display impaired proliferation relative to wild-type hepatocytes. These results suggest that wild-type hepatocyte transplantation may be therapeutic for AAT-Z liver disease and may provide an alternative to protein replacement for treating emphysema in AAT-ZZ individuals.

Changing concepts: Liver replacement for hereditary tyrosinemia and hepatoma*

In recent years there has been increased use of hepatic transplantation for the treatment of liver-based inborn errors of metabolism.1,2 In 1976, a 9-year-old girl with chronic hereditary tyrosinemia who had developed a 15-cm hepatoma in her cirrhotic liver underwent liver replacement with immunosuppression therapy with azathioprine, prednisone, and antilymphocyte globulin. The abnormal metabolic profile of tyrosinemia was promptly and completely corrected, but a pulmonary metastasis from the hepatoma was discovered shortly afterward. The new liver was rejected in 3 months, and the patient died during a second attempt at transplantation.3 We have had subsequent experience with four additional patients with the same diagnoses, in whom immunosuppression therapy after liver replacement was with cyclosporine and prednisone. These four recipients are well and metabolically normal 3 months to almost 3 years after transplantation and have no evidence of recurrent tumor. These observations suggest the desirability of liver transplantation earlier in the course of this disease. The point has been supported by experience with a fifth candidate whose proposed transplantation was interdicted by metastases to the diaphragm, which were discovered at the time of operation. This 4-year-old girl died 1½ months later. The four recipients, who received treatment in the cyclosporine era, were 2½ to 21 years of age. Each had cirrhosis and multiple abnormalities of liver function, including prolonged prothrombin time and low-grade hyperbilirubinemia (Table). The diagnosis had been made early in life by the demonstration at established metabolic centers of hypertyrosinemia, tyrosinuria, and marked excretion of tyrosine metabolites in the urine, which were managed with a diet low in tyrosine and phenylalanine. Table Clinical features In three of the patients, elevations of α-fetoprotein (Table) originally aroused suspicion of hepatoma development. However, a definite mass was detectable with computed tomography and other radiographic techniques only in the oldest (patient 1). This patient underwent a right hepatic lobectomy at another hospital, at which time the main portal vein was accidentally tied off; the hepatoma was thought to be cleanly removed. After the right-sided lobectomy, she developed very severe liver failure and was bedridden until the time of transplantation 2 months later. There was no residual tumor in the hepatic remnant. In patient 2 the diagnosis of hepatoma had been suspected after a routine ultrasound examination, and was confirmed by open liver biopsy. Patients 2, 3, and 4 had multiple small hepatomas in all parts of the excised livers. However, the surgical margins were free of tumor. Although the livers were cirrhotic, they were relatively soft. The transplantation procedures were by well-standardized techniques,1,4 except in the child who had undergone right hepatic lobectomy, whose portal vein was thrombosed from the site of surgical ligature back to the confluence of the splenic and superior mesenteric veins. In this recipient a cloaca was fashioned at the superior mesenteric–splenic venous junction, to which a free inferior vena caval graft from the liver donor was anastomosed. The donor portal vein was anastomosed, in turn, to the proximal end of this graft.5 Cyclosporine and prednisone were given intravenously or orally from the time of operation, with rapid weaning from prednisone to maintenance doses, presently 2.5 to 7.5 mg/day. Despite therapy, one of the recipients (patient 2) slowly rejected the graft, and retransplantation was carried out without incident 18 months after the primary procedure. She is well 15½ months after retransplantation. The other three recipients also are well after 3, 7, and 17 months, respectively. The α-fetoprotein levels, which ranged from 4600 to 25,000 ng/ml before liver replacement (or before hepatic resection in patient 1) fell to within the normal range within a few days or weeks, and have remained normal. There has been no evidence of recurrent hepatoma in any patient, and all four now have normal liver function. The metabolic abnormalities characteristic of tyrosinemia were normalized immediately after transplantation, even though the patients were given a regular diet. Detailed studies of amino acid metabolism have been or are being carried out in the referring centers (Table) and will be described separately. It is now thought that hereditary tyrosinemia is caused by fumarylacetoacetate hydrolase deficiency.6–8 In other liver-based inborn errors of metabolism with or without a specific and identifiable enzyme defect, the metabolic phenotype of the graft has remained permanently that of the donor.1,2 Thus the metabolic amelioration in our patients with tyrosinemia should be for the lifetime of the grafts. The use of liver transplantation for “metabolic engineering” has been a tantalizing prospect for a number of years, but the poor results with liver replacement discouraged the wide application of this approach until recently. With the advent of immunosuppression therapy with cyclosporine and steroids, the prognosis after liver replacement has improved so dramatically, particularly in pediatric recipients, that reluctance to go forward with this aggressive therapy has diminished.1 Furthermore, the increasingly recognized risk of hepatoma formation9 is an additional and potent reason to consider liver transplantation at an earlier time and under semielective conditions. In the early days of liver transplantation, efforts to treat hepatomas that could not be excised by conventional techniques resulted in an incidence of tumor recurrence so high that the potential value of the operation was vitiated.1,4 With better patient selection in more recent times, this incidence of recurrence has been reduced,1 and in patients with hepatomas incidental to tyrosinemia, α1-antitrypsin deficiency, sea-blue histiocyte syndrome, or biliary atresia, the incidence of recurrence has been zero. Thus, the threat of late metastases in the four surviving patients with tyrosinemia is not as great as might have been predicted from the older literature.

Patients Beware: Commercialized Stem Cell Treatments on the Web

A report by the International Society for Stem Cell Research (ISSCR)s Task Force on Unproven Stem Cell Treatments outlines development of resources for patients, their families, and physicians seeking information on stem cell treatments.