Stem Cells and Hepatocyte Transplantation

Introduction

Regenerative medicine is a much overused phrase but at its core is the concept that the therapy being administered would work with the innate healing and regenerative properties of the host tissue to facilitate recovery from illness or injury. Because of its regenerative capacity, the liver offers more opportunities for regenerative therapy than perhaps any other internal organ. The complexity and scope of the metabolic and synthetic processes provided by the liver range from the storage of glycogen and release of glucose to metabolism of drugs, toxins, and endogenous hormones, metabolism and excretion of ammonia ,and the production and secretion of bile acids, serum lipoproteins, clotting factors, albumin, and protease inhibitors. These examples underscore the large number and vital nature of the processes conducted in and by the liver. These processes are so critical that a minimum of liver function must be maintained for an individual to survive. The standard therapy for metabolic or end-stage liver disease has been orthotopic liver transplantation (OLT). Experimental therapies involving a regenerative approach of the supplementation of liver function by the incorporation of donor cells into the native organ rather than total replacement of the liver have received scientific and medical interest, and hepatocyte transplantation is now an accepted albeit still experimental therapy for liver disease.

Since the first report of the transplantation into a Gunn rat of rat hepatocytes that were proficient in bilirubin conjugation in 1977 by Groth et al., a body of literature has been generated testing the hypothesis that hepatocyte transplantation could be corrective of liver disease in the absence of whole organ transplantation. Preclinical studies using small animals as recipients of the transplanted hepatocytes were highly supportive of this hypothesis. On the basis of approximately 25 years of preclinical studies with different animal models, different transplantation techniques and a variety of regimens to precondition the liver before transplant to enhance hepatocyte engraftment and subsequent liver repopulation, clinical studies were initiated in the early 1990s. Clinical studies have for the most part validated the efficacy of hepatocyte transplants in a variety of human liver diseases. However, the levels of disease correction observed in the animal models were not always replicated in subsequent clinical trials. The general hypothesis that isolated hepatocytes could be transplanted into a patient with a liver disease and that the cell transplant would have a positive impact on the clinical condition of the patient has been well supported by the data. Despite there being room for improvement in the cell source and repopulation of the recipient liver with donor cells, hepatocyte transplantation remains a viable option for the treatment of liver disease in patients. There are at least seven active hepatocyte transplant groups in Europe, Asia, and the United States, and there may be more than one active group in China.

As originally conceived, hepatocyte transplantation was considered to be beneficial and was tested for three applications:

• 1.

  Bridge a patient to whole liver transplantation

• 2.

  Support of patients in acute liver failure

• 3.

  Correction of a metabolic liver disease

These applications were based on preclinical studies showing efficacy of hepatocyte transplants for the support of liver function in chronic liver disease, acute liver failure, and monogenetic defects in critical liver metabolic functions.

The concept of “bridging” a patient is a process where hepatocyte transplantation is used as a temporary measure to support liver function in a patient who is listed for whole organ transplantation but is deteriorating significantly and is not expected to live more than a few days. In general, these patients have end-stage liver disease with decompensated cirrhosis. They are frequently coagulopathic and encephalopathic. Hepatocyte transplants are performed not with the hope of reversing the disease process but simply to sustain the patient for hours to days until a liver can be identified for OLT. Because of the low-level engraftment of hepatocytes into cirrhotic liver, and the likelihood of shunting of infused cells around the liver parenchyma resulting in whole body distribution of transplanted cells, hepatocytes were frequently transplanted into the spleen of the patients rather than the liver. Evidence of clinical improvement was frequently observed in these patients after the cellular therapy, and will be discussed in greater detail below.

In a manner similar to the bridging technique, hepatocyte transplants have been used in cases of acute liver failure when an organ was not immediately available for OLT. Because it is impossible to introduce sufficient numbers of hepatocytes into the vascular system (e.g., in the range of 20% to 30% of liver mass) and obtain high levels of engraftment into liver parenchyma, hepatocyte transplants for acute liver failure are usually conducted with the intent to bridge the patient to OLT. However, in some reported cases the patients recovered liver function following the hepatocyte transplant and could be discharged without whole organ transplantation. As these are case reports rather than controlled clinical trials, it is not possible to determine if the cellular therapy was the cause of the improvement.

The most common use of hepatocyte transplants is for the correction of metabolic liver diseases. These transplants are usually conducted on pediatric patients with monogenetic defects in genes that encode critical liver functions such as ammonia or bilirubin metabolism. Loss-of-function mutations in these critical liver genes can result in life-threatening metabolic crises within a few hours of birth. In the immediate neonatal period, these patients are too small and fragile for a whole organ transplant. Cellular therapy is initiated to provide the enzymatic/metabolic function missing in the native liver. Unlike the first two applications, where the original intent of the treatment was to bridge the patient to OLT, with metabolic liver disease, the original intent of the cellular therapy was to correct the defect in liver function without whole organ transplant. Outcomes for all of these applications will be discussed in subsequent sections of this chapter.

The theoretical benefits of hepatocyte transplants over whole organ transplants are considerable, and are shown in Table 6-1 .

Hepatocyte transplants are usually conducted with cells isolated from a donor liver that is not being used for whole organ transplant. The donor liver tissue could result from an organ rejected for transplant or one simply reduced in size before transplantation. Thus sources of liver tissue for cell transplants are more numerous than those for OLT. The timing of a hepatocyte transplant is more flexible than that of an organ transplant. With proper cryopreservation techniques, cells from a viable, functional organ could be cryopreserved or cold-stored for up to 48 hours for a patient transplant at a later time. Although not all donor livers provide cells that can be effectively cryopreserved, there is a theoretical advantage to hepatocyte transplants in that they could be routinely and immediately available for patient transplants. Hepatocyte transplantation is minimally invasive and is usually conducted through a small catheter inserted into a blood vessel feeding an organ such as the liver or spleen, whereas OLT requires massive surgery and incurs significant financial costs for the procedure. Because hepatocyte transplant is a minimally invasive procedure, the complications associated with it are minor as compared with those associated with OLT, and the recovery time after the procedure is also minimal. Once a catheter has been placed, subsequent hepatocyte transplants can be conducted on a patient while the patient is awake, and most patients older than 2 years can be discharged home when the procedure has been completed without pain, discomfort, or sequelae. With OLT, the recipient's native liver is entirely removed, and the entirety of liver support is transferred to the transplanted organ. With hepatocyte transplantation, the native liver is maintained and the donor hepatocytes need only support liver function not entirely replace it. With patients in acute liver failure, partial support provided by hepatocyte transplants could sustain them and provide critical time for their native liver to regenerate.

In cases of metabolic liver disease, replacement of 5% to 15% of the metabolic activity or function missing in the patient is usually sufficient to substantially correct the symptoms of the disease, and the replacement of the entire recipient liver with donor hepatocytes is not required. Hepatocyte transplant has been conducted on the first day of life in a baby in whom ornithine transcarbamylase (OTC) deficiency was diagnosed prenatally, whereas OLT is usually reserved for patients of approximately 6 months of age. For life-threatening diseases such as urea cycle defects, cellular therapy of the disease can be initiated far earlier than OLT, thus providing an opportunity to at least partially correct the metabolic defect and transform a life-threatening disease into one more easily managed.

Differences between OLT and hepatocyte transplants becomes even more significant in the event of graft loss. With OLT, graft loss requires immediate retransplantation with additional costs, morbidity, and mortality. The loss of the hepatocyte cell graft only returns the patient to his or her clinical condition before the cell transplant and is not necessarily life threatening as the patient can usually be sustained by traditional medical therapy.

There is no evidence that prior hepatocyte transplantation interferes with a subsequent OLT. Fisher et al. reported that there was no evidence of sensitization of the patient to the donor cells or the eventual liver when an OLT was performed following a hepatocyte transplant. Although there are many theoretical benefits to hepatocyte transplants over OLT, to date no patients have achieved a long-term complete correction of their metabolic liver disease by hepatocyte transplants. Successful OLT is frequently curative of the liver defect, whereas hepatocyte transplants, as currently performed, are only partially corrective. Future improvements in the availability of cells and conditioning regimens that enhance hepatocyte engraftment and repopulation of the native liver will be required before this cellular therapy is completely corrective of a metabolic liver disease on a long-term basis.

Preclinical and Background Studies Enabling Clinical Translation

The clinical application of hepatocyte transplant procedures was supported by approximately 25 years of preclinical studies. It is not our intent here to review the entirety of the preclinical literature that provided the theoretical basis for later clinical translation; however, some specific reports will be cited that were either the initial studies in that area or were highly informative for the understanding of the mechanisms by which hepatocytes survive, engraft, and repopulate the liver and how donor cells integrate into native liver structure and function. More complete reviews of the older preclinical literature are available.

Although earlier studies of the correction of liver diseases were attempted with transplants of liver tumor cell lines or slices or fragments of liver, the first reported transplantation of isolated hepatocytes by Groth et al. could only be attempted after methods to isolate viable hepatocytes from liver tissue had been established. An article critical to the hepatocyte field is that of Berry and Friend from 1969: they reported methods for the routine and successful isolation of large numbers of viable hepatocytes from rat liver by perfusion of the existing liver vessels with a solution containing a crude mixture of proteolytic enzymes, including collagenases. A second article enabling hepatocyte transplants came from a parallel cell therapy field. In 1972 Ballinger and Lacy reported the transplantation of isolated islets of Langerhans into diabetic rats. This was the first report of the partial correction of a defect in a visceral organ by a cellular therapy, and supported the hypothesis that cellular transplants could be corrective of solid organ diseases.

Given the known ability of the liver to restore liver mass and function following tissue loss, acute liver failure became an obvious target for cell therapy research and application. From both animal and clinical studies it was known that large amounts of liver tissue could be destroyed by chemical or immunologic means or by surgical procedures and the subject could still survive, thus demonstrating that 100% of liver mass or function was not required for survival. Similarly, from genetic and functional studies of metabolic liver disease patients with mutations in genes encoding critical liver functions, it was understood that one does not require 100% of the enzyme activity or metabolic function of the entire liver to remain free of the symptoms of the disease. Because total liver replacement with donor hepatocytes would not be required, metabolic liver diseases were also the early targets for hepatocyte transplant studies. In the first published preclinical account of isolated hepatocyte transplantation, Groth et al. reported the correction of hyperbilirubinemia in the Gunn rat following hepatocyte infusion. The Gunn rat is deficient in uridine diphosphate glucuronosyltransferase 1 family member A1, the enzyme responsible for the conjugation of bilirubin, and is a useful animal model for Crigler-Najjar syndrome type 1, the human disease resulting from the same enzyme deficiency. These investigators reported a maximum 35% decrease in bilirubin levels in treated animals by day 28 after transplant.

Demetriou et al. were the first to examine the effects of hepatocyte transplantation on acute liver failure and also the first to use human hepatocytes in transplant protocols. In their initial studies, they performed 90% hepatectomy of rats to model small-for-size acute liver failure. All rats that did not receive transplants died within 48 hours, whereas 40% of the rats that received hepatocyte-covered microcarrier beads survived. Cell infusions were not effective when initiated after the hepatectomy, and were effective only when they were infused before the surgical procedure. These initial studies did, however, provide a proof of concept that the transplantation of isolated hepatocytes could support liver function during episodes of acute liver failure. Using the microcarrier approach to cell transplantation, Moscioni et al. were the first to report that the transplantation of isolated human hepatocytes could provide long-term synthetic function. Athymic Gunn rats and athymic analbuminemic Nagase rats received infusions of microcarrier-attached human hepatocytes. In the Gunn rats, bilirubin levels were significantly reduced and the production of bilirubin conjugates was demonstrated. The Nagase rat produces extremely low levels of circulating albumin. Following infusion of the human hepatocytes, albumin levels increased more than 40-fold and remained high for more than 30 days. These early studies provided proof that human hepatocytes could produce and secrete plasma proteins following transplantation.

Integration of Hepatocytes in the Recipient's Liver

Much of our understanding of the integration of hepatocytes following transplantation was derived from preclinical studies with small animals. After hepatocytes are introduced into the portal blood flow, there is a complex set of actions and reactions between the host and the donor cells. Gupta et al. were instrumental in describing the steps involved in cell integration. Although it is described in four general steps, there are no boundaries between the steps and it is one continual process. To fully integrate and incorporate donor cells into the recipient liver, the following four steps must occur:

• 1.

  Temporarily fill and occlude small portal veins

• 2.

  Traverse the endothelial barrier

• 3.

  Integrate into hepatic chords

• 4.

  Remodel microenvironment and reestablish intercellular connections

Pictures illustrating these steps can be found in Koenig et al.

Initial Clinical Studies

Given the extensive body of preclinical literature concerning hepatocyte transplants to treat liver disease, two groups published the first reports of hepatocyte transplants in patients. Mito et al. isolated hepatocytes from patients with end-stage cirrhosis into whom autologous hepatocytes were infused following the resection, whereas Fisher et al. isolated and transplanted allogeneic hepatocytes. Because of the cirrhotic nature of the liver, with the autologous transplants Fischer et al. transplanted the hepatocytes into the spleen. In a few recipients these authors reported that they could detect donor cells in the spleen out to several months after transplant. Although one patient returned to work following the procedure, the authors concluded that they could not discern any clinical benefit from the procedure. There are no other reports of autologous transplants, with the exception of gene therapy for familial hypercholesterolemia, and all subsequent investigators used allogeneic hepatocytes as the source of cells.

In the initial report by Fisher et al., and in later follow-up reports with additional patients, the spleen was chosen as the site for the transplantation of hepatocytes. Some details will be given concerning these initial transplants, as they are in many ways typical of the first allogeneic hepatocyte transplants conducted later by other groups. In the report of the first five patients who received allogeneic hepatocyte transplants, the causes differed but the clinical status of the patients before transplant was similar. In the initial group, there were two patients with acute liver failure, one due to phenytoin toxicity and a second due to hepatitis B. In addition there was one patient with total parenteral nutrition–induced liver failure secondary to necrotizing enterocolitis. The final two patients had decompensated end-stage liver disease, one due to α ₁ -antitrypsin deficiency and the other due to hepatitis C. All five patients had grade 4 encephalopathy, and failure of three or four organ systems, including liver, lung, renal, and brain failure and in the case of the total parenteral nutrition patient, intestinal failure. Four additional patients with an equal severity of disease as judged by the grade of encephalopathy, prothrombin times, and factor VII levels served as controls. Control patients were selected within the same period as the treated group but were not provided with hepatocyte transplants because of a lack of donor cells of the appropriate blood type, or failure to obtain informed consent from the donor families. All patients received continuous dextrose and fresh frozen plasma infusions, enteral lactulose administration, controlled hyperventilation, and hemofiltration or venous dialysis for renal failure. Patients were intubated when hepatic encephalopathy progressed from grade 3 to grade 4. Intracranial pressures were continuously monitored. All nine patients were listed for OLT. Patients maintained by traditional medical therapy (the four control patients) showed improvement but not normalization of the ammonia levels, and showed no improvement in intracranial pressure during the observation period. All four control patients displayed cardiovascular instability requiring catecholamine support to maintain blood pressure and cerebral perfusion. Despite aggressive therapy, all control patients died within 3 days of cardiopulmonary failure and/or brain stem herniation.

In the five patients who received hepatocyte transplants, cerebral perfusion was well maintained and they achieved cardiovascular stability that allowed the removal of medical therapy within 24 hours to 36 hours of hepatocyte transplant. Lactulose, mannitol, and hyperventilation were withdrawn, and the patients were maintained with renal-dose dopamine. Sustained and significant decreases in intracranial pressures and increases in the cerebral perfusion pressures were reported in the hepatocyte-treated group after transplant despite the withdrawal of the medical therapies. Three of the five hepatocyte-treated patients were successfully sustained for 2 days to 10 days following hepatocyte transplant and were bridged to OLT. The fourth patient remained hemodynamically stable, showed improvements in cerebral perfusion pressure and a decrease in intracranial pressure after hepatocyte transplant, and awoke from the coma on day 4 following the hepatocyte transplant and moved, causing displacement of the intracranial monitor, inducing a fatal subdural hematoma. The remaining patient, a 6-month-old patient with total parenteral nutrition–induced liver failure, showed substantial reductions in ammonia levels but cerebral perfusion pressures continued to decline and life support was withdrawn 7 days after cell transplant. In all of these cases, hepatocytes that were previously isolated from deceased donors and cryopreserved were used for the transplants.

These initial studies revealed clinical end points that were partially or completely corrected by hepatocyte transplants. The initial studies demonstrated that hepatocytes could be isolated from deceased donors and subsequently cryopreserved, and could be safely transplanted into the spleen of critically ill patients without infection or serious pulmonary complications. Importantly, the infusion of hepatocytes directly or indirectly induced significant reductions in ammonia levels and intracranial pressures, with a resulting increase in cerebral perfusion pressures and blood flow. Corrections in ammonia levels and intracranial pressures were also observed in later studies.

Support for the hypothesis that donor cells contributed to the improvements described above was also provided. Clusters of hepatocytes were detected in the spleen of a patient who underwent splenectomy at the time of OLT. Synthetic support in the case of the α ₁ -antitrypsin-deficient patient was demonstrated by the presence of circulating MM phenotype protein after transplant. A 34% increase in α ₁ -antitrypsin levels in the blood and specifically an increase in the M phenotype protein in a ZZ phenotype patient was observed when MM phenotype hepatocytes were infused.

An improvement of ammonia levels, prothrombin times, cerebral perfusion, and decreases in encephalopathy scores were also observed in a second group of five acute liver failure patients who received allogeneic hepatocyte transplants. In that study, none of the patients were listed for OLT. In three of the five patients contraindications related to active polysubstance abuse precluded their listing for OLT. Of the remaining two patients, one had hepatitis B virus–induced acute liver failure and the other patient had herpes simplex virus 2–induced hepatitis. As with the first five patients, all of the five patients in this report were encephalopathic, requiring ventilation, and were dialysis dependent and cerebral edema was confirmed in all patients. Two patients died within 18 hours of the hepatocyte transplant, one of disseminated herpes simplex virus 2 infection, and the other was irreversibly intoxicated by the ingestion of chloroform along with alcohol. The study authors reported stability during the first 48 hours in the remaining patients, but thereafter steady improvements in encephalopathy scores and coagulation parameters. All three surviving patients were subsequently weaned off ventilator support and were returned to oral nutrition. Survival of the three remaining patients was extended to 14 days, 20 days, or 52 days. Longer than expected survival of nontransplant candidates was also observed in two additional patients with acute decompensation following chronic liver disease due to long-term alcohol abuse, where the hepatocyte transplant recipients showed rapid although temporary (33 days or 50 days) improvement in liver function to the point where both patients were discharged from the hospital. In the latter two cases both patients finally died of renal failure when they refused further dialysis therapy. By 1999 there was a review listing the causes of the liver disease and the outcomes of the first 30 patients to receive deceased donor allogeneic hepatocyte transplants, with 16 deaths and 14 patients surviving, most of whom were bridged to successful OLT within 10 days.