Review of hepatocyte transplantation.

BACKGROUND: Hepatocyte transplantation is a promising treatment for several liver diseases and can also be used as a "bridge" to liver transplantation in cases of liver failure. Although the first animal experiments with this technique began in 1967, it was first applied in humans only in 1992. Unfortunately, unequivocal evidence of transplanted human hepatocyte function has been obtained in only one patient with Crigler-Najjar syndrome type I and, even then, the amount of bilirubin-UDP-glucuronosyltransferase enzyme activity derived from the transplanted cells was not sufficient to eliminate the patient's eventual need for organ transplantation. METHODS: A literature review was carried out using MEDLINE and library searches. RESULTS: This review considers the following: (1) alternatives or bridges to orthotopic liver transplantation (OLT); (2) solutions to the shortage of organs-the shortage of organ donors has impeded the development of human hepatocyte transplantation, and immortalized hepatocytes in particular could provide an unlimited supply of transplantable cells in a nearly future; (3) future directions. We review these efforts along with hepatocyte transplantation over the last 13 years.

Quality control for clinical islet transplantation: organ procurement and preservation, the islet processing facility, isolation, and potency tests.

Pancreatic islet transplantation has become one of the ideal treatments for patients with type 1 diabetes mellitus due to improvements in isolation techniques and immunosuppression regimens. In order to ensure the safety and rights of patients, isolated islets need to meet the criteria for regulation as both a biological product and a drug product. For the constant success of transplantation, therefore, all investigators involved in clinical islet transplantation must strive to ensure the safety, purity, and potency of islets in all the phases of clinical islet isolation and transplantation. In this review, we summarize the quality control for clinical islet isolation and transplantation, and the latest topics of pre-transplant islet assessment.

Ductal injection of preservation solution increases islet yields in islet isolation and improves islet graft function.

For islet transplantation, it is important to obtain an available islet mass adequate for diabetes reversal from a single donor pancreas. A recent report demonstrated that the use of M-Kyoto solution instead of UW solution improved islet yields in the two-layer method for pancreas preservation. The present study investigated whether the ductal injection of a large volume of preservation solution (UW and M-Kyoto solution) before pancreas storage improves islet yields. Islet yield both before and after purification was significantly higher in the ductal injection (+) group compared with the ductal injection (-) group. TUNEL-positive cells in the ductal injection (+) group were significantly decreased in comparison to the ductal injection (-) group. The ductal injection of preservation solution increased the ATP level in the pancreas tissue and reduced trypsin activity during the digestion step. Annexin V and PI assays showed that the ductal injection prevents islet apoptosis. In a transplant model, the ductal injection improved islet graft function. These findings suggest that the ductal injection of preservation solution, especially the M-Kyoto solution, leads to improved outcomes for pancreatic islet transplantation. Based on these data, this technique is now used for clinical islet transplantation from non-heart-beating donor pancreata or living donor pancreas.

Secretory unit of islet in transplantation (SUIT) and engrafted islet rate (EIR) indexes are useful for evaluating single islet transplantation.

The evaluation of engraftment is important to assess the success of islet transplantation, but it is complex because islet transplantation usually requires two or more donors to achieve euglycemia. Islet transplantation from NHBDs was evaluated using new assessment forms for the secretory unit of islet in transplantation (SUIT) and engrafted islet rate (EIR) indexes. Insulin independence was obtained when the SUIT index was more than 28, which might indicate that 28% of the beta-cell mass of a normal subject is required for insulin independence. Because the average EIR for a single transplantation is about 30, the percentage of engrafted islets following one transplantation is about 30%, assuming that a normal subject has 1 million islet equivalents. Although few cultured islet transplants have been performed, the increase of the SUIT and EIR indexes in patients who received cultured islets was significantly lower than in patients who received fresh islets, suggesting that fresh islets may be more effective than cultured islets. The SUIT and EIR indexes are thus considered to be useful values for evaluating islet transplantation, especially for single islet transplantation.

Immortalized hepatocytes using human artificial chromosome.

The shortage of organ donors has impeded the development of human hepatocyte transplantation. Immortalized hepatocytes could provide an unlimited supply of transplantable cells. To determine whether immortalized hepatocytes could provide global metabolic support in end-stage liver disease, rat hepatocyte clones were developed by transduction with the gene encoding the Simian virus 40 T antigen (SVT) using the human artificial minichromosome (HAC). The SVLT sequence was excised by FRT recombination. Following HAC infusion, the transduced hepatocytes express SVT, blasticidine resistance (BS), and the PGK promoter TK gene. Forty-six cell clones were obtained and at least partially characterized, as previously described, for albumin, alpha-1-antitrypsin, glucose-6-phosphatase (G6Pase), dipeptidylpeptidase 4 (Dpp4), gamma-glutamyltransferase 1 (Ggt), SVT, and beta-actin expression using RT-PCR. Clones were also assessed for albumin secretion into the culture medium using ELISA. All of the cell line secreted approximately 10 mg/dl of albumin, which is equivalent to the amount secreted by primary hepatocytes. In further experiments, this cell line will be used for transplantable cells or artificial organ using HAC. These results represent an important step toward the development of immortalized hepatocytes.

Analysis of donor- and isolation-related variables from non-heart-beating donors (NHBDs) using the Kyoto islet isolation method.

Recently, we demonstrated that islet transplantation from non-heart-beating donors (NHBDs) using the Kyoto islet isolation method (KIIM) successfully reversed patients' diabetes state. In this study, we evaluated the effects of donor- and isolation-related variables on islet isolation results from NHBDs by KIIM. Twenty-one islet preparations from the pancreata of NHBDs were isolated by KIIM. Islet preparations that met transplantation criteria and achieved improved patient diabetes control after transplantation were defined as successful isolations. Potential risk factors deemed to affect islet isolation results, such as age, gender, body mass index, hospital stay, donors' blood biochemical tests, a modified pancreata procurement method, and isolation and purification procedure-related variables, were analyzed. Seventeen out of 21 islet isolations (81%) were successful isolations. Postpurification islet yield was 447,639 +/- 39,902 islet equivalents (IE) in the successful isolation group and 108,007 +/- 31,532 IE in the failure group. Donor age was significantly younger in the success group (41.9 +/- 4.0 years old in the success group vs. 57.5 +/- 2.2 years old in the failure group, p = 0.003). Chronic pancreatitis significantly decreased islet yields (p = 0.006). Phase I time was significantly shorter (p = 0.010) and undigested tissue volume was significantly smaller (p = 0.020) in the success group. Purity was in positive correlation to postpurification islet yield, while donor age was in reverse correlation to postpurification islet yield. KIIM enables us to perform islet transplantation from NHBDs; however, the decision to use pancreata from older donors or those with chronic pancreatitis requires careful consideration.

[Navigation surgery for pancreatic disease using multislice computed tomography].

In navigation surgery, preoperatively acquired image data are used so that surgical instruments can be guided inside the body while their location is displayed on a computer monitor. It is used in cranial nerve and spinal surgery. In the field of abdominal surgery, however, surgical manipulations in the target area cause major changes in the displayed images compared with those obtained preoperatively, and therefore, with the exception of certain organs, navigation surgery is difficult to apply. In general, this type of surgery aims to use intraoperative image information to improve surgical precision, carry out the preoperative plan accurately, and avoid dangerous areas. Three-dimensional images of the vascular architecture obtained with multislice computed tomography (MS-CT) make it possible to visualize arteries, the portal vein, bile duct, and even the pancreatic duct from any angle, which cannot be done with conventional angiography. Accurate positional relationships in the affected region can be determined preoperatively by manipulating multiplanar reconstruction images at a work station. MS-CT is extremely useful in navigation for safe performance of all types of pancreatectomy.

Comparison of M-Kyoto solution and histidine-tryptophan-ketoglutarate solution with a trypsin inhibitor for pancreas preservation in islet transplantation.

The use of University of Wisconsin (UW) preservation solution in islet transplantation has some disadvantages, including inhibition of collagenase activity for pancreatic digestion. Histidine-tryptophan-ketoglutarate (HTK) solution has demonstrated an efficacy similar to UW solution for organ preservation in clinical pancreas transplantation. Recently, we reported that islet yield from porcine pancreata was significantly gtreater when they were preserved using M-Kyoto solution compared with UW solution. Here, we compared HTK solution with ulinastatin (M-HTK) and M-Kyoto solution for islet yield. In porcine islet isolation, islet yield after purification was significantly greater in the M-Kyoto/perfluorochemical (PFC) group compared with the M-HTK/PFC group. The M-Kyoto/PFC group had a significantly lower ADP/ATP ratio compared with the M-HTK/PFC group, suggesting that different islet yields might be due to the differences as energy sources of the solutions used. In conclusion, M-Kyoto/PFC solution is better for pancreas preservation before islet isolation than M-HTK/PFC solution.

Intrasplenic hepatocyte transplantation prolonged the survival in Nagase analbuminemic rats with liver failure induced by common bile duct ligation.

It has already been established that hepatocyte transplantation (HTx) in animal models, such as both chemically and surgically induced acute liver failure, liver-based metabolic disease, and cirrhosis, resulted in significant improvement of liver function and survival. However, the efficacy of hepatocyte transplantation in secondary cholestatic liver disease is not well known. In this study, we transplanted hepatocytes into the spleen of Nagase analbuminemic rats (NARs) with common bile duct ligation (CBDL) to evaluate the function of transplanted hepatocytes by both of serum albumin levels and total bilirubin levels. CBDL was carried out on NARs to induce liver failure. Lewis rat hepatocytes were transplanted in NARs 7 days after CBDL. Animals, in groups of four, underwent the following interventions: group 1--intrasplenic transplantation of 30 x 106 primary Lewis rat hepatocytes in NARs with CBDL (n=4), group 2--intrasplenic injection of 0.5 ml DMEM in NARs with CBDL (n=4); group 3--CBDL only (n=4); group 4--intrasplenic transplantation of 30 x 106 primary Lewis rat hepatocytes in NARs (n=4). Both bilirubin levels and albumin levels in NARs with CBDL were significantly improved post-HTx. Animals receiving hepatocyte transplantation survived longer than animals in nontransplant control groups. This study indicates that hepatocytes can be transplanted to temporarily provide life-supporting liver-specific metabolic function and prolong the survival in recipient rats with liver failure induced by CBDL.

Insulin independence after living-donor distal pancreatectomy and islet allotransplantation.

Rising demand for islet transplantation will lead to severe donor shortage in the near future, especially in countries where cadaveric organ donation is scarce. We undertook a successful transplantation of living-donor islets for unstable diabetes. The recipient was a 27-year-old woman who had had brittle, insulin-dependent diabetes mellitus for 12 years. The donor, who was a healthy 56-year-old woman and mother of the recipient, underwent a distal pancreatectomy. After isolation, 408 114 islet equivalents were transplanted immediately. The transplants functioned immediately and the recipient became insulin-independent 22 days after the operation. The donor had no complications and both women showed healthy glucose tolerance. Transplantation of living-donor islets from the distal pancreas can be sufficient to reverse brittle diabetes.

A new mouse model for intraportal islet transplantation with limited hepatic lobe as a graft site.

Intraportal site is the standard for grafting in clinical islet transplantation. In the mouse model, the whole liver has been used as the grafting site to mimic clinical islet transplantation. However, this model lacks the potency to directly assess the contribution of the islet graft to diabetes control. Only demonstrating the immediate recurrence of diabetes in a surviving recipient after the removal of the islet graft can validate this assessment. In this study, we develop a mouse model of intraportal islet transplantation equipped with the potency of this assessment by injecting islets selectively into the right hepatic lobe under temporal clamp of the left portal vein. The mouse of this model survives after the right hepatectomy by which the islet graft is removed. This model can be applied to investigate both the specific graft-recipient interaction in the liver and the islet graft contribution to the control of diabetes.

Successful islet transplantation from nonheartbeating donor pancreata using modified Ricordi islet isolation method.

BACKGROUND: Current success of islet transplantation has led to donor shortage and the need for marginal donor utilization to alleviate this shortage. The goal of this study was to improve the efficacy of islet transplantation using nonheartbeating donors (NHBDs). METHODS: First, we used porcine pancreata for the implementation of several strategies and applied to human pancreata. These strategies included ductal injection with trypsin inhibitor for protection of pancreatic ducts, ET-Kyoto solution for pancreas preservation, and Iodixanol for islet purification. RESULTS: These strategies significantly improved both porcine and human islet isolation efficacy. Average 399,469+/-36,411 IE human islets were obtained from NHBDs (n=13). All islet preparations met transplantation criteria and 11 out of 13 cases (85%) were transplanted into six type 1 diabetic patients for the first time in Japan. All islets started to secrete insulin and all patients showed better blood glucose control without hypoglycemic loss of consciousness. The average HbA1c levels of the six recipients significantly improved from 7.5+/-0.4% at transplant to 5.1+/-0.2% currently (P<0.0003). The average insulin amounts of the six recipients significantly reduced from 49.2+/-3.3 units at transplant to 11+/-4.4 units (P<0.0005) and five out of six patients reduced to less than half dose. The first patient is now insulin free, the first such case in Japan. CONCLUSION: This demonstrates that our current protocol makes it feasible to use NHBDs for islet transplant into type 1 diabetic patients efficiently.

Procurement of the human pancreas for pancreatic islet transplantation from marginal cadaver donors.

BACKGROUND: Recent advances in pancreatic islet transplantation (PIT) have contributed significantly to the treatment of patients with type 1 diabetes. The specific aim of this study was to develop an effective technique for the procurement of pancreas for PIT from nonheart-beating-donor (NHBDs). METHODS: Between January 2004 and August 2004, eight human pancreata were procured and processed for isolation of islets at a cell processing center. After confirmation of brain death status, a double balloon catheter was inserted to prevent warm ischemic damage to the donor pancreas by using an in situ regional organ cooling system that was originally developed for procurement of kidneys. The catheter position of the cooling system was modified specifically for the pancreas and kidney. Furthermore, we worked in cooperation with a kidney procurement team to protect the pancreas during kidney procurement. RESULTS: Warm ischemic time could be controlled with the modified in situ regional cooling system at 3.0 +/- 0.8 min (mean +/- SE). The operations for procurement of the kidneys and pancreata lasted 45.6 +/- 3.6 min and 10.6 +/- 1.8 min, respectively. Islet yield per isolation was 444,426 +/- 35,172 IE (islet equivalent). All eight cases met the criteria for PIT based on the Edmonton protocol. CONCLUSION: We developed a novel procurement technique in cooperation with our kidney procurement team. This protocol for the procurement of pancreas and kidney from a NHBD enabled us to transplant islets into a type 1 diabetic patient and kidney into a renal failure patient.

Follow-up study of the first successful living donor islet transplantation.

BACKGROUND: Islet transplantation has become an option for the treatment of insulin-dependent diabetes mellitus and is usually performed using brain-dead heartbeating donors. However, we have very limited number of such donors in Japan; therefore, it is not allowed to perform islet transplantation with brain-dead donors. In order to perform islet transplantation in Japan, we need to seek new donor resources. METHODS: We performed the first successful living-donor islet transplantation. In this case, the recipient had brittle diabetes with hypoglycemic unawareness. The donor was deemed qualified after undergoing both metabolic and preoperative assessments. Distal pancreatectomy was performed using open laparotomy and more than 400,000 islets were isolated and transplanted immediately. RESULTS: The recipient has been insulin independent posttransplant with positive C-peptide for more than one year. She no longer suffers from hypoglycemic unawareness and displayed a substantial improvement in hemoglobulin (Hb) A1C. The donor's clinical course was uneventful, which allowed her to return to her job within one month. She maintained normal fasting C-peptide and HbA1C levels during follow-up period. CONCLUSION: In our first case of living donor islet transplantation, both the donor and the recipient have been maintaining excellent glycemic control with no untreatable complications for more than one year.

Living donor islet transplantation, the alternative approach to overcome the obstacles limiting transplant.

We performed the world's first successful living donor islet transplantation for unstable diabetes. A total of 408,114 islet equivalents were isolated from half a living pancreas and transplanted immediately to the recipient who was a 27-year-old female. The donor was a 56-year-old female in good health, mother of the recipient. The islets functioned immediately, and the recipient was weaned completely from insulin on the 22nd posttransplant day, and has maintained excellent glycemic control since. The donor was discharged on the 18th postoperative day with normal oral glucose tolerance test and without complications. Living donor islet transplantation could cure one insulin-dependent diabetes mellitus patients with a single donor. There are some advantages in the living donor islet transplantation: (a) living donor can alleviate the issue of donor shortage; (b) highly potent islets can be isolated from a living donor; and (c) the recipient can be treated with immunosuppressant and controlled blood glucose level tightly prior to the transplantation. These are important factors in overcoming the obstacles limiting islet transplantation. We believe that the living donor islet transplantation may become an additional option in treating insulin-dependent diabetes.

Temporal decline in sirolimus elimination immediately after pancreatic islet transplantation.

Pancreatic islet transplantation is a curable treatment for type 1 diabetes and has been put into practice in various countries. In this study, we analyzed the pharmacokinetic characteristics of sirolimus and tacrolimus in six Japanese patients with pancreatic islet transplants immediately after surgery, and monitored efficacy and toxicity. The patients were treated with immunosuppressive therapy based on the Edmonton protocol, that is, sirolimus and low-dose tacrolimus. Pharmacokinetic analyses were performed using the nonlinear mixed-effects modeling program NONMEM. Large inter- and intra-individual variability was observed in the pharmacokinetics of sirolimus and tacrolimus. A model with increased apparent clearance in the postoperative period explained well the intra-individual variability in the pharmacokinetics of both drugs. The most frequent drug-induced toxicity was a decrease in the white blood cell count, and two of six patients required the administration of granulocyte colony-stimulating factor. Clinical laboratory tests immediately before the transplantation and cytochrome P450 3A5 genotype were not related to the high blood concentrations of sirolimus after the loading dose. From these results, the apparent clearance of sirolimus and tacrolimus might temporally decline immediately after pancreatic islet transplantation. A high trough concentration of sirolimus might increase the risk of hematological toxicy, and adjustment of the dosage for immunosuppressive treatment will be necessary in Japanese patients.

A single transplantation of the islets can produce glycemic stability and reduction of basal insulin requirement.

We investigated glycemic stability and insulin requirement 1 month after a single transplantation of the islets from non-heart-beating donors or a living donor. Overall blood glucose levels decreased immediately after transplantation. The M-value and mean amplitude of glycemic excursions (MAGE) decreased significantly from 53.0 (range, 8.9-91.0) to 4.2 (0.6-8.8, P<0.05) and from 8.5 mM (4.8-11.7) to 3.3 mM (2.0-4.5, P<0.05), respectively. The values after transplantation were lower than the first quartile of 102 type 2 diabetic control patients. The estimated HbA1c level decreased significantly from 7.9% (5.7-10.9) to 5.4% (4.7-5.9, P<0.05). The supplement of basal insulin decreased 43% from 0.31 units/kg/day (0.16-0.37) to 0.18 units/kg/day (0-0.22, P<0.05), while that of stimulated insulin did not decrease significantly, from 0.28 units/kg/day (0.13-0.51) to 0.21 units/kg/day (0-0.41). Thus, only one islet transplantation can be sufficient to attain metabolic stability, probably by effective supply of basal insulin secretion, sufficient to avoid life-threatening severe hypoglycemia and prevent or delay the progress of secondary complications of diabetes by decreasing the HbA1c level.

Effective islet isolation method with extremely high islet yields from adult pigs.

Achieving good islet isolation is one of the most important factors for successful islet transplantation. Porcine pancreas is suitable for islet isolation research due to its anatomical and physiological similarities to human pancreas. In this study, we evaluated a new porcine islet isolation method designed to maximize islet yield and compared it with our previous open pan method and the standard method using a Ricordi chamber (Ricordi method). We performed 15 porcine islet isolations, five each with the new method, the open pan method, and the Ricordi method. The new method features several important improvements. Pancreata remain uncut and are kept intact during collagenase intraductal injection, a large filtration chamber to handle whole pancreata, low concentration of collagenase (Liberase HI) for digestion, and large plastic containers for large-scale islet purification. All isolated islets were assessed for yield, purity, viability and in vitro function. Islets isolated with this new method were transplanted under the kidney capsules of SCID mice with chemically induced diabetes for in vivo functional assessment (n = 8). With the new method, we obtained on average more than 1,000,000 islet equivalents (IE) (1,236,266 +/- 213,486 IE) (mean +/- SE) before purification and 800,000 IE (879,815 +/- 222,729 IE) after purification from one adult pig. Islet yield per pancreas was significantly higher compared with our previous open pan method (30,666 +/- 11,532 IE, p < 0.01) and the Ricordi method (317,073 +/- 86,093 IE, p < 0.05). All mice, transplanted with 1000 islets from the new method, returned to normoglycemia within 4 days after transplantation. Our new method makes it possible to obtain extremely high porcine islet yield with good function. It should produce useful information for human islet isolation and transplantation, and might be applied to single donor clinical xenogeneic transplantation.

PDX-1 protein is internalized by lipid raft-dependent macropinocytosis.

PDX-1 plays a central role in regulating insulin gene transcription and differentiation of insulin-producing cells. It was previously reported that, due to its own Antennapedia-like protein transduction domain (PTD), exogenous PDX-1 protein can permeate cells and induces insulin gene expression in pancreatic ducts, thought to be islet progenitor cells. These data suggest that PDX-1 protein transduction could be a safe and valuable strategy for facilitating differentiation of progenitor cells into insulin-producing cells without requiring gene transfer technology. Here it is shown that after an initial ionic cell-surface interaction, PDX-1 proteins are rapidly internalized by lipid raft-dependent macropinocytosis. HeLa cells were treated with both FITC-conjugated PDX-1 PTD and FM 4-64, a general fluorescent marker of endocytosis. A punctate cytoplasmic distribution of PDX-1 PTD, which colocalized with FM 4-64, was observed in treated cells. Because expression of dominant-negative dynamin-1 did not block PDX-1 PTD uptake, PDX-1 protein transduction is independent on phagocytosis and clathrin- or caveolar-mediated endocytosis. Cells were pretreated with amiloride, a specific inhibitor of the Na+/H+ exchange required for macropinocytosis, or cytochalasin D, an F-actin elongation inhibitor. Treatment of cells with both macropinosome inhibitors resulted in the reduction in PDX-1 PTD transduction into vesicles, suggesting that PDX-1 PTD-mediated cellular entry occurs by lipid raft-mediated macropinocytosis. Taken together, these observations provide the mechanism of PDX-1 protein transduction and suggest that the protein transduction system could work for experimental and therapeutic strategies.

Route of hepatocyte delivery affects hepatocyte engraftment in the spleen.

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.