Kidney Versus Islet Allograft Survival After Induction of Mixed Chimerism With Combined Donor Bone Marrow Transplantation.

We have previously reported successful induction of transient mixed chimerism and long-term acceptance of renal allografts in MHC mismatched nonhuman primates. In this study, we attempted to extend this tolerance induction approach to islet allografts. A total of eight recipients underwent MHC mismatched combined islet and bone marrow (BM) transplantation after induction of diabetes by streptozotocin. Three recipients were treated after a nonmyeloablative conditioning regimen that included low-dose total body and thymic irradiation, horse Atgam (ATG), six doses of anti-CD154 monoclonal antibody (mAb), and a 1-month course of cyclosporine (CyA) (Islet A). In Islet B, anti-CD8 mAb was administered in place of CyA. In Islet C, two recipients were treated with Islet B, but without ATG. The results were compared with previously reported results of eight cynomolgus monkeys that received combined kidney and BM transplantation (Kidney A) following the same conditioning regimen used in Islet A. The majority of kidney/BM recipients achieved long-term renal allograft survival after induction of transient chimerism. However, prolonged islet survival was not achieved in similarly conditioned islet/BM recipients (Islet A), despite induction of comparable levels of chimerism. In order to rule out islet allograft loss due to CyA toxicity, three recipients were treated with anti-CD8 mAb in place of CyA. Although these recipients developed significantly superior mixed chimerism and more prolonged islet allograft survival (61, 103, and 113 days), islet function was lost soon after the disappearance of chimerism. In Islet C recipients, neither prolonged chimerism nor islet survival was observed (30 and 40 days). Significant improvement of mixed chimerism induction and islet allograft survival were achieved with a CyA-free regimen that included anti-CD8 mAb. However, unlike the kidney allograft, islet allograft tolerance was not induced with transient chimerism. Induction of more durable mixed chimerism may be necessary for induction of islet allograft tolerance.

Enrichment of human embryonic stem cell-derived NKX6.1-expressing pancreatic progenitor cells accelerates the maturation of insulin-secreting cells in vivo.

Human embryonic stem cells (hESCs) are considered a potential alternative to cadaveric islets as a source of transplantable cells for treating patients with diabetes. We previously described a differentiation protocol to generate pancreatic progenitor cells from hESCs, composed of mainly pancreatic endoderm (PDX1/NKX6.1-positive), endocrine precursors (NKX2.2/synaptophysin-positive, hormone/NKX6.1-negative), and polyhormonal cells (insulin/glucagon-positive, NKX6.1-negative). However, the relative contributions of NKX6.1-negative versus NKX6.1-positive cell fractions to the maturation of functional ß-cells remained unclear. To address this question, we generated two distinct pancreatic progenitor cell populations using modified differentiation protocols. Prior to transplant, both populations contained a high proportion of PDX1-expressing cells (~85%-90%) but were distinguished by their relatively high (~80%) or low (~25%) expression of NKX6.1. NKX6.1-high and NKX6.1-low progenitor populations were transplanted subcutaneously within macroencapsulation devices into diabetic mice. Mice transplanted with NKX6.1-low cells remained hyperglycemic throughout the 5-month post-transplant period whereas diabetes was reversed in NKX6.1-high recipients within 3 months. Fasting human C-peptide levels were similar between groups throughout the study, but only NKX6.1-high grafts displayed robust meal-, glucose- and arginine-responsive insulin secretion as early as 3 months post-transplant. NKX6.1-low recipients displayed elevated fasting glucagon levels. Theracyte devices from both groups contained almost exclusively pancreatic endocrine tissue, but NKX6.1-high grafts contained a greater proportion of insulin-positive and somatostatin-positive cells, whereas NKX6.1-low grafts contained mainly glucagon-expressing cells. Insulin-positive cells in NKX6.1-high, but not NKX6.1-low grafts expressed nuclear MAFA. Collectively, this study demonstrates that a pancreatic endoderm-enriched population can mature into highly functional ß-cells with only a minor contribution from the endocrine subpopulation.

Maturation and function of human embryonic stem cell-derived pancreatic progenitors in macroencapsulation devices following transplant into mice.

AIMS/HYPOTHESIS: Islet transplantation is a promising cell therapy for patients with diabetes, but it is currently limited by the reliance upon cadaveric donor tissue. We previously demonstrated that human embryonic stem cell (hESC)-derived pancreatic progenitor cells matured under the kidney capsule in a mouse model of diabetes into glucose-responsive insulin-secreting cells capable of reversing diabetes. However, the formation of cells resembling bone and cartilage was a major limitation of that study. Therefore, we developed an improved differentiation protocol that aimed to prevent the formation of off-target mesoderm tissue following transplantation. We also examined how variation within the complex host environment influenced the development of pancreatic progenitors in vivo. METHODS: The hESCs were differentiated for 14 days into pancreatic progenitor cells and transplanted either under the kidney capsule or within Theracyte (TheraCyte, Laguna Hills, CA, USA) devices into diabetic mice. RESULTS: Our revised differentiation protocol successfully eliminated the formation of non-endodermal cell populations in 99% of transplanted mice and generated grafts containing >80% endocrine cells. Progenitor cells developed efficiently into pancreatic endocrine tissue within macroencapsulation devices, despite lacking direct contact with the host environment, and reversed diabetes within 3 months. The preparation of cell aggregates pre-transplant was critical for the formation of insulin-producing cells in vivo and endocrine cell development was accelerated within a diabetic host environment compared with healthy mice. Neither insulin nor exendin-4 therapy post-transplant affected the maturation of macroencapsulated cells. CONCLUSIONS/INTERPRETATION: Efficient differentiation of hESC-derived pancreatic endocrine cells can occur in a macroencapsulation device, yielding glucose-responsive insulin-producing cells capable of reversing diabetes.

Maturation of human embryonic stem cell-derived pancreatic progenitors into functional islets capable of treating pre-existing diabetes in mice.

Diabetes is a chronic debilitating disease that results from insufficient production of insulin from pancreatic ß-cells. Islet cell replacement can effectively treat diabetes but is currently severely limited by the reliance upon cadaveric donor tissue. We have developed a protocol to efficiently differentiate commercially available human embryonic stem cells (hESCs) in vitro into a highly enriched PDX1+ pancreatic progenitor cell population that further develops in vivo to mature pancreatic endocrine cells. Immature pancreatic precursor cells were transplanted into immunodeficient mice with streptozotocin-induced diabetes, and glycemia was initially controlled with exogenous insulin. As graft-derived insulin levels increased over time, diabetic mice were weaned from exogenous insulin and human C-peptide secretion was eventually regulated by meal and glucose challenges. Similar differentiation of pancreatic precursor cells was observed after transplant in immunodeficient rats. Throughout the in vivo maturation period hESC-derived endocrine cells exhibited gene and protein expression profiles that were remarkably similar to the developing human fetal pancreas. Our findings support the feasibility of using differentiated hESCs as an alternative to cadaveric islets for treating patients with diabetes.

Quantitative analysis of cell composition and purity of human pancreatic islet preparations.

Despite improvements in outcomes for human islet transplantation, characterization of islet preparations remains poorly defined. This study used both light microscopy (LM) and electron microscopy (EM) to characterize 33 islet preparations used for clinical transplants. EM allowed an accurate identification and quantification of cell types with measured cell number fractions (mean±s.e.m.) of 35.6±2.1% ß-cells, 12.6±1.0% non-ß-islet cells (48.3±2.6% total islet cells), 22.7±1.5% duct cells, and 25.3±1.8% acinar cells. Of the islet cells, 73.6±1.7% were ß-cells. For comparison with the literature, estimates of cell number fraction, cell volume, and extracellular volume were combined to convert number fraction data to volume fractions applicable to cells, islets, and the entire preparation. The mathematical framework for this conversion was developed. By volume, ß-cells were 86.5±1.1% of the total islet cell volume and 61.2±0.8% of intact islets (including the extracellular volume), which is similar to that of islets in the pancreas. Our estimates produced 1560±20 cells in an islet equivalent (volume of 150-µm diameter sphere), of which 1140±15 were ß-cells. To test whether LM analysis of the same tissue samples could provide reasonable estimates of purity of the islet preparations, volume fraction of the islet tissue was measured on thin sections available from 27 of the clinical preparations by point counting morphometrics. Islet purity (islet volume fraction) of individual preparations determined by LM and EM analyses correlated linearly with excellent agreement (R²=0.95). However, islet purity by conventional dithizone staining was substantially higher with a 20-30% overestimation. Thus, both EM and LM provide accurate methods to determine the cell composition of human islet preparations and can help us understand many of the discrepancies of islet composition in the literature.

The use of an approved biodegradable polymer scaffold as a solid support system for improvement of islet engraftment.

The aim of this study was to investigate whether the use of a medically approved biodegradable scaffold as a solid support system would enhance graft survival following transplantation into the omental pouch in a preclinical large animal model. Six beagle dogs underwent total pancreatectomy followed by islet autotransplantation into the omental pouch. Four dogs received islets seeded in a biodegradable polymer scaffold and two received free islets without a scaffold. All four animals that received islets in the scaffold became normoglycemic without exogeneous insulin injection. One dog, transplanted with the largest number of islets, maintained a normal metabolic state until the graft was removed at 5 months posttransplant. In two out of the three that received a marginal islet mass, insulin independence was sustained up to 2 months. In contrast, two dogs transplanted with a similar marginal mass without the scaffold never became normoglycemic. Histological examination of the grafts in the scaffold showed numerous well-granulated, insulin-containing cells as well as glucagon-positive cells. These results indicate that biodegradable scaffolds may enhance survival and function of islet grafts. Manipulation of the microenvironment of transplanted islets may constitute the basis for new approaches to enhance islet engraftment.

Interference with tissue factor prolongs intrahepatic islet allograft survival in a nonhuman primate marginal mass model.

BACKGROUND: Tissue factor (TF) expression on islets can result in an instant blood-mediated inflammatory reaction (IBMIR) that contributes to early islet loss. We tested whether peritransplant protection of islets from IBMIR with a monoclonal anti-TF antibody (CNTO859) would enhance engraftment in our nonhuman primate marginal mass model. METHODS: Each of six pairs of cynomolgus monkeys (CM) with streptozotocin-induced diabetes was closely matched for metabolic control and was transplanted with 5,000 IEQ/kg allogeneic, ABO-compatible islets from the same donor under the cover of steroid-free immunosuppression. For each pair, experimental animals received islets cultured with 20 microg/mL anti-TF and were dosed with 6 mg/kg anti-TF intravenously, 10-25 min before islet infusion; control monkeys received an equal number of islets from the same preparation cultured without anti-TF and no in vivo treatment. RESULTS: Early fasting C-peptide (CP) values were different between (P<0.01), but not within, pairs and correlated with in vitro functional capacity of islets as assessed by perifusion (r=0.60; P=0.022). Compared to their matched controls, experimental animals had decreased posttransplant markers of coagulation, higher fasting CP levels (1 month posttransplant and end of study) and prolonged graft function. CONCLUSIONS: These data suggest that pretreatment of islets and the recipient with anti-TF may limit the effects of IBMIR, thereby enhancing islet engraftment and survival.

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.

Survival of microencapsulated adult pig islets in mice in spite of an antibody response.

The aim of this study was to assess the capacity of simple alginate capsules to protect adult pig islets in a model of xenotransplantation. Adult pig islets were microencapsulated in alginate, with either single alginate coats (SAC) or double alginate coats (DAC), and transplanted into the streptozotocin-induced diabetic B6AF1 mice. Normalization of glucose levels was associated with an improvement of the glucose clearance during intravenous glucose tolerance tests. After explantation, all mice became hyperglycemic, demonstrating the efficacy of the encapsulated pig islets. Explanted capsules were mainly free of fibrotic reaction and encapsulated islets were still functional, responding to glucose stimulation with a 10-fold increase in insulin secretion. However, a significant decrease in the insulin content and insulin responses to glucose was observed for encapsulated islets explanted from hyperglycemic mice. An immune response of both IgG and IgM subtypes was detectable after transplantation. Interestingly, there were more newly formed antibodies in the serum of mice transplanted with SAC capsules than in the serum of mice transplanted with DAC capsules. In conclusion, alginate capsules can prolong the survival of adult pig islets transplanted into diabetic mice for up to 190 days, even in the presence of an antibody response.

A simple and cost-effective method for the isolation of islets from nonhuman primates.

Recent advances in islet cell transplantation have led to insulin independence in a majority of islet transplant recipients. However, there exists a need to overcome the shortage of donor tissue and the necessity for life-long immunosuppression. Preclinical studies in large animal models are necessary to evaluate the safety and efficacy of alternative approaches for clinical islet transplantation. The nonhuman primate serves as an appropriate animal model for such investigations; however, a major impediment in performing such preclinical research has been the difficulty in isolating islets of sufficient quantity and quality. The current study describes a simple and cost-effective method to isolate nonhuman primate islets to support preclinical islet transplantation research. The results of islet isolations from 54 cynomolgus monkeys and 4 baboons are reported. The pancreas was infused with Liberase HI and subjected to static digestion. The digested tissue was shaken, filtered through a mesh screen, applied to a discontinuous gradient, and centrifuged in much the same manner as with conventional rodent islet isolations. Islets were collected from the two interfaces, washed, and transplanted. Following purification, cynomolgus monkey islet isolation yields were 50,100 +/- 3120 IE total or 8760 +/- 420 IE/g pancreas with the percent purity and viability of 90.8 +/- 0.9 and 90.7 +/- 0.7, respectively. Total insulin content of the isolated islets was 405 +/- 53 microg insulin with DNA content being and 976 +/- 117 microg DNA, corresponding to a ratio of 0.57 microg insulin/microg DNA. STZ-induced diabetes was reversed in both mouse and nonhuman primate recipients, which possessed significant levels of c-peptide following transplantation and well-granulated islet grafts. The technique yields sufficient numbers of pure and viable islets to support preclinical research to develop improved strategies to prevent the immune destruction of the transplanted islet graft.

Vascularized islet-cell transplantation in miniature swine. I. Preparation of vascularized islet kidneys.

BACKGROUND: Whereas clinical pancreatic transplantation has been highly successful in correcting the hyperglycemia of insulin-dependent diabetes mellitus (type 1), the results of islet transplantation have been disappointing. This discrepancy may be because of, at least in part, nonspecific loss of islets during the time required for revascularization. To test this hypothesis, we have designed composite kidney grafts containing vascularized autologous islets that can be used to compare the engraftment potential of vascularized versus nonvascularized islet tissue. METHODS: (1) Islet-cell isolation: miniature swine underwent either partial pancreatectomy to isolate autologous islets or total pancreatectomy to isolate minor antigen-mismatched islets. Islets were purified from excised pancreatic tissue by enzymatic digestion and discontinuous density gradient purification. Isolated islets were cultured for 3 days before transplant. (2) Creation of vascularized islet kidneys (IK): autologous islets alone (n=6), minor-mismatched islets alone (n=3), and minor-mismatched islets plus simultaneous autologous thymic tissue (n=3) were transplanted beneath the renal capsule of juvenile miniature swine. Minor antigen-mismatched islets were also transplanted into both the vascularized thymic graft of a thymokidney (to produce a thymo-islet kidney [TIK]) and the contralateral native kidney (n=3) and both the host thymus and beneath the renal capsule (n=2). All recipients receiving minor-mismatched islets were treated with a 12-day intravenous (IV) course of either cyclosporine A (CsA) at 10 mg/kg per day or FK506 at 0.15 mg/kg per day. (3) Assessment of Function: to evaluate the function of the transplanted islets, three animals bearing TIK and IK underwent total pancreatectomy 3 months following islet transplantation. RESULTS: (1) Islet-cell yields: an average of 254,960+/-51,879 (4,452+/-932 islet equivalents [IEQ]/gram of pancreas) and 374,410+/-9,548 (4,183+/-721 IEQ/gram of pancreas) viable islets were obtained by partial pancreatectomy and complete pancreatectomy, respectively. (2) Creation of IK: autologous islets engrafted indefinitely, whereas recipients of minor-mismatched islets alone rejected the islets within 2 months. However, when minor-mismatched islets were implanted into both the thymokidney and the contralateral kidney of animals bearing a thymokidney, the islets engrafted indefinitely in both sites (>3 months). Simultaneous implantation of islets into the host thymus and under the renal capsule also led to permanent engraftment of minor-mismatched islets. (3) Function of vascularized islets: three animals with both a TIK and an IK in place for 3 months underwent total pancreatectomy. All three animals maintained normoglycemia thereafter. In two of these animals, the IKs were removed 2 months after the pancreatectomy, and in both cases normoglycemia was maintained thereafter by the TIK. CONCLUSIONS: The implantation of islets beneath the autologous renal capsule permitted the establishment of a vascular supply and thereby supported normal islet-cell growth and function. The presence of thymic tissue beneath the autologous renal capsule facilitated the engraftment of minor-mismatched islets, and such grafts achieved results similar to autologous islet transplants. Therefore, the ability to create vascularized islet grafts may provide a strategy for successful islet transplantation across allogeneic and potentially across xenogeneic barriers.

Vascularized islet cell transplantation in miniature Swine: islet-kidney allografts correct the diabetic hyperglycemia induced by total pancreatectomy.

We have previously reported the preparation of vascularized islet-kidneys (IKs) by transplantation of islets under the autologous kidney capsule. Here, we compare the efficacy of transplanting vascularized versus nonvascularized islets into diabetic allogeneic swine recipients. In the vascularized islet transplantation (5,000 islet equivalents [IE]/kg), recipients received minor-mismatched (n = 4) or fully-mismatched (n = 2) IKs after pancreatectomy, with a 12-day course of cyclosporine A (CyA) or FK506, respectively. For the nonvascularized islet transplantation (7,000 IE/kg), three recipients received minor-mismatched islets alone and two recipients received minor-mismatched donor islets placed in a donor kidney on the day of transplantation. All recipients of nonvascularized islets were treated with a 12-day course of CyA. With vascularized islet transplantation, pancreatectomized recipients were markedly hyperglycemic pretransplant (fasting blood glucose >300 mg/dl). After composite IK transplantation, all recipients developed and maintained normoglycemia (<120 mg/dl) and stable renal function indefinitely (>3 months), and insulin therapy was not required. Major histocompatibility complex-mismatched recipients demonstrated in vitro donor-specific unresponsiveness. In contrast, recipients of nonvascularized islets remained hyperglycemic. In conclusion, IK allografts cured surgically induced diabetes across allogeneic barriers, whereas nonvascularized islet transplants did not. These data indicate that prevascularization of islet allografts is crucial for their subsequent engraftment and that composite IKs may provide a strategy for successful islet transplantation.