Jennifer Hollister-Lock

Size- and shape-dependent foreign body immune response to materials implanted in rodents and non-human primates

The efficacy of implanted biomedical devices is often compromised by host recognition and subsequent foreign body responses. Here, we demonstrate the role of the geometry of implanted materials on their biocompatibility in vivo. In rodent and non-human primate animal models, implanted spheres 1.5 mm and above in diameter across a broad spectrum of materials, including hydrogels, ceramics, metals, and plastics, significantly abrogated foreign body reactions and fibrosis when compared to smaller spheres. We also show that for encapsulated rat pancreatic islet cells transplanted into streptozotocin-treated diabetic C57BL/6 mice, islets prepared in 1.5 mm alginate capsules were able to restore blood-glucose control for up to 180 days, a period more than 5-fold longer than for transplanted grafts encapsulated within conventionally sized 0.5-mm alginate capsules. Our findings suggest that the in vivo biocompatibility of biomedical devices can be significantly improved by simply tuning their spherical dimensions.

Long-term glycemic control using polymer-encapsulated human stem cell–derived beta cells in immune-competent mice

The transplantation of glucose-responsive, insulin-producing cells offers the potential for restoring glycemic control in individuals with diabetes. Pancreas transplantation and the infusion of cadaveric islets are currently implemented clinically, but these approaches are limited by the adverse effects of immunosuppressive therapy over the lifetime of the recipient and the limited supply of donor tissue. The latter concern may be addressed by recently described glucose-responsive mature beta cells that are derived from human embryonic stem cells (referred to as SC-β cells), which may represent an unlimited source of human cells for pancreas replacement therapy. Strategies to address the immunosuppression concerns include immunoisolation of insulin-producing cells with porous biomaterials that function as an immune barrier. However, clinical implementation has been challenging because of host immune responses to the implant materials. Here we report the first long-term glycemic correction of a diabetic, immunocompetent animal model using human SC-β cells. SC-β cells were encapsulated with alginate derivatives capable of mitigating foreign-body responses in vivo and implanted into the intraperitoneal space of C57BL/6J mice treated with streptozotocin, which is an animal model for chemically induced type 1 diabetes. These implants induced glycemic correction without any immunosuppression until their removal at 174 d after implantation. Human C-peptide concentrations and in vivo glucose responsiveness demonstrated therapeutically relevant glycemic control. Implants retrieved after 174 d contained viable insulin-producing cells.

Gene expression of VEGF and its receptors Flk-1/KDR and Flt-1 in cultured and transplanted rat islets.

Background. Vascular endothelial growth factor (VEGF) and its two receptor tyrosine kinases, Flk-1/KDR and Flt-1, may play an important role in mediating the revascularization of transplanted pancreatic islets. Methods. Using semiquantitative multiplex reverse-transcribed polymerase chain reaction we determined the gene expression of VEGF and its receptors in cultured and transplanted rat islets. Results. After exposure of islet cells to hypoxia in vitro, increases were found in the gene expression of the VEGF120 and VEGF164 isoforms, with simultaneous increases in VE-cadherin, Flk-1/KDR, and Flt-1. In vivo studies consisted of analysis of islet grafts transplanted into both normal and diabetic recipients. Expression of both VEGF120 and VEGF164 in grafts was up-regulated for the first 2–3 days after transplantation, with the response being more prolonged in the diabetic rats. These increases were followed by reduced expression of VEGF on days 5, 7, and 9. Increases in the expression of VE-cadherin in islet grafts in normal and diabetic recipients tended to parallel VEGF expression, with the increases in both probably being caused by hypoxia. The early increases of VEGF expression were followed by a rise in the expression of VEGF receptors, which probably represents the early stages of angiogenesis. Graft expression of Flk-1/KDR and Flt-1 was enhanced at 3 and 5 days in the normoglycemic recipients, while in the diabetic recipients increases were found later on days 5, 7, and 14. Conclusions. The delayed expression of VEGF receptors in the diabetic recipients could reflect impaired angiogenesis caused by the diabetic milieu; this delay could contribute to the less outcomes of grafts transplanted into a hyperglycemic environment.

Reversal of hyperglycemia in mice after subcutaneous transplantation of macroencapsulated islets.

BACKGROUND Macroencapsulated islets can reverse hyperglycemia in diabetic animals when transplanted i.p. or into the fat pad. The s.c. space is an attractive site for such transplantation because macrocapsules can be implanted with local anesthesia and be easily removed or reloaded with fresh islets. METHODS Immunoprotective 20 microl ported devices were transplanted under the skin of Streptozocin-diabetic nude mice. Devices were loaded with 1200 rat islets in culture medium or in alginate. Empty devices were implanted for 2 weeks and then loaded with islets. Normal mice and mice with islets transplanted under the renal capsule or under the skin were used as controls. Seven weeks after transplantation, an intraperitoneal glucose tolerance test (IPGTT) was performed, followed by implant removal. RESULTS Three weeks after transplantation, normal blood glucose levels were observed in all animals. Compared with those of normal controls, IPGTTs showed accelerated blood glucose clearance in mice transplanted with islets either within devices or beneath the kidney capsule. Fasted transplanted mice were hypoglycemic before glucose injection and 2 hr later. After removal of the implants, all recipient mice returned to hyperglycemia. Histological evaluation revealed viable islet cells and a network of close vascular structures outside the devices. CONCLUSIONS Macroencapsulated islets transplanted into the s.c. space were able to survive and regulate blood glucose levels in mice. The observed differences in glucose metabolism between normal and transplanted mice may be attributed to the site of transplantation and to the use of rat islets, which have a different set point for glucose induced insulin release.

Adenosine kinase inhibition selectively promotes rodent and porcine islet β-cell replication

Diabetes is a pathological condition characterized by relative insulin deficiency, persistent hyperglycemia, and, consequently, diffuse micro- and macrovascular disease. One therapeutic strategy is to amplify insulin-secretion capacity by increasing the number of the insulin-producing β cells without triggering a generalized proliferative response. Here, we present the development of a small-molecule screening platform for the identification of molecules that increase β-cell replication. Using this platform, we identify a class of compounds [adenosine kinase inhibitors (ADK-Is)] that promote replication of primary β cells in three species (mouse, rat, and pig). Furthermore, the replication effect of ADK-Is is cell type-selective: treatment of islet cell cultures with ADK-Is increases replication of β cells but not that of α cells, PP cells, or fibroblasts. Short-term in vivo treatment with an ADK-I also increases β-cell replication but not exocrine cell or hepatocyte replication. Therefore, we propose ADK inhibition as a strategy for the treatment of diabetes.

Differentiation and expansion of beta cell mass in porcine neonatal pancreatic cell clusters transplanted into nude mice.

Neonatal porcine pancreas has considerable capacity for growth and differentiation, making it an attractive potential source of islet tissue for xenotransplantation. Pancreases from 1–3-day-old newborn pigs were digested with collagenase and cultured for 8 days. The resulting cellular aggregates are called porcine neonatal pancreatic cell clusters (NPCCs). The mean yield of NPCCs from a newborn pig was 28,200 ± 1700 islet equivalents. Cytokeratin 7 (CK7) was used as a marker for the immunostaining of pancreatic duct cells. In neonatal pancreas, 18% of the insulin-positive cells co-stained for CK7, thus being protodifferentiated. NPCCs also contained protodifferentiated cells; insulin/PP and insulin/somatostatin co-stained cells were more common than insulin/glucagon cells. Between 1 and 8 days of culture, the DNA content of the NPCCs fell to 16% and the insulin content to 33% of the starting value, mainly due to the preferential loss of exocrine cells. Transplantation of 2000 or 4000 NPCCs into diabetic nude mice typically normalized glucose values in 10–20 weeks. Mice with successful grafts had lower fasting blood glucose levels than normal mice and accelerated glucose clearance after an IP glucose load. The starting NPCCs consisted of 17% insulin-staining cells, but the grafts of mice with reversed diabetes consisted of 94% beta cells, with some co-stained for CK7, indicating that the grafts still contained immature cells. The mass of insulin-producing cells rose from 0.22 ± 0.08 mg 1 week after transplantation to 4.34 ± 0.27 mg in mice sacrificed at 27–35 weeks. In summary, NPCCs contain mostly islet precursor cells, which when transplanted into nude mice undergo striking differentiation and beta cell expansion.

Rat islet cell aggregates are superior to islets for transplantation in microcapsules

Aims/hypothesisIslet transplantation is a promising treatment for type 1 diabetes but is hampered by a shortage of donor human tissue and early failure. Research on islet cell transplantation includes finding new sources of cells and immunoisolation to protect from immune assault and tumourigenic potential. Small islet cell aggregates were studied to determine if their survival and function were superior to intact islets within microcapsules because of reduced oxygen transport limitation and inflammatory mediators.MethodsIslet cell aggregates were generated by dispersing rat islets into single cells and allowing them to re-aggregate in culture. Rat islets and islet cell aggregates were encapsulated in barium alginate capsules and studied when cultured in low (0.5% or 2%) or normal (20%) oxygen, or transplanted into mice.ResultsEncapsulated islet cell aggregates were able to survive and function better than intact islets in terms of oxygen-consumption rate, nuclei counts, insulin-to-DNA ratio and glucose-stimulated insulin secretion. They also had reduced expression of pro-inflammatory genes. Islet cell aggregates showed reduced tissue necrosis in an immunodeficient transplant model and a much greater proportion of diabetic xenogeneic transplant recipients receiving islet cell aggregates (tissue volume of only 85 islet equivalents) had reversal of hyperglycaemia than recipients receiving intact islets.Conclusions/interpretationThese aggregates were superior to intact islets in terms of survival and function in low-oxygen culture and during transplantation and are likely to provide more efficient utilisation of islet tissue, a finding of importance for the future of cell therapy for diabetes.

Function and survival of macroencapsulated syngeneic islets transplanted into streptozocin-diabetic mice

BACKGROUND Macroencapsulation is a strategy to protect transplanted islets from rejection and autoimmune attack. This study addresses questions about the survival and function of macroencapsulated syngeneic islets. METHODS Planar immunobarrier membrane diffusion devices were used for syngeneic islet transplantation. After being mixed with a 1% alginate solution, a total of 250, 500, 750 or 1000 islets were loaded into the devices, which were implanted into the epididymal fat pad(s) of streptozocin diabetic mice. RESULTS The success rate for restoration of normoglycemia at week 4 was highest for the recipients receiving two devices, each with 500 islets. Loading 750 or 1000 islets provided no improvement over loading 500 islets in a single device. Devices containing 250 islets were rarely successful. There was a striking tendency of transplants to either bring glucose levels into the near normal range or to fail with marked hyperglycemia. After an overnight fast at 1 and 4 weeks, but not at 12 weeks, hypoglycemia was found. The insulin content of devices from animals with normalized glucose values was higher than the insulin content in failed devices. Islet volume was maintained for 12 weeks, and fibrosis did not increase. CONCLUSIONS A relatively small mass of macroencapsulated islet tissue can survive and function well enough to normalize glucose levels for at least 12 weeks. Maintenance of glucose levels in the near-normal range seems to have a beneficial influence on graft success. The finding of fasting hypoglycemia raises important clinical questions about islet dysfunction. Important limitations in the requirements for islet packing density in macroencapsulation have been defined. New approaches for improving islet packing density must be developed to make diffusion-dependent macroencapsulation more practical.

Thyroid Hormone Promotes Postnatal Rat Pancreatic β-Cell Development and Glucose-Responsive Insulin Secretion Through MAFA

Neonatal β cells do not secrete glucose-responsive insulin and are considered immature. We previously showed the transcription factor MAFA is key for the functional maturation of β cells, but the physiological regulators of this process are unknown. Here we show that postnatal rat β cells express thyroid hormone (TH) receptor isoforms and deiodinases in an age-dependent pattern as glucose responsiveness develops. In vivo neonatal triiodothyronine supplementation and TH inhibition, respectively, accelerated and delayed metabolic development. In vitro exposure of immature islets to triiodothyronine enhanced the expression of Mafa, the secretion of glucose-responsive insulin, and the proportion of responsive cells, all of which are effects that were abolished in the presence of dominant-negative Mafa. Using chromatin immunoprecipitation and electrophoretic mobility shift assay, we show that TH has a direct receptor-ligand interaction with the Mafa promoter and, using a luciferase reporter, that this interaction was functional. Thus, TH can be considered a physiological regulator of functional maturation of β cells via its induction of Mafa.

Core-Shell Hydrogel Microcapsules for Improved Islets Encapsulation

Islets microencapsulation holds great promise to treat type 1 diabetes. Currently used alginate microcapsules often have islets protruding outside capsules, leading to inadequate immuno-protection. A novel design of microcapsules with core-shell structures using a two-fluid co-axial electro-jetting is reported. Improved encapsulation and diabetes correction is achieved in a single step by simply confining the islets in the core region of the capsules.