Cell delivery systems: Toward the next generation of cell therapies for type 1 diabetes.

Immunoprotection and oxygen supply are vital in implementing a cell therapy for type 1 diabetes (T1D). Without these features, the transplanted islet cell clusters will be rejected by the host immune system, and necrosis will occur due to hypoxia. The use of anti-rejection drugs can help protect the transplanted cells from the immune system; yet, they also may have severe side effects. Cell delivery systems (CDS) have been developed for islet transplantation to avoid using immunosuppressants. CDS provide physical barriers to reduce the immune response and chemical coatings to reduce host fibrotic reaction. In some CDS, there is architecture to support vascularization, which enhances oxygen exchange. In this review, we discuss the current clinical and preclinical studies using CDS without immunosuppression as a cell therapy for T1D. We find that though CDS have been demonstrated for their ability to support immunoisolation of the grafted cells, their functionality has not been fully optimized. Current advanced methods in clinical trials demonstrate the systems are partly functional, physically complicated to implement or inefficient. However, modifications are being made to overcome these issues.

Australian Public Attitudes on Gene Editing of the Human Embryo.

Technology is now available which facilitates gene editing and has recently been applied internationally to embryos in the laboratory. A 2002 law in Australia prohibits making heritable changes in embryos, regardless of whether the treated embryo is discarded thereafter. We sought to begin to understand public opinion in Australia about this matter, using a questionnaire given to the audience attending a Q and A panel of experts. We found majority support for allowing heritable changes for health purposes. If this is confirmed in a larger survey of the population, we suggest the existing law should be reviewed.

Clinical use of GLP-1 agonists and DPP4 inhibitors.

Type 2 diabetes is a growing problem, with 387 million people currently affected, and 592 million by 2035. Whilst diet and exercise are the corner stones of treatment, oral hypoglycaemic agents are often needed to achieve glycaemic control, thereby reducing the chance of long term diabetic complications. Biguanides and sulfonylureas have been the standard tablets used for this disorder, until 2005-7 when glucagon-like peptide-1 (GLP-1) agonists and dipeptidyl peptidase-4 (DPP4) inhibitors became available. Their major advantage over sulfonylureas is that they are weight lowering or weight neutral, and have a very low incidence of hypoglycaemia. GLP-1 agonists are injectables, whereas the DPP4 inhibitors are administered orally. Both agents are best used in combination with other hypoglycaemic medication, especially metformin and sodium glucose co-transporter 2 (SGLT2) inhibtors. Usage is increasing, being roughly equal to that of sulfonylureas, but less than that of metformin. Side effects appear to be minimal.

Challenges with Cell-based Therapies for Type 1 Diabetes Mellitus.

Type 1 diabetes (T1D) is a chronic, lifelong metabolic disease. It is characterised by the autoimmune-mediated loss of insulin-producing pancreatic ß cells in the islets of Langerhans (ß-islets), resulting in disrupted glucose homeostasis. Administration of exogenous insulin is the most common management method for T1D, but this requires lifelong reliance on insulin injections and invasive blood glucose monitoring. Replacement therapies with beta cells are being developed as an advanced curative treatment for T1D. Unfortunately, this approach is limited by the lack of donated pancreatic tissue, the difficulties in beta cell isolation and viability maintenance, the longevity of the transplanted cells in vivo, and consequently high costs. Emerging approaches to address these limitations are under intensive investigations, including the production of insulin-producing beta cells from various stem cells, and the development of bioengineered devices including nanotechnologies for improving islet transplantation efficacy without the need for recipients taking toxic anti-rejection drugs. These emerging approaches present promising prospects, while the challenges with the new techniques need to be tackled for ultimately clinical treatment of T1D. This review discussed the benefits and limitations of the cell-based therapies for beta cell replacement as potential curative treatment for T1D, and the applications of bioengineered devices including nanotechnology to overcome the challenges associated with beta cell transplantation.

In vitro evaluation of a hybrid drug-delivery nanosystem for fibrosis prevention in cell therapy for Type 1 diabetes.

Background: Implantation of insulin-secreting cells has been trialed as a treatment for Type 1 diabetes mellitus; however, the host immunogenic response limits their effectiveness. Methodology: The authors developed a core-shell nanostructure of upconversion nanoparticle-mesoporous silica for controlled local delivery of an immunomodulatory agent, MCC950, using near-infrared light and validated it in in vitro models of fibrosis. Results: The individual components of the nanosystem did not affect the proliferation of insulin-secreting cells, unlike fibroblast proliferation (p < 0.01). The nanosystem is effective at releasing MCC950 and preventing fibroblast differentiation (p < 0.01), inflammation (IL-6 expression; p < 0.05) and monocyte adhesion (p < 0.01). Conclusion: This MCC950-loaded nanomedicine system could be used in the future together with insulin-secreting cell implants to increase their longevity as a curative treatment for Type 1 diabetes mellitus.

This work describes a new drug-delivery system that can release an immunomodulatory drug in a controlled manner and prevent fibrosis, which is part of the immune response when a foreign body is implanted. This system can be particularly useful for insulin-secreting cell implants, used to replace multiple daily injections of insulin and improve the quality of life of people with Type 1 diabetes mellitus. By preventing the immune response that leads to fibrosis, the longevity of these cellular implants can be extended without the need for frequent replacement procedures. This innovative nanosystem can release the required amount of immunomodulatory drug, which could be stimulated with the use of special light, hence showing the ability for local and extended delivery. This type of system has the potential to reduce the side effects associated with oral daily administration of immunomodulatory agents in people with Type 1 diabetes mellitus.

Smart Drug-Delivery System of Upconversion Nanoparticles Coated with Mesoporous Silica for Controlled Release.

Drug-delivery vehicles have garnered immense interest in recent years due to unparalleled progress made in material science and nanomedicine. However, the development of stimuli-responsive devices with controllable drug-release systems (DRSs) is still in its nascent stage. In this paper, we designed a two-way controlled drug-release system that can be promoted and prolonged, using the external stimulation of near-infrared light (NIR) and protein coating. A hierarchical nanostructure was fabricated using upconversion nanoparticles (UCNPs)-mesoporous silica as the core-shell structure with protein lysozyme coating. The mesoporous silica shell provides abundant pores for the loading of drug molecules and a specific type of photosensitive molecules. The morphology and the physical properties of the nanostructures were thoroughly characterized. The results exhibited the uniform core-shell nanostructures of ~four UCNPs encapsulated in one mesoporous silica nanoparticle. The core-shell nanoparticles were in the spherical shape with an average size of 200 nm, average surface area of 446.54 m2/g, and pore size of 4.6 nm. Using doxorubicin (DOX), a chemotherapy agent as the drug model, we demonstrated that a novel DRS with capacity of smart modulation to promote or inhibit the drug release under NIR light and protein coating, respectively. Further, we demonstrated the therapeutic effect of the designed DRSs using breast cancer cells. The reported novel controlled DRS with dual functionality could have a promising potential for chemotherapy treatment of solid cancers.

Prevascularized Retrievable Hybrid Implant to Enhance Function of Subcutaneous Encapsulated Islets.

Replacement of pancreatic ß-cells is one of the most promising treatment options for treatment of type 1 diabetes (T1D), even though, toxic immunosuppressive drugs are required. In this study, we aim to deliver allogeneic ß-cell therapies without antirejection drugs using a bioengineered hybrid device that contains microencapsulated ß-cells inside 3D polycaprolactone (PCL) scaffolds printed using melt electrospin writing (MEW). Mouse ß-cell (MIN6) pseudoislets and QS mouse islets are encapsulated in alginate microcapsules, without affecting viability and insulin secretion. Microencapsulated MIN6 cells are then seeded within 3D MEW scaffolds, and these hybrid devices implanted subcutaneously in streptozotocin-treated diabetic NOD/SCID and BALB/c mice. Similar to NOD/SCID mice, blood glucose levels (BGL) are lowered from 30.1 to 4.8 mM in 25-41 days in BALB/c. In contrast, microencapsulated islets placed in prevascularized MEW scaffold 3 weeks after implantation in BALB/c mice normalize BGL (<12 mM) more rapidly, lasting for 60-105 days. The lowering of glucose levels is confirmed by an intraperitoneal glucose tolerance test. Vascularity within the implanted grafts is demonstrated and quantified by 3D-doppler ultrasound, with a linear increase over 4 weeks (r = 0.65). Examination of the device at 5 weeks shows inflammatory infiltrates of neutrophils, macrophages, and B-lymphocytes on the MEW scaffolds, but not on microcapsules, which have infrequent profibrotic walling. In conclusion, we demonstrate the fabrication of an implantable and retrievable hybrid device for vascularization and enhancing the survival of encapsulated islets implanted subcutaneously in an allotransplantation setting without immunosuppression. This study provides proof-of-concept for the application of such devices for human use, but, will require modifications to allow translation to people with T1D. Impact statement The retrievable 3D printed PCL scaffold we have produced promotes vascularization when implanted subcutaneously and allows seeded microencapsulated insulin-producing cells to normalize blood glucose of diabetic mice for at least 2 months, without the need for antirejection drugs to be administered. The scaffold is scalable for possible human use, but will require modification to ensure that normalization of blood glucose levels can be maintained long term.

Encapsulated pancreatic progenitors derived from human embryonic stem cells as a therapy for insulin-dependent diabetes.

BACKGROUND: Cellular-based therapies for insulin-dependent diabetes are potential means of achieving and maintaining normal blood glucose levels (BGL) without the need for insulin administration. Islets isolated from donor pancreases have been the most common tissue used to date, but supply is a limiting factor. The use of human embryonic stem cells (hESC) as a therapy became a possibility with the report that these cells could be differentiated to pancreatic progenitors (PP) over 12 days in vitro. Conversion of PP to glucose-responsive insulin-secreting cells can be achieved by transplanting the progenitors in vivo where cell maturation occurs. To date this step has not been shown under in vitro conditions. METHODS: Prior to transplanting, cells are encapsulated in alginate to prevent the immune cells of recipient attacking the graft. The alginate capsules have pores with a molecular weight cut-off of 250 kDa. These are too small to allow entry of immune cells, but large enough for passage of nutrients and insulin. RESULTS: Encapsulated insulin-producing cells survive and function when transplanted, and have been shown to normalize BGL when allografted into diabetic mice. As few as 750 encapsulated human islets are sufficient to normalize BGL of diabetic non-obese diabetic severe combined immunodeficient (NOD/SCID) recipient mice for at least 2 months. The safety of transplanting encapsulated human islets as demonstrated by the lack of major adverse events and infection was recently shown in a first-in-human clinical trial. Finally, fetal porcine islet-like cell clusters, which are akin to PP derived from ESC, mature and normalize BGL of diabetic recipient mice with the same efficiency as non-encapsulated clusters placed under the kidney capsule. CONCLUSION: Transplanting encapsulated PP, derived from hESCs, into diabetic recipients is the strategy that is now being explored in the Australia Diabetes Therapy Project.

Effects of high-density lipoproteins on pancreatic beta-cell insulin secretion.

OBJECTIVE: Type 2 diabetes is characterized by impaired beta-cell secretory function, insulin resistance, reduced high-density lipoprotein (HDL) levels, and increased cardiovascular risk. Given the current interest in therapeutic interventions that raise HDLs levels, this study investigates the effects of HDLs on insulin secretion from beta-cells. METHODS AND RESULTS: Incubation of Min6 cells and primary islets under basal or high-glucose conditions with either apolipoprotein (apo) A-I or apoA-II in the lipid-free form, as a constituent of discoidal reconstituted HDLs (rHDLs), or with HDLs isolated from human plasma increased insulin secretion up to 5-fold in a calcium-dependent manner. The increase was time and concentration dependent. It was also K(ATP) channel and glucose metabolism dependent under high-glucose, but not low-glucose, conditions. The lipid-free apolipoprotein-mediated increase in insulin secretion was ATP binding cassette (ABC) transporter A1 and scavenger receptor-B1 dependent. The rHDL-mediated increase in insulin secretion was ABCG1 dependent. Exposure of beta-cells to lipid-free apolipoproteins also increased insulin mRNA expression and insulin secretion without significantly depleting intracellular insulin or cholesterol levels. CONCLUSIONS: These results establish that lipid-free and lipid-associated apoA-I and apoA-II increase beta-cell insulin secretion and indicate that interventions that raise HDLs levels may be beneficial in type 2 diabetes.

Effect of prolonged gelling time on the intrinsic properties of barium alginate microcapsules and its biocompatibility.

Pericapsular fibrotic overgrowth (PFO) may be attributed to an immune response against microcapsules themselves or to antigen shedding through microcapsule pores from encapsulated islet tissue. Modification of microcapsules aimed at reducing pore size should prevent PFO and improve graft survival. This study investigated the effect of increased gelling time (20 vs. 2 min) in barium chloride on intrinsic properties of alginate microcapsules and tested their biocompatibility in vivo. Prolonged gelling time affected neither permeability nor size of the microcapsules. However, prolonged gelling time for 20 min produced brittle microcapsules compared to 2 min during compression test. Encapsulation of human islets in both types of microcapsules affected neither islet viability nor function. The presence of PFO when transplanted into a large animal model such as baboon and its absence in small animal models such as rodents suggest that the host immune response towards alginate microcapsules is species rather than alginate specific.

Encapsulation and Transplantation of Pancreatic Progenitor Cells.

Type 1 diabetes, characterized by autoimmune destruction of pancreatic beta cells, affects 41 million people worldwide. Beta cell replacement therapies have immense potential as a treatment option because pancreatic progenitors derived from human pluripotent stem cells can provide a near limitless supply of transplantable tissue. The key limitation of this approach is the need for lifelong use of immunosuppressive drugs that have undesirable side effects. Microencapsulation is an option for providing protection for transplanted cells from mechanical stress and immune attack. Traditionally, pluripotent cells are differentiated on a 2D matrix before being transferred into an immunoisolation device. Here, we describe a method of differentiating pluripotent stem cells into pancreatic progenitors while the cells are encapsulated in alginate microspheres. This method provides several advantages including the need for fewer steps compared to the traditional approach, protection against mechanical/physical damage during differentiation in bioreactors, and immune-protection of cells once transplanted into the host.

Islet cell transplantation.

PURPOSE OF REVIEW: The transplantation of human islets has come a long way since the first diabetic person became insulin independent in 1989. The advent of a steroid-free immunosuppressive protocol in 2000 resulted in most recipients becoming insulin independent and remaining so for a year. However, beta-cell function declines thereafter. Strategies to enhance the islet mass transplanted and preserve beta-cell function are necessary. RECENT FINDINGS: This review covers recent advances in determining the selection of appropriate enzymes for islet isolation, use of pancreases from heart-dead donors and techniques for predicting the functional capacity of isolated islets prior to transplantation. Changing the transplantation site away from the liver, where many islets are destroyed by an inflammatory process, is reviewed, and the possibility of seeding islets onto three-dimensional biodegradable scaffolds discussed. A method of preventing apoptosis of the beta cells prior to transplantation is detailed, as is the beneficial effect of using exenatide, after transplantation. Novel techniques to image islets are discussed, and this requires the labelling of the islets prior to implantation. Enhancing the vascularization of islets is shown to enhance functional outcomes. Encapsulation of the islets should obviate the need for using antirejection drugs, and it may be possible to expand beta cells in vitro. SUMMARY: The above strategies are likely to enhance the outcomes of clinical islet transplants.

Differentiation of transplanted microencapsulated fetal pancreatic cells.

BACKGROUND: Fetal beta cells are a potential form of cell therapy for type 1 diabetes. To protect transplanted cells from cellular immune attack, microencapsulation using barium alginate can be employed. Whether microencapsulated fetal pancreatic cells will differentiate as occurs with nonencapsulated fetal pancreatic cells is presently unknown. It is suggested that such differentiation would occur in encapsulated cells, similar to previous experiments conducted using encapsulated embryonic stem cells. METHODS: Streptozotocin-induced diabetic severe combined immunodeficient mice were transplanted with 5,000 to 38,000 fetal pig islet-like cell clusters (ICCs) within barium alginate microcapsules of diameter 300, 600, or 1000 microm. Viability, insulin secretion, and content of encapsulated cells were measured prior to transplantation. Blood glucose levels (BGL) were measured twice weekly and porcine C-peptide monthly. Encapsulated cells were recovered from mice at 6 months posttransplantation for analysis. RESULTS: Encapsulated cells became glucose responsive and normalized BGL within 13 to 68 days posttransplantation, with 5,000 to 10,000 ICCs required. Microcapsule diameter did not affect the time required to achieve normoglycemia. BGL remained normal for the 6-month duration of the experiments. After removal of grafts at 25 weeks posttransplantation, glucose stimulated insulin secretion of the explants was enhanced 96-fold, insulin content was enhanced 34-fold, and the percentage of insulin and glucagon positive cells increased 10-fold and threefold, respectively, from the time of transplantation. CONCLUSIONS: This study demonstrates that fetal pancreatic cells differentiate and function normally when placed within barium alginate microcapsules and transplanted.

Encapsulated Islet Transplantation: Where Do We Stand?

Transplantation of pancreatic islets encapsulated within immuno-protective microcapsules is a strategy that has the potential to overcome graft rejection without the need for toxic immunosuppressive medication. However, despite promising preclinical studies, clinical trials using encapsulated islets have lacked long-term efficacy, and although generally considered clinically safe, have not been encouraging overall. One of the major factors limiting the long-term function of encapsulated islets is the host's immunological reaction to the transplanted graft which is often manifested as pericapsular fibrotic overgrowth (PFO). PFO forms a barrier on the capsule surface that prevents the ingress of oxygen and nutrients leading to islet cell starvation, hypoxia and death. The mechanism of PFO formation is still not elucidated fully and studies using a pig model have tried to understand the host immune response to empty alginate microcapsules. In this review, the varied strategies to overcome or reduce PFO are discussed, including alginate purification, altering microcapsule geometry, modifying alginate chemical composition, co-encapsulation with immunomodulatory cells, administration of pharmacological agents, and alternative transplantation sites. Nanoencapsulation technologies, such as conformal and layer-by-layer coating technologies, as well as nanofiber, thin-film nanoporous devices, and silicone based NanoGland devices are also addressed. Finally, this review outlines recent progress in imaging technologies to track encapsulated cells, as well as promising perspectives concerning the production of insulin-producing cells from stem cells for encapsulation.

Co-encapsulation and co-transplantation of mesenchymal stem cells reduces pericapsular fibrosis and improves encapsulated islet survival and function when allografted.

Pericapsular fibrotic overgrowth (PFO) is associated with poor survival of encapsulated islets. A strategy to combat PFO is the use of mesenchymal stem cells (MSC). MSC have anti-inflammatory properties and their potential can be enhanced by stimulation with proinflammatory cytokines. This study investigated whether co-encapsulation or co-transplantation of MSC with encapsulated islets would reduce PFO and improve graft survival. Stimulating MSC with a cytokine cocktail of IFN-γ and TNF-α enhanced their immunosuppressive potential by increasing nitric oxide production and secreting higher levels of immunomodulatory cytokines. In vitro, co-encapsulation with MSC did not affect islet viability but significantly enhanced glucose-induced insulin secretion. In vivo, normoglycemia was achieved in 100% mice receiving islets co-encapsulated with stimulated MSC as opposed to 71.4% receiving unstimulated MSC and only 9.1% receiving encapsulated islets alone. Microcapsules retrieved from both unstimulated and stimulated MSC groups had significantly less PFO with improved islet viability and function compared to encapsulated islets alone. Levels of peritoneal immunomodulatory cytokines IL-4, IL-6, IL-10 and G-CSF were significantly higher in MSC co-encapsulated groups. Similar results were obtained when encapsulated islets and MSC were co-transplanted. In summary, co-encapsulation or co-transplantation of MSC with encapsulated islets reduced PFO and improved the functional outcome of allotransplants.

Modulation of Mitochondrial DNA Copy Number to Induce Hepatocytic Differentiation of Human Amniotic Epithelial Cells.

Mitochondrial deoxyribonucleic acid (mtDNA) copy number is tightly regulated during pluripotency and differentiation. There is increased demand of cellular adenosine triphosphate (ATP) during differentiation for energy-intensive cell types such as hepatocytes and neurons to meet the cell's functional requirements. During hepatocyte differentiation, mtDNA copy number should be synchronously increased to generate sufficient ATP through oxidative phosphorylation. Unlike bone marrow mesenchymal cells, mtDNA copy number failed to increase by 28 days of differentiation of human amniotic epithelial cells (hAEC) into hepatocyte-like cells (HLC) despite their expression of some end-stage hepatic markers. This was due to higher levels of DNA methylation at exon 2 of POLGA, the mtDNA-specific replication factor. Treatment with a DNA demethylation agent, 5-azacytidine, resulted in increased mtDNA copy number, reduced DNA methylation at exon 2 of POLGA, and reduced hepatic gene expression. Depletion of mtDNA followed by subsequent differentiation did not increase mtDNA copy number, but reduced DNA methylation at exon 2 of POLGA and increased expression of hepatic and pluripotency genes. We encapsulated hAEC in barium alginate microcapsules and subsequently differentiated them into HLC. Encapsulation resulted in no net increase of mtDNA copy number but a significant reduction in DNA methylation of POLGA. RNAseq analysis showed that differentiated HLC express hepatocyte-specific genes but also increased expression of inflammatory interferon genes. Differentiation in encapsulated cells showed suppression of inflammatory genes as well as increased expression of genes associated with hepatocyte function pathways and networks. This study demonstrates that an increase in classical hepatic gene expression can be achieved in HLC through encapsulation, although they fail to effectively regulate mtDNA copy number.