From insulin injections to islet transplantation: An overview of the journey.

When, in 1869, Paul Langerhans detected the "islands of tissue" in the pancreas, he took the first step on a journey towards islet transplantation as a treatment for type 1 diabetes. The route has embraced developments across biosciences, surgery, gene therapy and clinical research. This review highlights major milestones along that journey involving whole pancreas transplantation, islet transplantation, the creation of surrogate insulin-secreting cells and novel islet-like structures using genetic and bio-engineering technologies. To obviate the paucity of human tissue, pluripotent stem cells and non-ß-cells within the pancreas have been modified to create physiologically responsive insulin-secreting cells. Before implantation, these can be co-cultured with endothelial cells to promote vascularisation and with immune defence cells such as placental amnion cells to reduce immune rejection. Scaffolds to contain grafts and facilitate surgical placement provide further opportunities to achieve physiological insulin delivery. Alternatively, xenotransplants such as porcine islets might be reconsidered as opportunities exist to circumvent safety concerns and immune rejection. Thus, despite a long and arduous journey, the prospects for increased use of tissue transplantation to provide physiological insulin replacement are drawing ever closer.

The potential role of multifunctional human amniotic epithelial cells in pancreatic islet transplantation.

Pancreatic islet cell transplantation has proven efficacy as a treatment for type 1 diabetes mellitus, chiefly in individuals who are refractory to conventional insulin replacement therapy. At present its clinical use is restricted, firstly by the limited access to suitable donor organs but also due to factors associated with the current clinical transplant procedure which inadvertently impair the long-term functionality of the islet graft. Of note, the physical, biochemical, inflammatory, and immunological stresses to which islets are subjected, either during pretransplant processing or following implantation are detrimental to their sustained viability, necessitating repeated islet infusions to attain adequate glucose control. Progressive decline in functional beta (ß)-cell mass leads to graft failure and the eventual re-instatement of exogenous insulin treatment. Strategies which protect and/or preserve optimal islet function in the peri-transplant period would improve clinical outcomes. Human amniotic epithelial cells (HAEC) exhibit both pluripotency and immune-privilege and are ideally suited for use in replacement and regenerative therapies. The HAEC secretome exhibits trophic, anti-inflammatory, and immunomodulatory properties of relevance to islet graft survival. Facilitated by ß-cell supportive 3D cell culture systems, HAEC may be integrated with islets bringing them into close spatial arrangement where they may exert paracrine influences that support ß-cell function, reduce hypoxia-induced islet injury, and alter islet alloreactivity. The present review details the potential of multifunctional HAEC in the context of islet transplantation, with a focus on the innate capabilities that may counter adverse events associated with the current clinical transplant protocol to achieve long-term islet graft function.

Rotational culture and integration with amniotic stem cells reduce porcine islet immunoreactivity in vitro and slow xeno-rejection in a murine model of islet transplantation.

BACKGROUND: Pre-transplant modification of porcine islets may improve their suitability for clinical use in diabetes management by supporting graft function and reducing the potential for xeno-rejection. The present study investigates intra-graft incorporation of stem cells that secrete beta (ß)-cell trophic and immunomodulatory factors to preserve function and alter immune cell responsiveness to porcine islets. METHODS: Isolated porcine islets were maintained in a three-dimensional rotational cell culture system (RCCS) to facilitate aggregation with human amniotic epithelial cells (AECs). Assembled islet constructs were assessed for functional integrity and ability to avoid xeno-recognition by CD4+ T-cells using mixed islet:lymphocyte reaction assays. To determine whether stem cell-mediated modification of porcine islets provided a survival advantage over native islets, structural integrity was examined in a pig-to-mouse islet transplant model. RESULTS: Rotational cell culture system supported the formation of porcine islet:AEC aggregates with improved insulin-secretory capacity compared to unmodified islets, whilst the xeno-response of purified CD4+ T-cells to AEC-bearing grafts was significantly (P < 0.05) attenuated. Transplanted AEC-bearing grafts demonstrated slower rejection in immune-competent recipients compared to unmodified islets. CONCLUSIONS/INTERPRETATION: Rotational culture enables pre-transplant modification of porcine islets by integration with immunomodulatory stem cells capable of subduing xeno-reactivity to CD4+ T-cells. This reduces islet rejection and offers translational potential to widen availability and improve the clinical effectiveness of islet transplantation.

Low gravity rotational culture and the integration of immunomodulatory stem cells reduce human islet allo-reactivity.

Modification of human islets prior to transplantation may improve long-term clinical outcome in terms of diabetes management, by supporting graft function and reducing the potential for allo-rejection. Intragraft incorporation of stem cells secreting beta (ß)-cell trophic and immunomodulatory factors represents a credible approach, but requires suitable culture methods to facilitate islet alteration without compromising integrity. This study employed a three-dimensional rotational cell culture system (RCCS) to achieve modification, preserve function, and ultimately influence immune cell responsiveness to human islets. Islets underwent intentional dispersal and rotational culture-assisted aggregation with amniotic epithelial cells (AEC) exhibiting intrinsic immunomodulatory potential. Reassembled islet constructs were assessed for functional integrity, and their ability to induce an allo-response in discrete T-cell subsets determined using mixed islet:lymphocyte reaction assays. RCCS supported the formation of islet:AEC aggregates with improved insulin secretory capacity compared to unmodified islets. Further, the allo-response of peripheral blood mononuclear cell (PBMC) and purified CD4+ and CD8+ T-cell subsets to AEC-bearing grafts was significantly (p < 0.05) attenuated. Rotational culture enables pre-transplant islet modification involving their integration with immunomodulatory stem cells capable of subduing the allo-reactivity of T cells relevant to islet rejection. The approach may play a role in achieving acute and long-term graft survival in islet transplantation.

Pre-transplant signal induction for vascularisation in human islets.

Human islet transplant success is partially impaired by slow revascularisation. Our study investigated the potential for rotational cell culture (RC) of human islets combined with thiazolidinedione (TZD) stimulation of peroxisome proliferator-activated receptor gamma (PPARγ) to upregulate vascular endothelial growth factor (VEGF) expression in the islets. Four groups of human islets were studied: static culture (SC) with and without 25 mmol/L TZD and RC with and without 25 mmol/L TZD. These were assessed for insulin secretion and soluble VEGF-A release. Both proteins were quantified by enzyme-linked immunosorbent assay (ELISA), supported with qualitative immunofluorescence staining. RC + TZD increased insulin secretion by >20% (p < 0.05-0.001) in response to 16.7 mmol/L glucose and 16.7 mmol/L glucose + 10 mmol/L theophylline (G + T). This effect was seen at all time intervals compared with SC and without addition of TZD. Soluble VEGF-A release was significantly augmented by RC and TZD exposure with an increased effect of >30% (p < 0.001) at 72 h under both SC and RC conditions. RC supplemented with a TZD enhances and prolongs the release of insulin and soluble VEGF-A by isolated human islets.

Human amniotic epithelial cells induce localized cell-mediated immune privilege in vitro: implications for pancreatic islet transplantation.

Chronic systemic immunosuppression in cell replacement therapy restricts its clinical application. This study sought to explore the potential of cell-based immune modulation as an alternative to immunosuppressive drug therapy in the context of pancreatic islet transplantation. Human amniotic epithelial cells (AEC) possess innate anti-inflammatory and immunosuppressive properties that were utilized to create localized immune privilege in an in vitro islet cell culture system. Cellular constructs composed of human islets and AEC (islet/AEC) were bioengineered under defined rotational cell culture conditions. Insulin secretory capacity was validated by glucose challenge and immunomodulatory potential characterized using a peripheral blood lymphocyte (PBL) proliferation assay. Results were compared to control constructs composed of islets or AEC cultured alone. Studies employing AEC-conditioned medium examined the role of soluble factors, and fluorescence immunocytochemistry was used to identify putative mediators of the immunosuppressive response in isolated AEC monocultures. Sustained, physiologically appropriate insulin secretion was observed in both islets and islet/AEC constructs. Activation of resting PBL proliferation occurred on exposure to human islets alone but this response was significantly (p < 0.05) attenuated by the presence of AEC and AEC-conditioned medium. Mitogen (phytohaemagglutinin, 5 µg/ml)-induced PBL proliferation was sustained on contact with isolated islets but abrogated by AEC, conditioned medium, and the islet/AEC constructs. Immunocytochemical analysis of AEC monocultures identified a subpopulation of cells that expressed the proapoptosis protein Fas ligand. This study demonstrates that human islet/AEC constructs exhibit localized immunosuppressive properties with no impairment of ß-cell function. The data suggest that transplanted islets may benefit from the immune privilege status conferred on them as a consequence of their close proximity to human AEC. Such an approach may reduce the need for chronic systemic immunosuppression, thus making islet transplantation a more attractive treatment option for the management of insulin-dependent diabetes.

Preservation of glucose responsiveness in human islets maintained in a rotational cell culture system.

The diminution of glucose responsiveness in isolated human islets maintained under conventional static culture (CSC) conditions represents a major limitation for the long-term storage of islet tissue and precludes extensive study of beta (beta)-cell biology. In the present investigation, we examined the effect of culturing primary human islets in a rotational cell culture system (RCCS) to determine its' ability to sustain both the structural integrity and functional viability of these fragile cell constructs. Over a 10-day culture period both structural integrity and glucose-stimulated insulin release (GSIR) were preserved in islets maintained within the RCCS whilst those held under CSC conditions exhibited progressive fragmentation and rapid loss of secretory function. In addition, intentionally dissociated islet cells maintained within the RCCS demonstrated the ability to re-aggregate and form tight islet-like structures with enhanced secretory capacity compared to whole islets maintained in static culture. These findings suggest a novel use for the RCCS and illustrate its potential as an experimental tool for in vitro study of human islet/beta-cell physiology.