Selection for CD26- and CD49A+ Cells From Pluripotent Stem Cells-Derived Islet-Like Clusters Improves Therapeutic Activity in Diabetic Mice.

Background: Cell therapy of diabetes aims at restoring the physiological control of blood glucose by transplantation of functional pancreatic islet cells. A potentially unlimited source of cells for such transplantations would be islet cells derived from an in vitro differentiation of human pluripotent stem cells (hESC/hiPSC). The islet-like clusters (ILC) produced by the known differentiation protocols contain various cell populations. Among these, the ß-cells that express both insulin and the transcription factor Nkx6.1 seem to be the most efficient to restore normoglycemia in diabetes animal models. Our aim was to find markers allowing selection of these efficient cells. Methods: Functional Cell-Capture Screening (FCCS) was used to identify markers that preferentially capture the cells expressing both insulin and Nkx6.1, from hESC-derived ILC cells. In order to test whether selection for such markers could improve cell therapy in diabetic mouse models, we used ILC produced from a clinical-grade line of hESC by a refined differentiation protocol adapted to up-scalable bioreactors. Re-aggregated MACS sorted cells were encapsulated in microspheres made of alginate modified to reduce foreign body reaction. Implantation was done intraperitoneally in STZ-treated C57BL/6 immuno-competent mice. Results: CD49A (integrin alpha1) was identified by FCCS as a marker for cells that express insulin (or C-peptide) as well as Nkx6.1 in ILC derived by hESC differentiation. The ILC fraction enriched in CD49A + cells rapidly reduced glycemia when implanted in diabetic mice, whereas mice receiving the CD49A depleted population remained highly diabetic. CD49A-enriched ILC cells also produced higher levels of human C-peptide in the blood of transplanted mice. However, the difference between CD49A-enriched and total ILC cells remained small. Another marker, CD26 (DPP4), was identified by FCCS as binding insulin-expressing cells which are Nkx6.1 negative. Depletion of CD26 + cells followed by enrichment for CD49A + cells increased insulin+/Nkx6.1+ cells fraction to ~70%. The CD26 - /CD49A + enriched ILC exhibited improved function over non-sorted ILC or CD49A + cells in diabetic mice and maintain prolonged blood C-peptide levels. Conclusions: Refining the composition of ILC differentiated from hPSC by negative selection to remove cells expressing CD26 and positive selection for CD49A expressing cells could enable more effective cell therapy of diabetes.

Long-term viability and function of transplanted islets macroencapsulated at high density are achieved by enhanced oxygen supply.

Transplantation of encapsulated islets can cure diabetes without immunosuppression, but oxygen supply limitations can cause failure. We investigated a retrievable macroencapsulation device wherein islets are encapsulated in a planar alginate slab and supplied with exogenous oxygen from a replenishable gas chamber. Translation to clinically-useful devices entails reduction of device size by increasing islet surface density, which requires increased gas chamber pO2. Here we show that islet surface density can be substantially increased safely by increasing gas chamber pO2 to a supraphysiological level that maintains all islets viable and functional. These levels were determined from measurements of pO2 profiles in islet-alginate slabs. Encapsulated islets implanted with surface density as high as 4,800 islet equivalents/cm3 in diabetic rats maintained normoglycemia for more than 7 months and provided near-normal intravenous glucose tolerance tests. Nearly 90% of the original viable tissue was recovered after device explantation. Damaged islets failed after progressively shorter times. The required values of gas chamber pO2 were predictable from a mathematical model of oxygen consumption and diffusion in the device. These results demonstrate feasibility of developing retrievable macroencapsulated devices small enough for clinical use and provide a firm basis for design of devices for testing in large animals and humans.

The efficacy of an immunoisolating membrane system for islet xenotransplantation in minipigs.

Developing a device that protects xenogeneic islets to allow treatment and potentially cure of diabetes in large mammals has been a major challenge in the past decade. Using xenogeneic islets for transplantation is required in light of donor shortage and the large number of diabetic patients that qualify for islet transplantation. Until now, however, host immunoreactivity against the xenogeneic graft has been a major drawback for the use of porcine islets. Our study demonstrates the applicability of a novel immunoprotective membrane that allows successful xenotransplantation of rat islets in diabetic minipigs without immunosuppressive therapy. Rat pancreatic islets were encapsulated in highly purified alginate and integrated into a plastic macrochamber covered by a poly-membrane for subcutaneous transplantation. Diabetic Sinclair pigs were transplanted and followed for up to 90 days. We demonstrated a persistent graft function and restoration of normoglycemia without the need for immunosuppressive therapy. This concept could potentially offer an attractive strategy for a more widespread islet replacement therapy that would restore endogenous insulin secretion in diabetic patients without the need for immunosuppressive drugs and may even open up an avenue for safe utilization of xenogeneic islet donors.

Enhanced oxygen supply improves islet viability in a new bioartificial pancreas.

The current epidemic of diabetes with its overwhelming burden on our healthcare system requires better therapeutic strategies. Here we present a promising novel approach for a curative strategy that may be accessible for all insulin-dependent diabetes patients. We designed a subcutaneous implantable bioartificial pancreas (BAP)-the "ß-Air"-that is able to overcome critical challenges in current clinical islet transplantation protocols: adequate oxygen supply to the graft and protection of donor islets against the host immune system. The system consists of islets of Langerhans immobilized in an alginate hydrogel, a gas chamber, a gas permeable membrane, an external membrane, and a mechanical support. The minimally invasive implantable device, refueled with oxygen via subdermally implanted access ports, completely normalized diabetic indicators of glycemic control (blood glucose intravenous glucose tolerance test and HbA1c) in streptozotocin-induced diabetic rats for periods up to 6 months. The functionality of the device was dependent on oxygen supply to the device as the grafts failed when oxygen supply was ceased. In addition, we showed that the device is immuno-protective as it allowed for survival of not only isografts but also of allografts. Histological examination of the explanted devices demonstrated morphologically and functionally intact islets; the surrounding tissue was without signs of inflammation and showed visual evidence of vasculature at the site of implantation. Further increase in islets loading density will justify the translation of the system to clinical trials, opening up the potential for a novel approach in diabetes therapy.

Improvement of islet function in a bioartificial pancreas by enhanced oxygen supply and growth hormone releasing hormone agonist.

Islet transplantation is a feasible therapeutic alternative for metabolically labile patients with type 1 diabetes. The primary therapeutic target is stable glycemic control and prevention of complications associated with diabetes by reconstitution of endogenous insulin secretion. However, critical shortage of donor organs, gradual loss in graft function over time, and chronic need for immunosuppression limit the indication for islet transplantation to a small group of patients. Here we present a promising approach to address these limitations by utilization of a macrochamber specially engineered for islet transplantation. The s.c. implantable device allows for controlled and adequate oxygen supply and provides immunological protection of donor islets against the host immune system. The minimally invasive implantable chamber normalized blood glucose in streptozotocin-induced diabetic rodents for up to 3 mo. Sufficient graft function depended on oxygen supply. Pretreatment with the growth hormone-releasing hormone (GHRH) agonist, JI-36, significantly enhanced graft function by improving glucose tolerance and increasing ß-cell insulin reserve in rats thereby allowing for a reduction of the islet mass required for metabolic control. As a result of hypervascularization of the tissue surrounding the device, no relevant delay in insulin response to glucose changes has been observed. Consequently, this system opens up a fundamental strategy for therapy of diabetes and may provide a promising avenue for future approaches to xenotransplantation.

Immobilized microalgal cells as an oxygen supply system for encapsulated pancreatic islets: a feasibility study.

Recently, a novel technique for oxygen supply to immunoisolated islets, which adopts the photosynthetic capacity of microalgae to generate oxygen, has been described. Illuminated alga cells, co-immobilized with islets in one compartment, were capable of restoring glucose-stimulated insulin secretion during perifusion with anoxic medium. In the present study, a new model system for photosynthetic oxygen supply to encapsulated islets, containing two separate compartments-one for oxygen-producing alga cells and the other for insulin-secreting pancreatic islets-is described. No insulin response to increasing glucose concentrations was found when encapsulated islets alone were perifused with oxygen-free medium. However, when the perifused chamber contained not only encapsulated islets, but also illuminated algae, immobilized in alginate, the islets showed twice the amount of insulin secretion in response to a high level of glucose (P < 0.01). This finding suggests that the level of photosynthetic oxygen generated in the algal compartment was sufficient to support the functional activity of the islets. Such a technology may offer the potential application for oxygen supply to various transplanted immunoisolated cells.

Photosynthetic oxygen generator for bioartificial pancreas.

Immunoisolation of pancreatic islets interrupts their vascular connections and results in severe cell hypoxia and dysfunction. This process is believed to be the major obstacle to a successful cure of diabetes by implantation of bioartificial pancreas. Here we describe a new technology for microalga-based, photosynthetic oxygen supply to encapsulated islets, in which a thermophylic strain of the unicellular alga Chlorella was used as a natural photosynthetic oxygen generator. Following determinations of the optimal number of alga cells required for compensation of islet respiration, an appropriate number of islets and algae were co-encapsulated in alginate and perifused with oxygen-free medium at increasing glucose concentrations. No insulin response to glucose was obtained in islets alone, or upon inactivation of photosynthesis by darkness. However, under illumination, photosynthetic- dependent oxygen generation induced higher glucose-stimulated insulin response when compared to normoxic perifusion. Such photosynthetic oxygen generation may have a potential application in development of various bioartificial tissues, in particular the endocrine pancreas.