Therapeutic effects of a non-ß cell bioartificial pancreas in diabetic mice.

BACKGROUND: Cell-based insulin therapies can potentially improve glycemic regulation in insulin-dependent diabetic patients. Enteroendocrine cells engineered to secrete recombinant insulin have exhibited glycemic efficacy, but have been primarily studied as uncontrollable growth systems in immune incompetent mice. Furthermore, reports suggest that suboptimal insulin secretion remains a barrier to expanded application. METHODS: Genetic and tissue engineering strategies were applied to improve recombinant insulin secretion from intestinal L-cells on both a per-cell and per-graft basis. Transduction of insulin-expressing GLUTag L-cells with lentivirus carrying an additional human insulin gene-enhanced secretion twofold. We infected cells with lentivirus expressing a luciferase reporter gene to track cell survival in vivo. To provide a growth-controlled and immune protective environment without affecting secretory capacity, cells were microencapsulated in barium alginate. Approximately 9×10(7) microencapsulated cells were injected intraperitoneally in immune competent streptozotocin-induced diabetic mice for therapeutic efficacy evaluation. RESULTS: Graft insulin secretion was increased to 16 to 24 mU insulin per day. Transient normoglycemia was achieved in treated mice two days after transplantation, and endogenous insulin was sufficient to sustain body weights of treated mice receiving minimal supplementation. CONCLUSION: Glycemic efficacy of a bioartificial pancreas based on insulin-secreting enteroendocrine cells is insufficient as a standalone therapy, despite enhancement of graft insulin secretion capacity. Supplemental strategies to alleviate secretion limitations should be pursued.

Intravitreal cell-based production of glucagon-like peptide-1.

BACKGROUND: To examine the efficacy and safety of an intravitreal cell-based production of glucagon-like peptide-1 (GLP-1) by intravitreally implanted and encapsulated cells. METHODS: The experimental study included 12 Sprague-Dawley rats. Four cell beads with a diameter of 600 µm were intravitreally implanted. Each bead contained 3,000 GLP-1-secreting cells, which were encapsulated by a barium cross-linked sodium alginate matrix. At baseline and at each of the follow-up examinations at Day 3, Day 7, and Day 14, 4, 3, 3, and 2 animals, respectively, were killed. The concentration of active GLP-1 in the vitreous body samples was determined by enzyme-linked immunosorbent assay. The retinas were histologically examined. RESULTS: The active GLP-1 concentration in the vitreous samples increased significantly after baseline (<5 pM) to a peak at Day 3 (287 ± 196 pM) and at Day 7 (238 ± 55 pM), before it decreased at Day 14 (70 ± 8 pM). The histologic examinations did not show signs of apoptosis or tissue destruction. CONCLUSION: The intravitreal application of beads containing alginate-encapsulated cells producing GLP-1 resulted in an intraocular production of GLP-1 with a significant increase in the intraocular GLP-1 concentration, without observed cytotoxic effects. An intravitreal cell-based drug therapy with GLP-1 appears feasible.

A cell-based approach for diabetes treatment using engineered non-beta cells.

BACKGROUND: Implantation of insulin-secreting cells has the potential to provide tight glycemic regulation in diabetic subjects. Implantation of cadaveric human islets in immunosuppressed human patients is currently applied at a very small scale. To overcome the limitations of tissue availability and recipient immunosuppression, encapsulation of nonautologous cells and use of potentially autologous nonislet cells, the latter engineered for insulin secretion, are being pursued. This article reports on recent findings with the implantation of tissue constructs containing enteroendocrine cells stably expressing recombinant insulin in diabetic mice. The concept of a dual recombinant hepatic and enteroendocrine cell system, which may better approximate the secretory response of islets, is discussed. METHODS: Mouse GLUTag-INS cells engineered to secrete human insulin were developed and incorporated in tissue constructs as reported previously. Constructs were implanted intraperitoneally in diabetic mice, and blood glucose levels, animal weights, and plasma insulin levels were measured at various time points. RESULTS: GLUTag-INS-containing tissue constructs secreted insulin preimplantation and postexplantation, and human insulin was detected in the plasma of diabetic mice. However, normoglycemia was not restored. CONCLUSIONS: A variety of cell types and of encapsulation methods to enhance immune acceptance of insulin-secreting grafts are being pursued. Recombinant enteroendocrine cells show promise, but it is likely that they need to be combined with recombinant hepatic cells to achieve glycemic normalization.

Genetically engineered K cells provide sufficient insulin to correct hyperglycemia in a nude murine model.

A gene therapy-based treatment of type 1 diabetes mellitus requires the development of a surrogate beta cell that can synthesize and secrete functionally active insulin in response to physiologically relevant changes in ambient glucose levels. In this study, the murine enteroendocrine cell line STC-1 was genetically modified by stable transfection. Two clone cells were selected (STC-1-2 and STC-1-14) that secreted the highest levels of insulin among the 22 clones expressing insulin from 0 to 157.2 microIU/ml/10(6) cells/d. After glucose concentration in the culture medium was increased from 1 mM to 10 mM, secreted insulin rose from 40.3+/-0.8 to 56.3+/-3.2 microIU/ml (STC-1-2), and from 10.8+/-0.8 to 23.6+/-2.3 microIU/ml (STC-1-14). After STC-1-14 cells were implanted into diabetic nude mice, their blood glucose levels were reduced to normal. Body weight loss was also ameliorated. Our data suggested that genetically engineered K cells secrete active insulin in a glucose-regulated manner, and in vivo study showed that hyperglycemia could be reversed by implantation of the cells, suggesting that the use of genetically engineered K cells to express human insulin might provide a glucose-regulated approach to treat diabetic hyperglycemia.

Engineered enteroendocrine cells secrete insulin in response to glucose and reverse hyperglycemia in diabetic mice.

Type 1 diabetes is a metabolic disorder caused by loss of insulin-producing pancreatic beta-cells. Expression of insulin in non-beta-cells to create beta-cell surrogates has been tried to treat type 1 diabetes. Enteroendocrine K cells have characteristics similar to pancreatic beta-cells, such as a glucose-sensing system and insulin-processing proteases. In this study, we genetically engineered an enteroendocrine cell line (STC-1) to express insulin under the control of the glucose-dependent insulinotropic polypeptide promoter. We screened clones and chose one, Gi-INS-7, based on its high production of insulin. Gi-INS-7 cells expressed glucose transporter 2 (GLUT2) and glucokinase (GK) and secreted insulin in response to elevated glucose levels in vitro. To determine whether Gi-INS-7 cells can control blood glucose levels in diabetic mice, we transplanted these cells under the kidney capsule of streptozotocin (STZ)-induced diabetic mice and found that blood glucose levels became normal within 2 weeks of transplantation. In addition, glucose tolerance tests in mice that became normoglycemic after transplantation with Gi-INS-7 cells showed that exogenous glucose was cleared appropriately. These results suggest that engineered K cells may be promising surrogate beta-cells for possible therapeutic use for the treatment of type 1 diabetes.