Use of in vitro-generated, stem cell-derived islets to cure type 1 diabetes: how close are we?

Recent successes in treating type 1 diabetic patients with islet transplantation portends a future need for an increase in available islets. Ductal structures of the adult pancreas contain multipotent stem cells that, under the proper in vitro conditions, can both self-renew and differentiate into functional islets of Langerhans. In vitro-generated islets exhibit temporal changes in mRNA transcripts for islet-associated markers as well as regulated insulin responses following glucose challenge. When implanted into diabetic mice, in vitro-generated islets induce neovascularization and reverse insulin-dependent diabetes. The possibility of growing functional endocrine pancreas from stem cells provides new opportunities to produce large numbers of islets, even autologous islets, for use as implants.

Pancreatic stem cells: building blocks for a better surrogate islet to treat type 1 diabetes.

Type 1, insulin-dependent, diabetes is one of the more costly chronic diseases of children, adolescents and adults in Europe and North America. While routine insulin injections currently provide diabetic patients with their daily insulin requirements, blood glucose excursions are common, leading eventually to microvascular and macrovascular complications and early death. A 'cure' for Type 1 diabetes relies on replacement of the beta-cell mass which, today, is accomplished by pancreas transplants or islets of Langerhans implants. Recent advances in the isolation of stem cells that possess the capacity to differentiate to functional endocrine pancreas provide new opportunities to produce large numbers of islets, even autologous islets, that can be used as implants. We discuss briefly this new technology and its meaning for diabetes.

Pancreatic stem cells: a therapeutic agent that may offer the best approach for curing type 1 diabetes.

Type 1 (insulin-dependent) diabetes is one of the most costly chronic diseases of children and adolescents in North America and Europe. It occurs in genetically predisposed individuals when the immune system attacks and destroys specifically the insulin-producing beta cells of the pancreatic islets of Langerhans. Although routine insulin injections can provide diabetic patients their daily insulin requirements, non-compliance commonly results in blood glucose excursions that eventually lead to microvascular and macrovascular complications and early death. The only real 'cure' for type 1 diabetes is replacement of the beta-cell mass which, today, is either an ectopancreatic transplant or an islet of Langerhans implant. Two new developments may offer additional options: surrogate, non-endocrine cells genetically modified to secrete insulin in response to high blood glucose levels; and stem cells that possess the capacity to differentiate to endocrine pancreas. In this short review, we discuss the efforts currently being made to regulate pancreatic stem cell growth in order to produce large numbers of functional islets that can be used as implants. Hopefully, autologous stem cell-derived islet cell implants without lifelong immunosuppressive therapy may one day be realized.

Reversal of insulin-dependent diabetes using islets generated in vitro from pancreatic stem cells.

Ductal structures of the adult pancreas contain stem cells that differentiate into islets of Langerhans. Here, we grew pancreatic ductal epithelial cells isolated from prediabetic adult non-obese diabetic mice in long-term cultures, where they were induced to produce functioning islets containing alpha, beta and delta cells. These in vitro-generated islets showed temporal changes in mRNA transcripts for islet cell-associated differentiation markers, responded in vitro to glucose challenge, and reversed insulin-dependent diabetes after being implanted into diabetic non-obese diabetic mice. The ability to control growth and differentiation of islet stem cells provides an abundant islet source for beta-cell reconstitution in type I diabetes.

Immunization therapies in the prevention of diabetes.

Insulin-dependent diabetes (IDD), being an autoimmune disease, offers several opportunities for immunological interventions that may result either in the reduction of disease severity or in delaying diabetes onset. Among the various experimental preventative approaches, parenteral immunization with islet-specific autoantigens appears to be practically simpler and promising. We have previously shown that immunization with insulin, insulin B chain and B chain epitope (p9-23), but not insulin A chain, in incomplete Freund's adjuvant (IFA) and in alum (with B chain) delayed/prevented diabetes onset in NOD mice. Here we demonstrate the protective efficacy of affinity purified GAD65 in IFA. While both insulin B chain and GAD65 significantly delayed the onset of diabetes (P=0.001), a recently described tyrosine phosphatase (IA-2) antigen did not (P=0.38). Interestingly, B chain immunization reduced the incidence of cyclophosphamide (CY)-accelerated diabetes by about 50-55%. We also provide further evidence that B chain, upon increased adsorption to alum, could improve on its protective capacity in NOD mice.

Insulin in diabetes prevention.

Insulin-dependent diabetes (IDD) is a chronic immune-endocrine disease in which there is a progressive destruction of insulin-secreting pancreatic beta cells, caused primarily by autoreactive T cells. Many islet cell proteins including insulin, glutamic acid decarboxylase, and tyrosine phosphatase antigens (IA-2) are targeted by the autoimmune responses in IDD patients. Since its discovery 75 years ago, insulin has been the major player in the clinical management of hyperglycaemia in these patients. The morbidity and mortality associated with IDD derives mainly from the complications of the disease. However, routine insulin injections seldom achieve a consistent, near-normal glucose level, where multiple daily doses of the hormone involve considerable restrictions to a normal lifestyle. In terms of economics, the management of diabetes is expensive, and in the USA diabetes alone accounts for one seventh of the healthcare budget. These clinical, lifestyle and economic issues emphasize the need to investigate alternative preventative measures in IDD treatment. Recent reports suggest a pivotal role for insulin in various aspects of the immune system. In this study, insulin and B-chain were used to modulate autoimmune responses in non-obese diabetic mice, findings which have therapeutic implications in man.

Effect of oral and intravenous insulin and glutamic acid decarboxylase in NOD mice.

Islet cell antigens have been administered orally and intravenously (I.V.) to NOD mice to assess their abilities to protect from or delay the onset of diabetes, and thereby provide insights that may have therapeutic implications in human trials. Whereas we and others have observed a delay in the onset of diabetes in NOD mice that have been fed with insulin from early life, we report here for the first time that feedings with porcine GAD65 alone (p = 0.226) or in combination with insulin (p = 0.011), have anti-diabetic effects in a prolonged study period (>400 days). While antigen-specific inhibitions of in vitro lymphocytic proliferation responses were seen (p < 0.05), antibody levels were unaffected by oral antigen treatments. IFN-gamma mRNA levels were downregulated in the islet infiltrates following oral antigen treatments while IL-2 and TNF-beta were expressed in all instances. We also observed that I.V. human recombinant GAD65, and porcine GAD given at weaning, delayed diabetes onset (p = 0.004) while similar treatments with a variety of inactive insulin preparations were generally ineffective. These findings thus indicate varying effects of oral and I.V. autoantigen administrations on the development of diabetes in NOD mice, and describe the immunological processes induced by oral autoantigen treatments.

Immunotherapies in diabetes.

Insulin-dependent diabetes (IDD) is a serious, life-long disease replete with life-threatening complications that are not preventable through conventional insulin replacement therapies. The prolonged prodromal period of autoimmunity to beta cell antigens offers multiple intervention opportunities. These can target different steps that precede final destruction of insulin-secreting beta cells and clinical onset of the disease. All current and proposed immunotherapies are experimental procedures that have proven to be protective in animal models, especially the nonobese diabetic (NOD) mouse. This brief review deals with a selected list of nonspecific and autoantigen-specific immunotherapies that may bring hope in the near future to individuals at risk of developing the disease. None are yet proven to be effective in humans.

Quantitative thresholds of MHC class II I-E expressed on hemopoietically derived antigen-presenting cells in transgenic NOD/Lt mice determine level of diabetes resistance and indicate mechanism of protection.

Two homozygous lines of transgenic NOD/Lt mice expressing MHC class II I-E molecules at quantitatively different levels were utilized to study mechanisms of I-E-mediated diabetes prevention. In line 12, I-E expression on APC at levels comparable with that in BALB/cByJ controls conferred only partial diabetes resistance. In line 5, greater than normal I-E levels on APC correlated with nearly complete resistance. Levels of endogenously encoded I-Ag7 correlated inversely with transgene-induced I-E expression. T cell transfer experiments into NOD/severe combined immunodeficient mice demonstrated the presence of pathogenic T cells in I-E+ donors, and that continuous expression of I-E on hemopoietically derived APC was required to block their pathogenic function. T cells from transgenic and nontransgenic NOD/Lt mice primed in vivo against the beta cell autoantigen 65-kDa isoform of glutamic acid decarboxylase (GAD65) and two peptides derived from this protein proliferated when restimulated in vitro. However, reverse-transcription PCR and ELISA measurements of cytokine mRNA and protein levels showed that the GAD65-reactive T cells from both line 5 and line 12 mice produced higher levels of IL-4 and lower levels of IFN-gamma than similar T cells from standard NOD/Lt mice. Thus, the inverse relationship between I-E and I-Ag7 expression was associated with qualitative differences in T cell responses to putative beta cell autoantigens. Collectively, these data indicate quantitative increases in I-E expression on APC may block insulin-dependent diabetes mellitus by altering the balance of cytokines produced by beta cell autoreactive T cells.

Antigen based therapies to prevent diabetes in NOD mice.

Interventional approaches that have been successful in delaying insulin-dependent diabetes mellitus (IDDM) using antigen-based immunotherapies include parenteral immunization. It has potential for clinical application provided that effective adjuvants suitable for human use can be found. We have previously shown that immunization with insulin and insulin B chain but not A chain in incomplete Freund's adjuvant (IFA) prevented diabetes by reducing IFN-gamma mRNA in the insulitis lesions. In this paper we show that the insulin B chain peptide (p9-23) contain the most protective epitope. Immunization with selected GAD peptides was ineffective. Immunization with B chain but not A chain using alum as adjuvant delayed diabetes onset (P = 0.012), whereas administration of alum alone was not protective. When Diphtheria-Tetanus toxoid-Acellular Pertussis (DTP) vaccine was used as the adjuvant vehicle, DTP itself induced significant protection (P < 0.003) which was associated with a Th2-like cytokine producing insulitis profile, IL-4 driven IgG1 antibody responses to insulin, GAD in the periphery and an augmentation of the autoimmune response to GAD. The anti-diabetic effect of DTP was enhanced when given with insulin B chain. These results encourage consideration of an approach using alum/DTP and insulin B chain immunization in clinical trials.