Xeno-Transplantation of macro-encapsulated islets and Pluripotent Stem Cell-Derived Pancreatic Progenitors without Immunosuppression.

Islet transplantation effectively treats diabetes but relies on immune suppression and is practically limited by the number of cadaveric islets available. An alternative cellular source is insulin-producing cells derived from pluripotent cell sources. Three animal cohorts were used in the current study to evaluate whether an oxygen-providing macro-encapsulation device, 'ßAIR', could function in conjunction with human embryonic stem cells (hESCs) and their derivatives. The first cohort received macro-encapsulated undifferentiated hESCs, a second cohort received hESCs differentiated to a pancreatic progenitor state with limited endocrine differentiation. A reference cohort received human islets. Macro-encapsulation devices were implanted subcutaneously and monitored for up to 4 months. Undifferentiated pluripotent stem cells did not form teratoma but underwent cell death following implantation. Human C-peptide (hC- peptide) was detectable in host serum one week after implantation for both other cohorts. hC-peptide levels decreasing over time but remained detectable up to the end of the study. Key factors associated with mature endocrine cells were observed in grafts recovered from cohorts containing islets and hESC-derivatives including C-peptide, insulin, glucagon and urocortin 3. We conclude that the 'ßAIR' macroencapsulation device is compatible with both human islets and pluripotent derivatives, but has a limited capability of sustaining undifferentiated pluripotent cells.

Implementation of a simplified method of islet isolation for allogeneic islet transplantation in cynomolgus monkeys.

OBJECTIVES: The present study describes a simple and cost-effective islet isolation procedure. Using this method, allogeneic islets reverse diabetes in cynomolgus monkeys. METHODS: Pancreatic tissue from 11 cynomolgus monkeys were digested, collected, and purified using a simplified method. Islet quantification, purity, viability, and glucose static incubation were conducted immediately after isolation. Five streptozotocin-induced monkeys with diabetes were transplanted intrahepatically, and liver biopsies from 3 of these monkeys were taken at different time points for histologic study. RESULTS: The mean (SD) of viability, purity, and static glucose incubation stimulation index were 94.4% (2.3%), 91.8% (3.4%), and 2.6 (1.7), respectively. Monkeys who received a mean (SD) dose of 19,968 (2273) islet equivalent per kilogram (n = 4) from 2 to 3 donors who achieved prolonged normoglycemia (57-232 days), whereas the single monkey who received an islet dose of 8000 islet equivalent per kilogram did not experience diabetes reversal. Immunohistochemical assessment of the liver biopsies taken from the monkeys with normoglycemia revealed an insulin- and glucagon-positive islet graft for up to 6 months with minimal peri-islet inflammatory infiltration. CONCLUSIONS: This study demonstrates that cynomolgus monkey islets can be successfully and efficiently harvested using a simple isolation method, and these islets can restore normoglycemia in monkeys with diabetes.

Overexpression of E2F3 promotes proliferation of functional human ß cells without induction of apoptosis.

The mechanisms that control proliferation, or lack thereof, in adult human ß cells are poorly understood. Controlled induction of proliferation could dramatically expand the clinical application of islet cell transplantation and represents an important component of regenerative approaches to a functional cure of diabetes. Adult human ß cells are particularly resistant to common proliferative targets and often dedifferentiate during proliferation. Here we show that expression of the transcription factor E2F3 has a role in regulating ß-cell quiescence and proliferation. We found human islets have virtually no expression of the pro-proliferative G 1/S transcription factors E2F1-3, but an abundance of inhibitory E2Fs 4-6. In proliferative human insulinomas, inhibitory E2Fs were absent, while E2F3 is expressed. Using this pattern as a "roadmap" for proliferation, we demonstrated that ectopic expression of nuclear E2F3 induced significant expansion of insulin-positive cells in both rat and human islets. These cells did not undergo apoptosis and retained their glucose-responsive insulin secretion, showing the ability to reverse diabetes in mice. Our results suggest that E2F4-6 may help maintain quiescence in human ß cells and identify E2F3 as a novel target to induce proliferation of functional ß cells. Refinement of this approach may increase the islets available for cell-based therapies and research and could provide important cues for understanding in vivo proliferation of ß cells.

Use of glucagon-like peptide-1 agonists to improve islet graft performance.

Human islet transplantation is an effective and promising therapy for type I diabetes. However, long-term insulin independence is both difficult to achieve and inconsistent. De novo or early administration of incretin-based drugs is being explored for improving islet engraftment. In addition to its glucose-dependent insulinotropic effects, incretins also lower postprandial glucose excursion by inhibiting glucagon secretion, delaying gastric emptying, and can protect beta-cell function. Incretin therapy has so far proven clinically safe and tolerable with little hypoglycemic risk. The present review aims to highlight the new frontiers in research involving incretins from both in vitro and in vivo animal studies in the field of islet transplant. It also provides an overview of the current clinical status of incretin usage in islet transplantation in the management of type I diabetes.

Stroke in Spain: epidemiologic incidence and patterns; a health sentinel network study.

INTRODUCTION: Cerebrovascular disease is among the 4 main causes of mortality in Spain. The objective of this study was to estimate the incidence of stroke and to describe the principal risk factors and other clinical and epidemiologic patterns found in patients. METHODS: Doctors from the Spanish sentinel health network recorded the episodes of acute cerebrovascular diseases in 2005 in a population of 201,205 inhabitants older than 14 years. The information of the patients (age and sex) and the episode (e.g., duration, symptoms, origin, medical attention, risk factors) was collected on a standard form. RESULTS: The estimated incidence rate of stroke was 141 cases per 100,000 inhabitants (confidence interval [CI] 95%: 125-158), 134 (95% CI: 112-157) in women and 148 (95% CI: 124-172) in men. The incidence increases significantly from the age of 65 years and men younger than this have higher rates than women. In all, 12% of patients with stroke die within the first 24 hours. CONCLUSIONS: Incidence of cerebrovascular disease in Spain is below that recorded in other countries. There is no difference according to sex, but incidence among young and middle-aged men is greater than that among women. Significant variations from some population groups to others are observed, maybe because of the difference in the prevalence of risk factors.

Nicotinamide induces differentiation of embryonic stem cells into insulin-secreting cells.

The poly(ADP-ribose) polymerase (PARP) inhibitor, nicotinamide, induces differentiation and maturation of fetal pancreatic cells. In addition, we have previously reported evidence that nicotinamide increases the insulin content of cells differentiated from embryonic stem (ES) cells, but the possibility of nicotinamide acting as a differentiating agent on its own has never been completely explored. Islet cell differentiation was studied by: (i) X-gal staining after neomycin selection; (ii) BrdU studies; (iii) single and double immunohistochemistry for insulin, C-peptide and Glut-2; (iv) insulin and C-peptide content and secretion assays; and (v) transplantation of differentiated cells, under the kidney capsule, into streptozotocin (STZ)-diabetic mice. Here we show that undifferentiated mouse ES cells treated with nicotinamide: (i) showed an 80% decrease in cell proliferation; (ii) co-expressed insulin, C-peptide and Glut-2; (iii) had values of insulin and C-peptide corresponding to 10% of normal mouse islets; (iv) released insulin and C-peptide in response to stimulatory glucose concentrations; and (v) after transplantation into diabetic mice, normalized blood glucose levels over 7 weeks. Our data indicate that nicotinamide decreases ES cell proliferation and induces differentiation into insulin-secreting cells. Both aspects are very important when thinking about cell therapy for the treatment of diabetes based on ES cells.

Induction of differentiation of embryonic stem cells into insulin-secreting cells by fetal soluble factors.

Cell signals produced during pancreas embryogenesis regulate pancreatic differentiation. We show that the developing pancreas releases soluble factors responsible for in vitro endocrine pancreatic differentiation from embryonic stem cells (ESCs). A mouse D3 ESC line was transfected with a human insulin promoter/betageo/phosphoglycerate kinase-hygromycin-resistant construct. To direct differentiation, cells were cultured for 7 days to form embryoid bodies and then plated for an additional 7 days. During this 14-day period, besides eliminating leukemia inhibitory factor, cells were cultured in low serum concentration with the addition of conditioned media from embryonic day-16.5 pancreatic buds. Islet cell differentiation was studied by the following: (a) X-gal staining after neomycin selection, (b) BrdU (bro-modeoxyuridine) studies, (c) simple and double immunohistochemistry for insulin, C-peptide, and glucose transporter 2 (Glut-2), (d) reverse transcription-polymerase chain reaction for insulin and pancreas duodenum homeobox 1 (PDX-1), (e) insulin and C-peptide content and secretion assays, (f) intraperitoneal glucose tolerance test, (g) electrophysiology (patch-clamp studies in inside-out configuration), and (h) transplantation of differentiated cells under the kidney capsule of streptozotocin-diabetic mice. The differentiated ESCs showed the following: changes in the mRNA levels of insulin and PDX-1; coexpression of insulin, C-peptide, and Glut-2; glucose and tolbutamide-dependent insulin and C-peptide release; K-channel activity regulated by ATP; and normalization of blood glucose levels after transplantation into diabetic mice and hyperglycemia after graft removal. In this study, we establish a battery of techniques that could be used together to properly characterize islet cell differentiation. Moreover, identification of factors released by the developing pancreas may be instrumental in engineering beta cells from stem cells.