Michael J. Riedel

Maturation of Human Embryonic Stem Cell–Derived Pancreatic Progenitors Into Functional Islets Capable of Treating Pre-existing Diabetes in Mice

Diabetes is a chronic debilitating disease that results from insufficient production of insulin from pancreatic β-cells. Islet cell replacement can effectively treat diabetes but is currently severely limited by the reliance upon cadaveric donor tissue. We have developed a protocol to efficiently differentiate commercially available human embryonic stem cells (hESCs) in vitro into a highly enriched PDX1+ pancreatic progenitor cell population that further develops in vivo to mature pancreatic endocrine cells. Immature pancreatic precursor cells were transplanted into immunodeficient mice with streptozotocin-induced diabetes, and glycemia was initially controlled with exogenous insulin. As graft-derived insulin levels increased over time, diabetic mice were weaned from exogenous insulin and human C-peptide secretion was eventually regulated by meal and glucose challenges. Similar differentiation of pancreatic precursor cells was observed after transplant in immunodeficient rats. Throughout the in vivo maturation period hESC-derived endocrine cells exhibited gene and protein expression profiles that were remarkably similar to the developing human fetal pancreas. Our findings support the feasibility of using differentiated hESCs as an alternative to cadaveric islets for treating patients with diabetes.

Production of Functional Glucagon-Secreting α-Cells From Human Embryonic Stem Cells

OBJECTIVE Differentiation of human embryonic stem (hES) cells to fully developed cell types holds great therapeutic promise. Despite significant progress, the conversion of hES cells to stable, fully differentiated endocrine cells that exhibit physiologically regulated hormone secretion has not yet been achieved. Here we describe an efficient differentiation protocol for the in vitro conversion of hES cells to functional glucagon-producing α- cells. RESEARCH DESIGN AND METHODS Using a combination of small molecule screening and empirical testing, we developed a six-stage differentiation protocol for creating functional α-cells. An extensive in vitro and in vivo characterization of the differentiated cells was performed. RESULTS A high rate of synaptophysin expression (>75%) and robust expression of glucagon and the α-cell transcription factor ARX was achieved. After a transient polyhormonal state in which cells coexpress glucagon and insulin, maturation in vitro or in vivo resulted in depletion of insulin and other β-cell markers with concomitant enrichment of α-cell markers. After transplantation, these cells secreted fully processed, biologically active glucagon in response to physiologic stimuli including prolonged fasting and amino acid challenge. Moreover, glucagon release from transplanted cells was sufficient to reduce demand for pancreatic glucagon, resulting in a significant decrease in pancreatic α-cell mass. CONCLUSIONS These results indicate that fully differentiated pancreatic endocrine cells can be created via stepwise differentiation of hES cells. These cells may serve as a useful screening tool for the identification of compounds that modulate glucagon secretion as well as those that promote the transdifferentiation of α-cells to β-cells.

In Vivo Expression of HGF/NK1 and GLP-1 From dsAAV Vectors Enhances Pancreatic β-Cell Proliferation and Improves Pathology in the db/db Mouse Model of Diabetes

OBJECTIVE The purpose of the current study was to determine whether double-stranded adeno-associated virus (dsAAV)-mediated in vivo expression of β-cell growth factors, glucagon-like peptide-1 (GLP-1) and the NK1 fragment of hepatocyte growth factor (HGF/NK1) in β-cells, improves pathology in the db/db mouse model of type 2 diabetes. RESEARCH DESIGN AND METHODS The glucoregulatory actions of GLP-1 and full-length HGF are well characterized. Here, we test the ability of HGF/NK1 to induce proliferation of exogenous islets and MIN6 β-cells. In addition, we target both GLP-1 and HGF/NK1 to endogenous β-cells using dsAAV vectors containing the mouse insulin-II promoter. We compare the abilities of these gene products to induce islet proliferation in vitro and in vivo and characterize their abilities to regulate diabetes after AAV-mediated delivery to endogenous islets of db/db mice. RESULTS Recombinant HGF/NK1 induces proliferation of isolated islets, and dsAAV-mediated expression of both GLP-1 and HGF/NK1 induces significant β-cell proliferation in vivo. Furthermore, both GLP-1 and HGF/NK1 expressed from dsAAV vectors enhance β-cell mass and insulin secretion in vivo and significantly delay the onset of hyperglycemia in db/db mice. CONCLUSIONS A single treatment with dsAAV vectors expressing GLP-1 or HGF/NK1 enhances islet growth and significantly improves pathology in a mouse model of type 2 diabetes. This represents the first example of a successful use of HGF/NK1 for diabetes therapy, providing support for direct AAV-mediated in vivo delivery of β-cell growth factors as a novel therapeutic strategy for the treatment of type 2 diabetes.

DsAAV8-mediated expression of glucagon-like peptide-1 in pancreatic beta-cells ameliorates streptozotocin-induced diabetes

Glucagon-like peptide-1 (GLP-1) is an incretin hormone that performs a wide array of well-characterized antidiabetic actions, including stimulation of glucose-dependent insulin secretion, upregulation of insulin gene expression and improvements in beta-cell survival. GLP-1-receptor agonists have been developed for treatment of diabetes; however, the short biological half-lives of these peptide-based therapeutics requires that frequent injections be administered to maintain sufficient circulating levels. Thus, novel methods of delivering GLP-1 remain an important avenue of active research. It has recently been demonstrated that self-complimentary, double-stranded, adeno-associated virus serotype-8 (DsAAV8) can efficiently transduce pancreatic beta-cells in vivo, resulting in long-term transgene expression. In this study, we engineered a DsAAV8 vector containing a GLP-1 transgene driven by the mouse insulin-II promoter (MIP). Biological activity of the GLP-1 produced from this transgene was assessed using a luciferase-based bioassay. DsAAV8–MIP–GLP-1 was delivered via intraperitoneal injection and beta-cell damage induced by multiple low dose streptozotocin (STZ) administration. Glucose tolerance was assessed following intraperitoneal glucose injections and beta-cell proliferation measured by PCNA expression. Expression of GLP-1 in Min6 beta-cells resulted in glucose-dependent secretion of biologically active GLP-1. Intraperitoneal delivery of DsAAV8–MIP–GLP-1 to mice led to localized GLP-1 expression in beta-cells and protection against development of diabetes induced by multiple low-dose STZ administration. This protection was associated with significant increase in beta-cell proliferation. Results from this study indicate that expression and secretion of GLP-1 from beta-cells in vivo via DsAAV8 represents a novel therapeutic strategy for treatment of diabetes.

Inhibition of β-Cell Sodium-Calcium Exchange Enhances Glucose-Dependent Elevations in Cytoplasmic Calcium and Insulin Secretion

OBJECTIVE The sodium-calcium exchanger isoform 1 (NCX1) regulates cytoplasmic calcium (Ca2+c) required for insulin secretion in β-cells. NCX1 is alternatively spliced, resulting in the expression of splice variants in different tissues such as NCX1.3 and -1.7 in β-cells. As pharmacological inhibitors of NCX1 splice variants are in development, the pharmacological profile of β-cell NCX1.3 and -1.7 and the cellular effects of NCX1 inhibition were investigated. RESEARCH DESIGN AND METHODS The patch-clamp technique was used to examine the pharmacological profile of the NCX1 inhibitor KB-R7943 on recombinant NCX1.3 and -1.7 activity. Ca2+ imaging and membrane capacitance were used to assess the effects of KB-R7943 on Ca2+c and insulin secretion in mouse and human β-cells and islets. RESULTS NCX1.3 and -1.7 calcium extrusion (forward-mode) activity was ∼16-fold more sensitive to KB-R7943 inhibition compared with cardiac NCX1.1 (IC50s = 2.9 and 2.4 vs. 43.0 μmol/l, respectively). In single mouse/human β-cells, 1 μmol/l KB-R7943 increased insulin granule exocytosis but was without effect on α-cell glucagon granule exocytosis. KB-R7943 also augmented sulfonylurea and glucose-stimulated Ca2+c levels and insulin secretion in mouse and human islets, although KB-R7943 was without effect under nonstimulated conditions. CONCLUSIONS Islet NCX1 splice variants display a markedly greater sensitivity to pharmacological inhibition than the cardiac NCX1.1 splice variant. NCX1 inhibition resulted in glucose-dependent increases in Ca2+c and insulin secretion in mouse and human islets. Thus, we identify β-cell NCX1 splice variants as targets for the development of novel glucose-sensitive insulinotropic drugs for type 2 diabetes.

Splice Variant-Dependent Regulation of β-Cell Sodium-Calcium Exchange by Acyl-Coenzyme As

The sodium-calcium exchanger isoform 1 (NCX1) is intimately involved in the regulation of calcium (Ca(2+)) homeostasis in many tissues including excitation-secretion coupling in pancreatic beta-cells. Our group has previously found that intracellular long-chain acyl-coenzyme As (acyl CoAs) are potent regulators of the cardiac NCX1.1 splice variant. Despite this, little is known about the biophysical properties of beta-cell NCX1 splice variants and the effects of intracellular modulators on their important physiological function in health and disease. Here, we show that the forward-mode activity of beta-cell NCX1 splice variants is differentially modulated by acyl-CoAs and is dependent both upon the intrinsic biophysical properties of the particular NCX1 splice variant as well as the side chain length and degree of saturation of the acyl-CoA moiety. Notably, saturated long-chain acyl-CoAs increased both peak and total NCX1 activity, whereas polyunsaturated long-chain acyl-CoAs did not show this effect. Furthermore, we have identified the exon within the alternative splicing region that bestows sensitivity to acyl-CoAs. We conclude that the physiologically relevant forward-mode activity of NCX1 splice variants expressed in the pancreatic beta-cell are sensitive to acyl-CoAs of different saturation and alterations in intracellular acyl-CoA levels may ultimately lead to defects in Ca(2+)-mediated exocytosis and insulin secretion.

Cellular reprogramming of human amniotic fluid cells to express insulin.

Islet transplantation represents a potential cure for type 1 diabetes; however, a lack of sufficient donor material limits its clinical use. To address the shortfall of islet availability, surrogate insulin-producing cells are sought. Studies suggest that human amniotic fluid (hAF) contains multipotent progenitor cells capable of differentiating to all three germ layers. Here, we used high-content, live-cell imaging to assess the ability to reprogram hAF cells towards a beta cell phenotype. A fluorescent reporter system was developed where DsRed express (DSRE) expression is driven by the human insulin promoter. Using integrative lentiviral technology, we created stable reporter hAF cells that could be routinely monitored for insulin promoter activation. These cells were subjected to combinatorial high-content screening using adenoviral-mediated expression of up to six transcription factors important for beta cell development. Cells were monitored for DSRE expression which revealed an optimal combination of the transcription factors required to induce insulin gene expression in hAF cells. These optimally induced cells were examined for expression of additional beta cell transcription factors and proteins involved in glucose sensing and insulin processing. RT-qPCR revealed very low level expression of insulin that was ultimately insufficient to reverse streptozotocin-induced diabetes following sub-capsular kidney transplantation. High-content, live-cell imaging using fluorescent reporter cells provides a convenient method for repeated assessment of cellular reprogramming. hAF cells could be reprogrammed to express key beta cell proteins, however insulin gene expression was insufficient to reverse hyperglycemia in diabetic animals.

Treatment of diabetes with glucagon-like peptide-1 gene therapy.

Importance of the field: Glucagon-like peptide (GLP)-1 receptor agonists are in widespread clinical use for the treatment of diabetes. While effective, these peptides require frequent injections to maintain efficacy. Therefore, alternative delivery methods including gene therapy are currently being evaluated. Areas covered in this review: Here, we review the biology of GLP-1, evidence supporting the clinical use of the native peptide as well as synthetic GLP-1 receptor agonists, and the rationale for their delivery by gene therapy. We then review progress made in the field of GLP-1 gene therapy for both type 1 and type 2 diabetes. What the reader will gain: Efforts to improve the biological half-life of GLP-1 receptor agonists are discussed. We focus on the development of both viral and non-viral gene delivery methods, highlighting vector designs and the strengths and weaknesses of these approaches. We also discuss the utility of targeting regulated GLP-1 production to tissues including the liver, muscle, islet and gut. Take home message: GLP-1 is a natural peptide possessing several actions that effectively combat diabetes. Current delivery methods for GLP-1-based drugs are cumbersome and do not recapitulate the normal secretion pattern of the native hormone. Gene therapy offers a useful method for directing long-term production and secretion of the native peptide. Targeted production of GLP-1 using tissue-specific promoters and delivery methods may improve therapeutic efficacy, while also eliminating the burden of frequent injections.

Elevation in Intracellular Long-Chain Acyl-Coenzyme A Esters Lead to Reduced β-Cell Excitability via Activation of Adenosine 5′-Triphosphate-Sensitive Potassium Channels

Closure of pancreatic beta-cell ATP-sensitive potassium (K(ATP)) channels links glucose metabolism to electrical activity and insulin secretion. It is now known that saturated, but not polyunsaturated, long-chain acyl-coenyzme A esters (acyl-CoAs) can potently activate K(ATP) channels when superfused directly across excised membrane patches, suggesting a plausible mechanism to account for reduced beta-cell excitability and insulin secretion observed in obesity and type 2 diabetes. However, reduced beta-cell excitability due to elevation of endogenous saturated acyl-CoAs has not been confirmed in intact pancreatic beta-cells. To test this notion directly, endogenous acyl-CoA levels were elevated within primary mouse beta-cells using virally delivered overexpression of long-chain acyl-CoA synthetase-1 (AdACSL-1), and the effects on beta-cell K(ATP) channel activity and cell excitability was assessed using the perforated whole-cell and cell-attached patch-clamp technique. Data indicated a significant increase in K(ATP) channel activity in AdACSL-1-infected beta-cells cultured in medium supplemented with palmitate/oleate but not with the polyunsaturated fat linoleate. No changes in the ATP/ADP ratio were observed in any of the groups. Furthermore, AdACSL-1-infected beta-cells (with palmitate/oleate) showed a significant decrease in electrical responsiveness to glucose and tolbutamide and a hyperpolarized resting membrane potential at 5 mm glucose. These results suggest a direct link between intracellular fatty ester accumulation and K(ATP) channel activation, which may contribute to beta-cell dysfunction in type 2 diabetes.

dsAAV8-mediated gene transfer and β-cell expression of IL-4 and β-cell growth factors are capable of reversing early-onset diabetes in NOD mice

Type-I diabetes is a chronic disease mediated by autoimmune destruction of insulin-producing β-cells. Although progress has been made towards improving diabetes-associated pathologies and the quality of life for those living with diabetes, no therapy has been effective at eliminating disease manifestations or reversing disease progression. Here, we examined whether double-stranded adeno-associated virus serotype 8 (dsAAV8)-mediated gene delivery to endogenous β-cells of interleukin (IL)-4 in combination with β-cell growth factors can reverse early-onset diabetes in NOD mice. Our results demonstrate that a single treatment with dsAAV8 vectors expressing IL-4 in combination with glucagon-like peptide-1 or hepatocyte growth factor/NK1 under the regulation of the insulin promoter enhanced β-cell proliferation and survival in vivo, significantly delaying diabetes progression in NOD mice, and reversing disease in ∼10% of treated NOD mice. These results demonstrate the ability to reverse hyperglycemia in NOD mice with established diabetes by in vivo gene transfer to β-cells of immunomodulatory factors and β-cell growth factors.