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.     

Isolation,Culture, and Imaging of Human Fetal Pancreatic Cell Clusters

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning endocrine cells, as evidenced by a significant increase in circulating human C-peptide following glucose stimulation^1-9. However in vitro, genesis of insulin producing cells from human fetal ICCs is low¹⁰; results reminiscent of recent experiments performed with human embryonic stem cells (hESC), a renewable source of cells that hold great promise as a potential therapeutic treatment for type 1 diabetes. Like ICCs, transplantation of partially differentiated hESC generate glucose responsive, insulin producing cells, but in vitro genesis of insulin producing cells from hESC is much less robust^11-17. A complete understanding of the factors that influence the growth and differentiation of endocrine precursor cells will likely require data generated from both ICCs and hESC. While a number of protocols exist to generate insulin producing cells from hESC in vitro^11-22, far fewer exist for ICCs^10,23,24. Part of that discrepancy likely comes from the difficulty of working with human fetal pancreas. Towards that end, we have continued to build upon existing methods to isolate fetal islets from human pancreases with gestational ages ranging from 12 to 23 weeks, grow the cells as a monolayer or in suspension, and image for cell proliferation, pancreatic markers and human hormones including glucagon and C-peptide. ICCs generated by the protocol described below result in C-peptide release after transplantation under the kidney capsule of nude mice that are similar to C-peptide levels obtained by transplantation of fresh tissue⁶. Although the examples presented here focus upon the pancreatic endoderm proliferation and β cell genesis, the protocol can be employed to study other aspects of pancreatic development, including exocrine, ductal, and other hormone producing cells.     

Differentiation of insulin-producing cells from human neural progenitor cells

BackgroundSuccess in islet-transplantation-based therapies for type 1 diabetes, coupled with a worldwide shortage of transplant-ready islets, has motivated efforts to develop renewable sources of islet-replacement tissue. Islets and neurons share features, including common developmental programs, and in some species brain neurons are the principal source of systemic insulin.Methods and FindingsHere we show that brain-derived human neural progenitor cells, exposed to a series of signals that regulate in vivo pancreatic islet development, form clusters of glucose-responsive insulin-producing cells (IPCs). During in vitro differentiation of neural progenitor cells with this novel method, genes encoding essential known in vivo regulators of pancreatic islet development were expressed. Following transplantation into immunocompromised mice, IPCs released insulin C-peptide upon glucose challenge, remained differentiated, and did not form detectable tumors.ConclusionProduction of IPCs solely through extracellular factor modulation in the absence of genetic manipulations may promote strategies to derive transplantable islet-replacement tissues from human neural progenitor cells and other types of multipotent human stem cells.     

Production of pancreatic hormone-expressing endocrine cells from human embryonic stem cells

Of paramount importance for the development of cell therapies to treat diabetes is the production of sufficient numbers of pancreatic endocrine cells that function similarly to primary islets. We have developed a differentiation process that converts human embryonic stem (hES) cells to endocrine cells capable of synthesizing the pancreatic hormones insulin, glucagon, somatostatin, pancreatic polypeptide and ghrelin. This process mimics in vivo pancreatic organogenesis by directing cells through stages resembling definitive endoderm, gut-tube endoderm, pancreatic endoderm and endocrine precursor--en route to cells that express endocrine hormones. The hES cell-derived insulin-expressing cells have an insulin content approaching that of adult islets. Similar to fetal beta-cells, they release C-peptide in response to multiple secretory stimuli, but only minimally to glucose. Production of these hES cell-derived endocrine cells may represent a critical step in the development of a renewable source of cells for diabetes cell therapy.     

Induction of human umbilical cord blood-derived stem cells with embryonic stem cell phenotypes into insulin producing islet-like structure

Success in islet-transplantation-based therapies for type I diabetes, coupled with a worldwide shortage of transplant-ready islets, has motivated efforts to develop renewable sources of islet-replacement tissue. Embryonic stem cells (ESCs) have been successfully induced into insulin producing islet-like structure in several studies. However, the source of the ESCs has presented ethical and technical concerns. Here, we isolated a population of stem cells from human cord blood (UCB), which expressed embryo stage specific maker, SSEA-4, and the multi-potential stem cell marker, Oct4. Subsequently, we successfully induced them into insulin-producing islet-like structures, which co-express insulin and C-peptide. These findings might have a significant potential to advance human UCB derived stem-cell-based therapeutics for diabetes.     

Islet-like cell aggregates generated from human adipose tissue derived stem cells ameliorate experimental diabetes in mice

Background

Type 1 Diabetes Mellitus is caused by auto immune destruction of insulin producing beta cells in the pancreas. Currently available treatments include transplantation of isolated islets from donor pancreas to the patient. However, this method is limited by inadequate means of immuno-suppression to prevent islet rejection and importantly, limited supply of islets for transplantation. Autologous adult stem cells are now considered for cell replacement therapy in diabetes as it has the potential to generate neo-islets which are genetically part of the treated individual. Adopting methods of islet encapsulation in immuno-isolatory devices would eliminate the need for immuno-suppressants.

Methodology/Principal Findings

In the present study we explore the potential of human adipose tissue derived adult stem cells (h-ASCs) to differentiate into functional islet like cell aggregates (ICAs). Our stage specific differentiation protocol permit the conversion of mesodermic h-ASCs to definitive endoderm (Hnf3β, TCF2 and Sox17) and to PDX1, Ngn3, NeuroD, Pax4 positive pancreatic endoderm which further matures in vitro to secrete insulin. These ICAs are shown to produce human C-peptide in a glucose dependent manner exhibiting in-vitro functionality. Transplantation of mature ICAs, packed in immuno-isolatory biocompatible capsules to STZ induced diabetic mice restored near normoglycemia within 3–4 weeks. The detection of human C-peptide, 1155±165 pM in blood serum of experimental mice demonstrate the efficacy of our differentiation approach.

Conclusions

h-ASC is an ideal population of personal stem cells for cell replacement therapy, given that they are abundant, easily available and autologous in origin. Our findings present evidence that h-ASCs could be induced to differentiate into physiologically competent functional islet like cell aggregates, which may provide as a source of alternative islets for cell replacement therapy in type 1 diabetes.     

Toward beta cell replacement for diabetes

The discovery of insulin more than 90 years ago introduced a life‐saving treatment for patients with type 1 diabetes, and since then, significant progress has been made in clinical care for all forms of diabetes. However, no method of insulin delivery matches the ability of the human pancreas to reliably and automatically maintain glucose levels within a tight range. Transplantation of human islets or of an intact pancreas can in principle cure diabetes, but this approach is generally reserved for cases with simultaneous transplantation of a kidney, where immunosuppression is already a requirement. Recent advances in cell reprogramming and beta cell differentiation now allow the generation of personalized stem cells, providing an unlimited source of beta cells for research and for developing autologous cell therapies. In this review, we will discuss the utility of stem cell‐derived beta cells to investigate the mechanisms of beta cell failure in diabetes, and the challenges to develop beta cell replacement therapies. These challenges include appropriate quality controls of the cells being used, the ability to generate beta cell grafts of stable cellular composition, and in the case of type 1 diabetes, protecting implanted cells from autoimmune destruction without compromising other aspects of the immune system or the functionality of the graft. Such novel treatments will need to match or exceed the relative safety and efficacy of available care for diabetes.     

Generation of insulin-producing cells from PDX-1 gene-modified human mesenchymal stem cells

Islet cell replacement is considered as the optimal treatment for type I diabetes. However, the availability of human pancreatic islets for transplantation is limited. Here, we show that human bone marrow-derived mesenchymal stem cells (hMSCs) could be induced to differentiate into functional insulin-producing cells by introduction of the pancreatic duodenal homeobox-1 (PDX-1). Recombinant adenoviral vector was used to deliver PDX-1 gene into hMSCs. After being infected with Ad-PDX-1, hMSCs were successfully induced to differentiate into insulin-secreting cells. The differentiated PDX-1+ hMSCs expressed multiple islet-cell genes including neurogenin3 (Ngn3), insulin, GK, Glut2, and glucagon, produced and released insulin/C-peptide in a weak glucose-regulated manner. After the differentiated PDX-1+ hMSCs were transplanted into STZ-induced diabetic mice, euglycemia can be obtained within 2 weeks and maintained for at least 42 days. These findings validate the hMSCs model system as a potential basis for enrichment of human beta cells or their precursors, and a possible source for cell replacement therapy in diabetes.     

胰高血糖素样肽1与干细胞定向分化

糖尿病已经成为21世纪严重威胁人类健康的疾病之一。胰岛移植被认为是治疗Ⅰ型和部分Ⅱ型糖尿病的最有效方法。然而,供体组织来源的匮乏限制了其应用。随着细胞移植和组织工程的日益发展,干细胞研究为新型胰岛的来源开辟了新的途径。干细胞定向诱导分化的关键是筛选合适的诱导剂以及优化诱导微环境,使干细胞培养微环境尽可能接近体内正常细胞发育分化的微环境,从而有利于干细胞适宜生长及定向分化。最近研究证实,胰高血糖素样肽1（Glucagon- Like PeptideⅠ,GLP-1）在干细胞向胰岛样细胞诱导分化中具有显著作用。因此,为了更好地应用GLP-1在干细胞定向分化中的潜能、促进应用干细胞治疗糖尿病新疗法研究的进程及干细胞定向分化技术逐渐成熟,本文就胰高血糖素样肽-1及它诱导干细胞定向分化胰岛样细胞的研究进展作一阐述。     

Evidence for unimpaired pancreatic secretion and hepatic removal of insulin in healthy offspring of type 2 (noninsulin-dependent) diabetic couples

The aim of the present study was to investigate the secretion and the hepatic removal of insulin in a group of 14 unaffected offspring of 14 type 2 (noninsulin-dependent) diabetic couples compared to 14 healthy subjects without family history of diabetes mellitus. The two groups, each consisting of 5 obese and 9 nonobese subjects, were carefully matched for sex, age, and body weight. We examined glucose, insulin, and C-peptide levels, as well as C-peptide to insulin ratios and relations during the oral glucose tolerance test. Glucose concentrations and incremental areas were similar in the two groups, as well as insulin and C-peptide levels and areas. C-peptide to insulin molar ratios, both in fasting state and after glucose load, as well as relations between C-peptide and insulin incremental areas were not different. Our results suggest that the healthy offspring of type 2 diabetic couples have a normal response of beta-cell to oral glucose as well as a normal removal of insulin by the liver.     

Functional rest through intensive treatment with insulin and potassium channel openers preserves residual beta-cell function and mass in acutely diabetic BB rats.

Diazoxide and the diazoxide-analogue, NNC 55-0118, are potassium channel openers that interfere with insulin secretion from beta-cells. In vitro, we show that these two drugs inhibit insulin release from diabetes-resistant BB rat islets cultured at either low or high glucose concentration and cause an intracellular accumulation of insulin with high glucose. Preservation of beta-cells was investigated in newly diabetic BB rats treated with insulin implants from day 0-8 under oral diazoxide, NNC 55-0118 or solvent gavage once a day from day 0-7. Three of eight rats (37.5%) treated with diazoxide and three of ten (30%) treated with NNC 55-0118 retained near normal C-peptide responses when challenged with glucose/arginine on day 9, whereas none of eight (0%) solvent-treated rats showed a C-peptide response. Immunohistochemical staining for insulin and glucagon showed that all the C-peptide responding rats had insulin-positive cells in their islets. In contrast, islets from non-responding rats displayed marked inflammation or end-stage lesions. Furthermore, rats with C-peptide response and treated with NNC 55-0118 exhibited only minimal signs of islet inflammation, whereas C-peptide responding diazoxide-treated rats had low level islet inflammation. These results imply that it is conceivable to preserve residual beta-cells at diabetes onset by induction of target cell rest with potassium channel openers and continuous insulin treatment.     

For debate: Starling's curve of the pancreas--overuse of a concept?

It is commonly accepted that insulin secretion follows the pattern of an inverted U, also termed 'Starling's curve of the pancreas' during the natural history of hyperglycemia in glucose intolerance and type 2 diabetes. This concept is based on the cross-sectional observation that insulin concentrations initially increase when insulin sensitivity declines (as a consequence of obesity, for example) and decrease when glucose tolerance deteriorates (impaired glucose tolerance or overt type 2 diabetes). The initial increase in insulin concentrations has been viewed as 'hypersecretion' of insulin, thought to indicate that beta cell dysfunction is not etiological but secondary in nature. However, this view is oblivious to the now well-established fact that assessment of insulin secretion must account for individual insulin sensitivity. Here, we revisit the concept of Starling's curve of the pancreas based on first-phase C-peptide concentrations (hyperglycemic clamp) from subjects with normal glucose tolerance (n=66), impaired glucose tolerance (n=19) and mild type 2 diabetes (n=9). In absolute terms, first-phase C-peptide concentrations plotted against increasing fasting glucose concentrations indeed followed an inverted U. However, adjusted for direct and indirect measures of insulin sensitivity (insulin sensitivity index from the hyperglycemic clamp, body mass index, age and sex), first-phase C-peptide concentrations of the same individuals tended to decrease steadily. In conclusion, while the Starling curve exists for insulin concentrations, and perhaps also for insulin secretion, it does not hold for beta-cell function if that term were to imply appropriateness of insulin secretion (based on a formal test of glucose-stimulated insulin secretion) for the degree of insulin resistance, as it should.     

Redifferentiation of insulin-secreting cells after in vitro expansion of adult human pancreatic islet tissue

Cellular replacement therapy holds promise for the treatment of diabetes mellitus but donor tissue is severely limited. Therefore, we investigated whether insulin-secreting cells could be differentiated in vitro from a monolayer of cells expanded from human donor pancreatic islets. We describe a three-step culture protocol that allows for the efficient generation of insulin-producing cell clusters from in vitro expanded, hormone-negative cells. These clusters express insulin at levels of up to 34% that of average freshly isolated human islets and secrete C-peptide upon membrane depolarization. They also contain cells expressing the other major islet hormones (glucagon, somatostatin, and pancreatic polypeptide). The source of the newly differentiated endocrine cells could either be indigenous stem/progenitor cells or the proliferation-associated dedifferentiation and subsequent redifferentiation of mature endocrine cells. The in vitro generated cell clusters may be efficacious in providing islet-like tissue for transplantation into diabetic recipients.     

Mechanisms of endothelial dysfunction with development of type 1 diabetes mellitus: role of insulin and C-peptide

Complications associated with insulin-dependent diabetes mellitus (type-1diabetes) primarily represent vascular dysfunction that has its origin in the endothelium. While many of the vascular changes are more accountable in the late stages of type-1diabetes, changes that occur in the early or initial functional stages of this disease may precipitate these later complications. The early stages of type-1diabetes are characterized by a diminished production of both insulin and C-peptide with a significant hyperglycemia. During the last decade numerous speculations and theories have been developed to try to explain the mechanisms responsible for the selective changes in vascular reactivity and/or tone and the vascular permeability changes that characterize the development of type-1diabetes. Much of this research has suggested that hyperglycemia and/or the lack of insulin may mediate the observed functional changes in both endothelial cells and vascular smooth muscle. Recent studies suggest several possible mechanisms that might be involved in the observed decreases in vascular nitric oxide (NO) availability with the development of type-1 diabetes. In addition more recent studies have indicated a direct role for both endogenous insulin and C-peptide in the amelioration of the observed endothelial dysfunction. These results suggest a synergistic action between insulin and C-peptide that facilitates increase NO availability and may suggest new clinical treatment modalities for type-1 diabetes mellitus.     

C-peptide promotes lesion development in a mouse model of arteriosclerosis

Patients with insulin resistance and early type 2 diabetes exhibit an increased propensity to develop a diffuse and extensive pattern of arteriosclerosis. Typically, these patients show elevated serum levels of the proinsulin cleavage product C-peptide and immunohistochemical data from our group revealed C-peptide deposition in early lesions of these individuals. Moreover, in vitro studies suggest that C-peptide could promote atherogenesis. This study examined whether C-peptide promotes vascular inflammation and lesion development in a mouse model of arteriosclerosis. ApoE-deficient mice on a high fat diet were treated with C-peptide or control injections for 12 weeks and the effect on lesion size and plaque composition was analysed. C-peptide treatment significantly increased C-peptide blood levels by 4.8-fold without having an effect on glucose or insulin levels, nor on the lipid profile. In these mice, C-peptide deposition in atherosclerotic plaques was significantly increased compared with controls. Moreover, lesions of C-peptide-treated mice contained significantly more macrophages (1.6 ± 0.3% versus 0.7 ± 0.2% positive area; P < 0.01) and more vascular smooth muscle cells (4.8 ± 0.6% versus 2.4 ± 0.3% positive area; P < 0.01). Finally, lipid deposition measured by Oil-red-O staining in the aortic arch was significantly higher in the C-peptide group compared with controls. Our results demonstrate that elevated C-peptide levels promote inflammatory cell infiltration and lesion development in ApoE-deficient mice without having metabolic effects. These data obtained in a mouse model of arteriosclerosis support the hypothesis that C-peptide may have an active role in atherogenesis in patients with diabetes and insulin resistance.     

Pancreatic Insulin-Producing Cells Differentiated from Human Embryonic Stem Cells Correct Hyperglycemia in SCID/NOD Mice,an Animal Model of Diabetes

Background

Human pancreatic islet transplantation is a prospective curative treatment for diabetes. However, the lack of donor pancreases greatly limits this approach. One approach to overcome the limited supply of donor pancreases is to generate functional islets from human embryonic stem cells (hESCs), a cell line with unlimited proliferative capacity, through rapid directed differentiation. This study investigated whether pancreatic insulin-producing cells (IPCs) differentiated from hESCs could correct hyperglycemia in severe combined immunodeficient (SCID)/non-obese diabetic (NOD) mice, an animal model of diabetes.

Methods

We generated pancreatic IPCs from two hESC lines, YT1 and YT2, using an optimized four-stage differentiation protocol in a chemically defined culture system. Then, about 5–7×10⁶ differentiated cells were transplanted into the epididymal fat pad of SCID/NOD mice (n = 20). The control group were transplanted with undifferentiated hESCs (n = 6). Graft survival and function were assessed using immunohistochemistry, and measuring serum human C-peptide and blood glucose levels.

Results

The pancreatic IPCs were generated by the four-stage differentiation protocol using hESCs. About 17.1% of differentiated cells expressed insulin, as determined by flow cytometry. These cells secreted insulin/C-peptide following glucose stimulation, similarly to adult human islets. Most of these IPCs co-expressed mature β cell-specific markers, including human C-peptide, GLUT2, PDX1, insulin, and glucagon. After implantation into the epididymal fat pad of SCID/NOD mice, the hESC-derived pancreatic IPCs corrected hyperglycemia for ≥8 weeks. None of the animals transplanted with pancreatic IPCs developed tumors during the time. The mean survival of recipients was increased by implanted IPCs as compared to implanted undifferentiated hESCs (P<0.0001).

Conclusions

The results of this study confirmed that human terminally differentiated pancreatic IPCs derived from hESCs can correct hyperglycemia in SCID/NOD mice for ≥8 weeks.