C-peptide In Response Research Articles

Modulation of naïve mesenchymal stromal cells by extracellular vesicles derived from insulin-producing cells: an in vitro study

This study was to determine whether extracellular vesicles (EVs) derived from insulin-producing cells (IPCs) can modulate naïve mesenchymal stromal cells (MSCs) to become insulin-secreting. MSCs were isolated from human adipose tissue. The cells were then differentiated to generate IPCs by achemical-based induction protocol. EVs were retrieved from the conditioned media of undifferentiated (naïve) MSCs (uneducated EVs) and from that of MSC-derived IPCs (educated EVs) by sequential ultracentrifugation. The obtained EVs were co-cultured with naïve MSCs.The cocultured cells were evaluated by immunofluorescence, flow cytometry, C-peptide nanogold silver-enhanced immunostaining, relative gene expression and their response to a glucose challenge.Immunostaining for naïve MSCs cocultured with educated EVs was positive for insulin, C-peptide, and GAD65. By flow cytometry, the median percentages of insulin-andC-peptide-positive cells were 16.1% and 14.2% respectively. C-peptide nanogoldimmunostaining providedevidence for the intrinsic synthesis of C-peptide. These cells released increasing amounts of insulin and C-peptide in response to increasing glucose concentrations. Gene expression of relevant pancreatic endocrine genes, except for insulin, was modest. In contrast, the results of naïve MSCs co-cultured with uneducated exosomes were negative for insulin, C-peptide, and GAD65. These findings suggest that this approach may overcome the limitations of cell therapy.

Disturbances in dynamics of glucose, insulin and C-peptide in blood after a normalized intake of mixed meal in obesity and type 2 diabetes mellitus

The aim of the study is to investigate the dynamics of venous blood glucose, insulin, and C-peptide in response to intake a meal normalized to body mass in obese patients without and with type 2 diabetes mellitus. Venous blood samples were taken from 7 healthy subjects, 9 obese patients, and 10 obese patients with type 2 diabetes mellitus (mean period of diagnosed diabetes 7 years) before and 30, 60, 90, 120, and 180 min after a mixed meal (6 kcal/kg of body mass); additionally, 9 patients with obesity and type 2 diabetes mellitus and 3 healthy volunteers completed a hyperinsulinemic euglycemic clamp test. In patient groups the energy content of food did not differ, but was 1.8 times higher than in the control. An increase in glucose level one hour after a meal was maximal in patients with type 2 diabetes, but an increase in insulin and C-peptide — in obese patients, that related to impairment of insulin-dependent glucose uptake by tissues and of the rate of insulin secretion (dysfunction of â-cells) in patients. At the same time, an increase in the total area under the curve “C-peptide–time” shows that the maximum secretory response of â-cells is comparable in obese patients without and with type 2 diabetes mellitus. The absolute blood glucose level 90 minutes after a meal was closely correlated with the M-index — the marker of systemic sensitivity to insulin (rs = –0.82, p = 0.002). Our results characterize the features in the regulation of carbohydrate metabolism after intake a mixed meal, normalized to body mass, in people with the varying severity of metabolic disorders, and open up prospects for a wider application of this test in practice.

Generation of glucose sensitive insulin-secreting cells from human induced pluripotent stem cells on optimized polyethersulfone hybrid nanofibrous scaffold.

In the realm of diabetes treatment, various strategies have been tried, including islet transplantation and common drug therapies, but the limitations of these procedures and lack of responsive to the high number of patients have prompted researchers to develop a new method. In recent decades, the use of stem cells and three-dimonsional (3D) scaffold to produce insulin-secreting cells is one of the most promising new approaches. Meanwhile, human-induced pluripotent stem cells (iPSCs) propose due to advantages such as autologousness and high pluripotency in cell therapy. This study aimed to evaluate the differentiation of iPSCs into pancreatic islet insuli-producing cells (IPCs) on Silk/PES (polyethersulfone) nanofibers as a 3D scaffold and compare it with a two-dimonsional (2D) cultured group. Investigating the functional, morphological, molecular, and cellular characteristics of differentiated iPSCs on control cultures (without differentiation medium), 2D and 3D were measured by various methods such as electron microscopy, Q-PCR, immunofluorescence, western blot, and ELISA. This investigation revealed that differentiated cells on the 3D Silk/PES scaffold expressed pancreatic specific-markers such as insulin and pdx1 at higher levels than the control and 2D groups, with a significant difference between the two groups. All results of Q-PCR, immunocytochemistry, and western blot showed that IPCs in the silk/PES 3D group was more efficient than in the 2D group. In the face of these cases, the release of insulin and C-peptide in response to several concentrations of glucose in the 3D group was significantly higher than in the 2D culture. Finally, our findings displayed that optimized Silk/PES 3D scaffolds can enhance the differentiation of IPCs from iPSCs compared to the 2D culture group.

The microenvironment of silk/gelatin nanofibrous scaffold improves proliferation and differentiation of Wharton’s jelly-derived mesenchymal cells into islet-like cells

The use of umbilical cord-derived mesenchymal stem cells along with three-dimensional (3D) scaffolds in pancreatic tissue engineering can be considered as a treatment for diabetes. This study aimed to investigate the differentiation of Wharton’s jelly-derived mesenchymal stem cells (WJ-MSCs) into pancreatic islet-insulin producing cells (IPCs) on silk/gelatin nanofibers as a 3D scaffold. Mesenchymal markers were evaluated at the mesenchymal stem cells (MSCs) level by flow cytometry. WJ-MSCs were then cultured on 3D scaffolds and treated with a differential medium. Immunocytochemical assays showed efficient differentiation of WJ-MSCs into IPCs. Also, Real-time PCR results showed a significant increase in the expression of pancreatic genes in the 3D culture group compared to the two-dimensional (2D) culture group. Despite these cases, the secretion of insulin and C-peptide in response to different concentrations of glucose in the 3D group was significantly higher than in the 2D culture. The results of our study showed that silk/gelatin scaffold with WJ-MSCs could be a good option in the production of IPCs in regenerative medicine and pancreatic tissue engineering.

A saponin from astragalus promotes pancreatic ductal organoids differentiation into insulin-producing cells

BackgroundIslet transplantation is an effective treatment for the type 1 and severe type 2 diabetes, but it is restricted by the severe lack of pancreas donors. In vitro differentiation of pancreatic progenitors into insulin-secreting cells is one of the hopeful strategies in the cell transplantation therapy of diabetes. Isoastragaloside I is one of the saponin molecules found in Astragalus membranaceus, which has been demonstrated to alleviate insulin resistance and glucose intolerance in obese mice. Study designWe established mouse pancreatic ductal organoids (mPDOs) with progenitor characteristics and an insulin promoter-driven EGFP reporter system to screen astragalus saponin components for monomers that can promote insulin-producing cell differentiation. MethodsmPDOs treated with or without astragalus saponin monomers were investigated by the insulin promoter-driven EGFP reporter, quantitative PCR, immunofluorescence and flow cytometry to evaluate the expression of endocrine progenitor and β-cell markers. ResultsIsoastragaloside I significantly promoted the expression of β-cell differentiation genes, which was demonstrated by the activation of the insulin promoter-driven EGFP reporter, as well as the significant increase of mRNA levels of the endocrine progenitor marker Ngn3 and the β-cell markers insulin1 and insulin2. Immunostaining studies indicated that the β-cell-specific C-peptide was upregulated in isoastragaloside I-treated mPDOs. FACS analysis revealed that the ratio of C-peptide-secreting cells in isoastragaloside I-treated mPDOs was over 40%. Glucose tolerance tests demonstrated that the differentiated mPDOs could secrete C-peptide in response to glucose stimulation. ConclusionsWe discover a novel strategy of inducing pancreatic ductal progenitors to differentiate into insulin-producing cells using isoastragaloside I. This approach can be potentially applied to β-cell transplantation in diabetes therapies.

Intermittent cold exposure improves glucose homeostasis despite exacerbating diet‐induced obesity in mice housed at thermoneutrality

Ambient cold exposure is often regarded as a promising anti-obesity treatment in mice. However, most preclinical studies aimed at treating obesity via cold-induced thermogenesis have been confounded by subthermoneutral housing temperatures. Therefore, the ability of ambient cold to combat diet-induced obesity in mice housed under humanized thermoneutral conditions is currently unknown. Moreover, mammals such as mice are rarely exposed to chronic ambient cold without reprieve, yet mice are often subjected to experimental conditions of chronic rather than intermittent cold exposure (ICE), despite ICE being more physiologically relevant. In the present study, we provide novel evidence that thermoneutral housing uncouples the effects of ICE on glucose and energy homeostasis suggesting that ICE, despite improving glucose tolerance, is not an effective obesity treatment when mice are housed under humanized thermoneutral conditions. The present study examines whether a physiologically relevant model of ambient cold exposure, intermittent cold exposure (ICE), could ameliorate the metabolic impairments of diet-induced obesity in male and female mice housed under humanized thermoneutral conditions. Male and female C57BL/6J mice housed at thermoneutrality (29°C) were fed a low-fat diet or high-fat diet for 6weeks before being weight matched into groups that remained unperturbed or underwent ICE for 4 weeks (4°C for 60minday-1 ; 5daysweek-1 ) when being maintained on their respective diets. ICE induced rapid and persistent hyperphagia exacerbating rather than attenuating high-fat diet-induced obesity over time. These ICE-induced increases in adiposity were found to be energy intake-dependent via pair-feeding. Despite exacerbating high-fat diet-induced obesity, ICE improved glucose tolerance, independent of diet, in a sex-specific manner. The effects of ICE on glucose tolerance were not attributed to improvements in whole-body insulin tolerance, tissue specific insulin action, nor differences in markers of hepatic insulin clearance or pancreatic beta cell proliferation. Instead, ICE increased serum concentrations of insulin and C-peptide in response to glucose, suggesting that ICE may improve glucose tolerance by potentiating pancreatic glucose-stimulated insulin secretion. These data suggest that ICE, despite improving glucose tolerance, is not an effective obesity treatment in mice housed under humanized conditions.

Intermittent Ambient Cold Exposure Is Not an Effective Obesity Treatment in Mice Housed at Thermoneutrality

Chronic ambient cold exposure has been regarded as a promising obesity treatment given it potently increases energy expenditure. However, few mammals are chronically exposed to cold ambient conditions without reprieve highlighting the need to determine whether more physiologically relevant interventions, such as intermittent ambient cold exposure, could be utilized to treat obesity. Moreover, most preclinical cold exposure work to date has been conducted at housing temperatures below thermoneutrality, unintentionally confounding metabolic outcomes. As such, this study examined whether intermittent ambient cold exposure could abrogate the metabolic impairments of diet-induced obesity, in mice housed under humanized thermoneutral conditions. Eight-week-old male and female C57Bl/6J mice (n ≥ 40/sex) were singly housed, acclimated to thermoneutrality (30 °C), and then weight matched into low-fat (LFD; 10% kcal from fat) or high-fat (HFD; 45% kcal from fat) diet fed groups. Male and female mice were given six weeks to feed on their respective diets before being weight-matched into groups that remained unperturbed (CTRL) or underwent intermittent ambient cold exposure (ICE), respectively. ICE consisted of exposure to 4 °C for one hour/day, five days/week, for four weeks. Mice remained on their respective diets for the entire four-week intervention period, during which time glucose and insulin tolerance were assessed. Following the intervention mice were sedated with sodium pentobarbital and sacrificed by exsanguination of the heart. The epididymal and inguinal white adipose tissue depots (eWAT and iWAT, respectively), the interscapular brown adipose tissue depot (BAT), as well as the liver, triceps, and hypothalamus were excised, snap-frozen in liquid nitrogen, and stored at -80 °C. ICE induced hyperphagia that was rapid in onset and persistent, occurring with every exposure independent of diet. Over time, this exacerbated rather than attenuated HFD-induced obesity in both male and female mice, increasing BAT, eWAT, and iWAT mass as well as cellular lipid deposition. Pair-feeding revealed that these ICE-induced increases in adiposity were energy intake dependent, as the pair-fed, but not ad libitum fed, HFD-ICE group was protected from ICE-induced increases in body mass. Despite exacerbating HFD-induced obesity, ICE improved glucose tolerance, independent of diet, in a sex-dependent manner. The effects of ICE on glucose tolerance were not attributed to improvements in whole-body insulin tolerance, tissue specific insulin action, nor to differences in hepatic insulin clearance. Instead, ICE increased serum concentrations of insulin and C-peptide in response to glucose, suggesting that ICE may improve glucose tolerance by potentiating pancreatic glucose-stimulated insulin secretion. Taken together, this data suggests that intermittent ambient cold exposure, despite improving glucose tolerance, is not an effective obesity treatment in mice housed under humanized conditions.

PDX1- /NKX6.1+ progenitors derived from human pluripotent stem cells as a novel source of insulin-secreting cells.

Beta cell replacement strategies are a promising alternative for diabetes treatment. Human pluripotent stem cells (hPSCs) serve as a scalable source for producing insulin-secreting cells for transplantation therapy. We recently generated novel hPSC-derived pancreatic progenitors, expressing high levels of the transcription factor NKX6.1, in the absence of PDX1 (PDX1- /NKX6.1+ ). Herein, our aim was to characterize this novel population and assess its ability to differentiate into insulin-secreting beta cells in vitro. Three different hPSC lines were differentiated into PDX1- /NKX6.1+ progenitors, which were further differentiated into insulin-secreting cells using two different protocols. The progenitors and beta cells were extensively characterized. Transcriptome analysis was performed at different stages and compared with the profiles of various pancreatic counterparts. PDX1- /NKX6.1+ progenitors expressed high levels of nestin, a key marker of pancreatic islet-derived progenitors, in the absence of E-cadherin, similar to pancreatic mesenchymal stem cells. At progenitor stage, comparison of the two populations showed downregulation of pancreatic epithelial genes and upregulation of neuronal development genes in PDX1- /NKX6.1+ cells in comparison to the PDX1+ /NKX6.1+ cells. Interestingly, on further differentiation, PDX1- /NKX6.1+ cells generated mono-hormonal insulin+ cells and activated pancreatic key genes, such as PDX1. The transcriptome profile of PDX1- /NKX6.1+ -derived beta (3D-beta) was closely similar to those of human pancreatic islets and purified hPSC-derived beta cells. Also, the 3D-beta cells secreted C-peptide in response to increased glucose concentrations indicating their functionality. These findings provide a novel source of insulin-secreting cells that can be used for beta cell therapy for diabetes.

The expression of cytochrome P4502J2 gene and 14, 15 epoxyeicosatrienoic acid level influence the amount of insulin secreted from human mesenchymal stem cell-derived insulin-producing cells

BackgroundInsulin-producing cells differentiated from human mesenchymal stem cells demonstrate limited glucose-stimulated insulin secretion. Cytochrome P450 2J2 and its product epoxyeicosatrienoic acids regulate β-cell function in the human pancreas. The aim of this study is to explore the expression pattern of cytochrome P450 2J2 gene and 14, 15 epoxyeicosatrienoic level along the differentiation of human bone marrow-derived mesenchymal stem cells into insulin-producing cells.ResultsThe differentiated insulin-producing cells express high levels of pancreatic duodenal homeobox-1 and insulin gene mRNA. It secretes increasing amounts of C-peptide in response to increasing glucose concentrations than undifferentiated cells. The differentiated insulin-producing cells were found to express reduced amounts of cytochrome P450 2J2 gene mRNA and significant low level of 14, 15 epoxyeicosatrienoic acid than the undifferentiated cells. A strong positive correlation between 14, 15 epoxyeicosatrienoic concentrations and C-peptide released from the differentiated insulin-producing cells was noticed.ConclusionsCytochrome P4502J2 and its product 14, 15 epoxyeicosatrienoic might affect insulin secretion from differentiated insulin-producing cells.

The effect of insulin administration on c-peptide in critically ill patients with type 2 diabetes

BackgroundIn critically ill patients with permissive hyperglycemia, it is uncertain whether exogenous insulin administration suppresses or enhances c-peptide secretion (a marker of pancreatic beta-cell response). We aimed to explore this effect in patients with type 2 diabetes.MethodsWe prospectively enrolled a cohort of 45 critically ill patients with type 2 diabetes managed according to a liberal glucose protocol (target blood glucose 10–14 mmol/l). We recorded the administration of insulin and oral hypoglycemic agents and measured plasma c-peptide as surrogate marker of endogenous insulin secretion on the first two consecutive days in ICU.ResultsOverall, 20 (44.4%) patients required insulin to achieve target blood glucose. Insulin-treated patients had higher glycated hemoglobin A1c, more premorbid insulin-requiring type 2 diabetes, and greater blood glucose levels but lower c-peptide levels on admission. Premorbid insulin-requiring diabetes was independently associated with lower admission c-peptide, whereas greater plasma creatinine was independently associated with higher levels. Increases in c-peptide were positively correlated with an increase in blood glucose both in patients who did (r = 0.54, P = 0.01) and did not (r = 0.56, P = 0.004) receive insulin. However, insulin administration was independently associated with a greater increase in c-peptide (P = 0.04). This association was not modified by the use of oral insulin secretagogues.ConclusionsC-peptide, a marker of beta-cell response, responds to and is influenced by glycemia and renal function in critically ill patients with type 2 diabetes. In addition, in our cohort, exogenous insulin administration was associated with a greater increase in c-peptide in response to hyperglycemia.Trial Registration Australian New Zealand Clinical Trials Registry (ACTRN12615000216516).

Pancreatic Endoderm-Derived From Diabetic Patient-Specific Induced Pluripotent Stem Cell Generates Glucose-Responsive Insulin-Secreting Cells.

Human-induced pluripotent stem cells (hiPSCs) can potentially serve as an invaluable source for cell replacement therapy and allow the creation of patient- and disease-specific stem cells without the controversial use of embryos and avoids any immunological incompatibility. The generation of insulin-producing pancreatic β-cells from pluripotent stem cells in vitro provides an unprecedented cell source for personal drug discovery and cell transplantation therapy in diabetes. A new five-step protocol was introduced in this study, effectively induced hiPSCs to differentiate into glucose-responsive insulin-producing cells. This process mimics in vivo pancreatic organogenesis by directing cells through stages resembling definitive endoderm, primitive gut-tube endoderm, posterior foregut, pancreatic endoderm, and endocrine precursor. Each stage of differentiation were characterized by stage-specific markers. The produced cells exhibited many properties of functional β-cells, including expression of critical β-cells transcription factors, the potency to secrete C-peptide in response to high levels of glucose and the presence of mature endocrine secretory granules. This high efficient differentiation protocol, established in this study, yielded 79.18% insulin-secreting cells which were responsive to glucose five times higher than the basal level. These hiPSCs-derived glucose-responsive insulin-secreting cells might provide a promising approach for the treatment of type I diabetes mellitus. J. Cell. Physiol. 232: 2616-2625, 2017. © 2016 Wiley Periodicals, Inc.

V-Maf Musculoaponeurotic Fibrosarcoma Oncogene Homolog A Synthetic Modified mRNA Drives Reprogramming of Human Pancreatic Duct-Derived Cells Into Insulin-Secreting Cells.

β-Cell replacement therapy represents the most promising approach to restore glucose homeostasis in patients with type 1 diabetes. This study shows an innovative and robust in vitro system for large-scale production of β-like cells from human pancreatic duct-derived cells (HDDCs) using a nonintegrative RNA-based reprogramming technique. V-Maf musculoaponeurotic fibrosarcoma oncogene homolog A overexpression was efficient and sufficient to induce β-cell differentiation and insulin secretion from HDDCs in response to glucose stimulation, allowing the cells to mitigate hyperglycemia in diabetic SCID-beige mice. The data describe a new, reliable, and fast procedure in adult human pancreatic cells to generate clinically relevant amounts of new β cells with the potential to reverse diabetes.

Umbilical Cord Derived Mesenchymal Stem Cells Useful in Insulin Production - Another Opportunity in Cell Therapy.

Background and ObjectivesType 1 Diabetes Mellitus (T1DM) is an autoimmune disorder resulting out of T cell mediated destruction of pancreatic beta cells. Immunomodulatory properties of mesenchymal stem cells may help to regenerate beta cells and/or prevent further destruction of remnant, unaffected beta cells in diabetes. We have assessed the ability of umbilical cord derived MSCs (UCMSCs) to differentiate into functional islet cells in vitro.Methods and ResultsWe have isolated UCMSCs and allowed sequential exposure of various inducing agents and growth factors. We characterized these cells for confirmation of the presence of islet cell markers and their functionality. The spindle shaped undifferentiated UCMSCs, change their morphology to become triangular in shape. These cells then come together to form the islet like structures which then grow in size and mature over time. These cells express pancreatic and duodenal homeobox −1 (PDX-1), neurogenin 3 (Ngn-3), glucose transporter 2 (Glut 2) and other pancreatic cell markers like glucagon, somatostatin and pancreatic polypeptide and lose expression of MSC markers like CD73 and CD105. They were functionally active as demonstrated by release of physiological insulin and C-peptide in response to elevated glucose concentrations.ConclusionsPancreatic islet like cells with desired functionality can thus be obtained in reasonable numbers from undifferentiated UCMSCs invitro. This could help in establishing a “very definitive source” of islet like cells for cell therapy. UCMSCs could thus be a game changer in treatment of diabetes.

Human induced pluripotent stem cells differentiate into insulin-producing cells able to engraft in vivo.

New sources of insulin-secreting cells are strongly required for the cure of diabetes. Recent successes in differentiating embryonic stem cells, in combination with the discovery that it is possible to derive human induced pluripotent stem cells (iPSCs) from somatic cells, have raised the possibility that patient-specific beta cells might be derived from patients through cell reprogramming and differentiation. In this study, we aimed to obtain insulin-producing cells from human iPSCs and test their ability to secrete insulin in vivo. Human iPSCs, derived from both fetal and adult fibroblasts, were differentiated in vitro into pancreas-committed cells and then transplanted into immunodeficient mice at two different stages of differentiation (posterior foregut and endocrine cells). IPSCs were shown to differentiate in insulin-producing cells in vitro, following the stages of pancreatic organogenesis. At the end of the differentiation, the production of INSULIN mRNA was highly increased and 5 ± 2.9 % of the cell population became insulin-positive. Terminally differentiated cells also produced C-peptide in vitro in both basal and stimulated conditions. In vivo, mice transplanted with pancreatic cells secreted human C-peptide in response to glucose stimulus, but transplanted cells were observed to lose insulin secretion capacity during the time. At histological evaluation, the grafts resulted to be composed of a mixed population of cells containing mature pancreatic cells, but also pluripotent and some neuronal cells. These data overall suggest that human iPSCs have the potential to generate insulin-producing cells and that these differentiated cells can engraft and secrete insulin in vivo.

FOXO1 inhibition yields functional insulin-producing cells in human gut organoid cultures.

Generation of surrogate sources of insulin-producing β-cells remains a goal of diabetes therapy. While most efforts have been directed at differentiating embryonic or induced pluripotent stem (iPS) cells into β-like-cells through endodermal progenitors, we have shown that gut endocrine progenitor cells of mice can be differentiated into glucose-responsive, insulin-producing cells by ablation of transcription factor Foxo1. Here we show that FOXO1 is present in human gut endocrine progenitor and serotonin-producing cells. Using gut organoids derived from human iPS cells, we show that FOXO1 inhibition using a dominant-negative mutant or lentivirus-encoded shRNA promotes generation of insulin-positive cells that express all markers of mature pancreatic β-cells, release C-peptide in response to secretagogues, and survive in vivo following transplantation into mice. The findings raise the possibility of using gut-targeted FOXO1 inhibition or gut organoids as a source of insulin-producing cells to treat human diabetes.

Insulin secretion measured by stimulated C-peptide in long-established Type 1 diabetes in the Diabetes Control and Complications Trial (DCCT)/ Epidemiology of Diabetes Interventions and Complications (EDIC) cohort: a pilot study.

To evaluate whether clinically relevant concentrations of stimulated C-peptide in response to a mixed-meal tolerance test can be detected after almost 30 years of diabetes in people included in the Diabetes Control and Complications Trial/Epidemiology of Diabetes Interventions and Complications cohort. Mixed-meal tolerance tests were performed in a sample of 58 people. C-peptide levels were measured using a chemiluminescent immunoassay. This sample size assured a high probability of detecting C-peptide response if the true prevalence was at least 5%, a level that would justify the subsequent assessment of C-peptide in the entire cohort. Of the 58 participants, 17% showed a definite response, defined as one or more post-stimulus concentrations of C-peptide > 0.03 nmol/l, and measurable concentrations were found in all participants. These results show that a stimulated C-peptide response can be measured in some people with long-term Type 1 diabetes. Further investigation of all participants in the Diabetes Control and Complications Trial/Epidemiology of Diabetes Interventions and Complications study will help relate long-term residual C-peptide response to glycaemia over time and provide insight into the relevance of this response in terms of insulin dose, severe hypoglycaemia, retinopathy, nephropathy and macrovascular disease. Establishing the clinical relevance of long-term C-peptide responses is important in understanding the impact that therapy to preserve or improve β-cell function may have in patients with long-term Type 1 diabetes.

Generation of functional insulin-producing cells in the gut by Foxo1 ablation

Restoration of regulated insulin secretion is the ultimate goal of type 1 diabetes therapy. Here we show that, surprisingly, somatic ablation of Foxo1 in Neurog3+ enteroendocrine progenitor cells gives rise to gut insulin-positive cells (Ins+) that express markers of mature β-cells, and secrete bioactive insulin as well as C-peptide in response to glucose and sulfonylureas. Lineage tracing experiments show that gut Ins+ cells arise cell-autonomously from Foxo1-deficient cells. Inducible Foxo1 ablation in adult mice also results in the generation of gut Ins+ cells. Following ablation by the β-cell toxin, streptozotocin, gut Ins+ cells regenerate and produce insulin, reversing hyperglycemia in mice. The data indicate that Neurog3+ enteroendocrine progenitors require active Foxo1 to prevent differentiation into Ins+ cells. Foxo1 ablation in gut epithelium may provide an approach to restore insulin production in type 1 diabetes.

The importance of beta cell characterisation: generating human beta cells by differentiating human embryonic stem cells

Exogenous insulin administration continues to be the most common means of correcting the absolute insulin deficiency in patients with type 1 diabetes mellitus. Nevertheless, the best available insulin therapy does not control blood glucose levels in a physiological manner, leading to several serious metabolic and neurovascular complications [1]. Beta cell replacement offers an attractive alternative, potentially enabling precise glycaemic control. Clinical islet transplantation, for instance, has proven effective in improving glucose homeostasis, reducing episodes of hypoglycaemia and achieving insulin independence [2]. However, the shortage of donor islet tissues is a major restriction for widespread application of beta cell replacement therapies. The recent developments in stem cell research suggest that human embryonic stem cells (hESCs) hold great promise for fulfilling this need for an unlimited supply of readily available insulin-producing cells. Embryonic stem cells are pluripotent cells derived from the inner cell mass of blastocysts. They exhibit the capacity to develop into the three embryonic germ layers (endoderm, mesoderm and ectoderm). To generate beta cells from hESCs, current approaches have been guided by knowledge of pancreatic development. By recapitulating these developmental signals, several groups have been able to differentiate hESCs into insulinpositive cells [3–6]. Jiang et al. utilised a differentiation protocol omitting serum from the culture medium and reported the presence of islet-like clusters that secreted Cpeptide in response to glucose [3]. However, C-peptidepositive cells only accounted for 2–8% of all cells in the islet-like cluster [3]. When transplanted into chemically induced diabetic mice, the islet-like clusters prolonged survival and secreted C-peptide in response to a glucose challenge [4]. Novocell (now known as ViaCyte, San Diego, CA, USA) developed another differentiation protocol in which hESC-derived insulin positive cells could secrete C-peptide but only in the presence of secretagogues like KCl [5]. When hESC-derived pancreatic progenitors were transplanted into athymic mice, they reported that these cells could also produce human insulin and C-peptide levels comparable to 3,000 human islets [6]. While the results reported by the ViaCyte group were impressive, Matveyenko et al. had difficulty replicating these results in athymic rats [7]. In addition, the recipient mice responded poorly to glucose tolerance testing, which suggests that insulin secretion was not glucose dependent. Clearly, improvements to directed differentiation attempts are necessary to overcome two significant hurdles that G. S. Korbutt : T. Y. Yeung :C. E. Ellis Department of Surgery, Alberta Diabetes Institute, Edmonton, AB, Canada

Small molecules induce efficient differentiation into insulin-producing cells from human induced pluripotent stem cells

Human induced pluripotent stem (hiPS) cells have potential uses for drug discovery and cell therapy, including generation of pancreatic β-cells for diabetes research and treatment. In this study, we developed a simple protocol for generating insulin-producing cells from hiPS cells. Treatment with activin A and a GSK3β inhibitor enhanced efficient endodermal differentiation, and then combined treatment with retinoic acid, a bone morphogenic protein inhibitor, and a transforming growth factor-β (TGF-β) inhibitor induced efficient differentiation of pancreatic progenitor cells from definitive endoderm. Expression of the pancreatic progenitor markers PDX1 and NGN3 was significantly increased at this step and most cells were positive for anti-PDX1 antibody. Moreover, several compounds, including forskolin, dexamethasone, and a TGF-β inhibitor, were found to induce the differentiation of insulin-producing cells from pancreatic progenitor cells. By combined treatment with these compounds, more than 10% of the cells became insulin positive. The differentiated cells secreted human c-peptide in response to various insulin secretagogues. In addition, all five hiPS cell lines that we examined showed efficient differentiation into insulin-producing cells with this protocol.

Insulin-producing cells from human pancreatic islet-derived progenitor cells following transplantation in mice

Stem/progenitor cells hold promise for alleviating/curing type 1 diabetes due to the capacity to differentiate into functional insulin-producing cells. The current study aims to assess the differentiation potential of human pancreatic IPCs (islet-derived progenitor cells). IPCs were derived from four human donors and subjected to more than 2000-fold expansion before turning into ICCs (islet-like cell clusters). The ICCs expressed ISL-1 Glut2, PDX-1, ngn3, insulin, glucagon and somatostatin at the mRNA level and stained positive for insulin and glucagon by immunofluorescence. Following glucose challenge in vitro, C-peptide was detected in the sonicated ICCs, instead of in the conditioned medium. To examine the function of the cells in vivo, IPCs or ICCs were transplanted under the renal capsule of immunodeficient mice. One month later, 19 of 28 mice transplanted with ICCs and 4 of 14 mice with IPCs produced human C-peptide detectable in blood, indicating that the in vivo environment further facilitated the maturation of ICCs. However, among the hormone-positive mice, only 9 of 19 mice with ICCs and two of four mice with IPCs were able to secrete C-peptide in response to glucose.