Roux-en-Y gastric bypass promotes expression of PDX-1 and regeneration of β-cells in Goto-Kakizaki rats

AIM:To study the effects of Roux-en-Y gastric bypass(RYGB) on the expression of pancreatic duodenal homeobox-1(PDX-1) and pancreatic β-cell regeneration/neogenesis,and their possible mechanisms in diabetics.METHODS:Three groups of randomly selected nonobese diabetic Goto-Kakizaki(GK) rats were subjected to RYGB,sham-RYGB and sham-operation(sham-op) surgery,respectively.The rats were euthanized at postoperative 1,2,4 and 12 wk.Their pancreases were resected and analyzed using reverse transcription polymerase chain reaction to detect the mRNA of PDX-1.Anti-PDX-1 immunohistochemical(IHC) staining and Western blotting were used to detect the protein of PDX-1.Double IHC staining of anti-Brdu and-insulin was performed to detect regenerated β-cells.The index of double Brdu and insulin positive cells was calculated.RESULTS:In comparison with sham-RYGB and sham-op groups,a significant increase in the expressions of PDX-1 mRNA in RYGB group was observed at all experimental time points(1 wk:0.378 ± 0.013 vs 0.120 ± 0.010,0.100 ± 0.010,F = 727.717,P < 0.001;2 wk:0.318 ± 0.013 vs 0.110 ± 0.010,0.143 ± 0.015,F = 301.509,P < 0.001;4 wk:0.172 ± 0.011 vs 0.107 ± 0.012,0.090 ± 0.010,F = 64.297,P < 0.001;12 wk:0.140 ± 0.007 vs 0.120 ± 0.010,0.097 ± 0.015,F = 16.392,P < 0.001);PDX-1 protein in RYGB group was also increased significantly(1 wk:0.61 ± 0.01 vs 0.21 ± 0.01,0.15 ± 0.01,F = 3031.127,P < 0.001;2 wk:0.55 ± 0.00 vs 0.15 ± 0.01,0.17 ± 0.01,F = 3426.455,P < 0.001;4 wk:0.39 ± 0.01 vs 0.18 ± 0.01,0.22 ± 0.01,F = 882.909,P < 0.001;12 wk:0.41 ± 0.01 vs 0.20 ± 0.01,0.18 ± 0.01,F = 515.833,P < 0.001).PDX-1 mRNA and PDX-1 protein production showed no statistical significance between the two sham groups.Many PDX-1 positive cells could be found in the pancreatic islets of the rats in RYGB group at all time points.In addition,the percentage of Brdu-insulin double staining positive cells was higher in RYGB group than in the other two groups(1 wk:0.22 ± 0.13 vs 0.03 ± 0.06,0.03 ± 0.06,P < 0.05;2 wk:0.28 ± 0.08 vs 0.00 ± 0.00,0.03 ± 0.06,P < 0.05;4 wk:0.24 ± 0.11 vs 0.07 ± 0.06,0.00 ± 0.00,P < 0.001;12 wk:0.20 ± 0.07 vs 0.03 ± 0.06,0.00 ± 0.00,P < 0.05).CONCLUSION:RYGB can increase the expression of pancreatic PDX-1 and induce the regeneration of β-cells in GK rats.The associated regeneration of islet cells may be a possible mechanism that how RYGB could improve type 2 diabetes mellitus.

Glucose regulates LXRα subcellular localization and function in rat pancreatic β-cells

Liver X receptors(LXRs)are members of the nuclear receptor superfamily,which have been implicated in lipid ho-meostasis and more recently in glucose metabolism.Here,we show that glucose does not change LXRα protein level,but affects its localization in pancreatic β-cells.LXRα is found in the nucleus at 8 mM glucose and in the cytoplasm at4.2 mM.Addition of glucose translocates LXRα from the cytoplasm into the nucleus.Moreover,after the activation ofLXR by its synthetic non-steroidal agonist(T0901317),insulin secretion and glucose uptake are increased at 8 mM anddecreased at 4.2 mM glucose in a dose-dependent manner.Furthermore,at low glucose condition,okadaic acid reversedLXRα effect on insulin secretion,suggesting the involvement of glucose signaling through a phosphorylation-dependentmechanism.

Upregulation of α-ENaC induces pancreatic β-cell dysfunction,ER stress,and SIRT2 degradation

Islet beta cells(β-cells)produce insulin in response to high blood glucose levels,which is essential for preserving glucose homeostasis.Voltage-gated ion channels inβ-cells,including Na+,K+,and Ca2+channels,aid in the release of insulin.The epithelial sodium channel alpha subunit(α-ENaC),a voltage-independent sodium ion channel,is also expressed in human pancreatic endocrine cells.However,there is no reported study on the function of ENaC in theβ-cells.In the current study,we found thatα-ENaC was expressed in human pancreatic glandule and pancreatic isletβ-cells.In the pancreas of db/db mice and high-fat diet-induced mice,and in mouse isletβ-cells(MIN6 cells)treated with palmitate,α-ENaC expression was increased.Whenα-ENaC was overexpressed in MIN6 cells,insulin content and glucose-induced insulin secretion were significantly reduced.On the other hand,palmitate injured isletβ-cells and suppressed insulin synthesis and secretion,but increasedα-ENaC expression in MIN6 cells.However,α-ENaC knockout(Scnn1a−/−)in MIN6 cells attenuatedβ-cell disorder induced by palmitate.Furthermore,α-ENaC regulated the ubiquitylation and degradation of sirtuin 2 inβ-cells.α-ENaC also modulatedβ-cell function in correlation with the inositol-requiring enzyme 1 alpha/X-box binding protein 1(IRE1α/XBP1)and protein kinase RNA-like endoplasmic reticulum kinase/C/EBP homologous protein(PERK/CHOP)endoplasmic reticulum stress pathways.These results suggest thatα-ENaC may play a novel role in insulin synthesis and secretion in theβ-cells,and the upregulation ofα-ENaC promotes isletβ-cell dysfunction.In conclusion,α-ENaC may be a key regulator involved in isletβ-cell damage and a potential therapeutic target for type 2 diabetes mellitus.

The zinc transporter Slc39a5 controls glucose sensing and insulin secretion in pancreatic β-cells via Sirt1- and Pgc-1α-mediated regulation of Glut2

Zinc levels are high in pancreatic β-cells, and zinc is involved in the synthesis, processing and secretion of insulin in these cells. However, precisely how cellular zinc homeostasis is regulated in pancreatic β-cells is poorly understood. By screening the expression of 14 Slc39a metal importer family member genes, we found that the zinc transporter Slc39a5 is significantly downregulated in pancreatic β-cells in diabetic db/db mice, obese ob/ob mice and high-fat diet-fed mice. Moreover,β-cell-specific Slc39a5 knockout mice have impaired insulin secretion. In addition, Slc39a5-deficient pancreatic islets have reduced glucose tolerance accompanied by reduced expression of Pgc-1α and its downstream target gene Glut2. The down-regulation of Glut2 in Slc39a5-deficient islets was rescued using agonists of Sirt1, Pgc-1α and Ppar-γ. At the mechanistic level, we found that Slc39a5-mediated zinc influx induces Glut2 expression via Sirt1-mediated Pgc-1α activation. These findings suggest that Slc39a5 may serve as a possible therapeutic target for diabetes-related conditions.

Antioxidant Effects of Alperujo Extract (Arbequina and Frantoio Varieties) on MIN6 <i>β</i>-Cells Subjected to Stress with Glucose or H₂O₂

Alperujo, an antioxidant-rich by-product of olive oil extraction, could protect β-cells against oxidative damage. Our goal was to study the antioxidant effects of an alperujo extract (AE) on MIN6 β-cells challenged with glucose or hydrogen peroxide. MIN6 β-cells were challenged with glucose (100 mM) or H2O2 (0.15 mM), with or without AE (20 μM phenol). Reactive oxygen species, intracellular iron (Fe), insulin, glucose uptake, and mRNA gene expression of Uncoupling Protein-2 (UCP-2), Thioredoxin (TRDX), p47phox, and the ratio Bax/Bcl-2 were measured. ROS increased when the stressors were incubated with AE (p < 0.05 and p < 0.01, respectively). Intracellular Fe increased in glucose presence (100 mM p < 0.001). Insulin secretion improved when cells were pre-incubated with AE (p < 0.001) and glucose uptake increased when cells were pre-incubated with AE for 3 days and then further treated with glucose (p < 0.001). After 3 days of AE alone, mRNA relative expression of UCP-2 and TRDX increased (p < 0.001) and after 5 days p47phox, also increased. The Bax/Bcl-2 ratio tended to decrease in the samples pre-incubated with AE. The Alperujo extract,in vitro, had a pro-oxidant behavior, however pre-incubating MIN6 β-cells with AE tended to protect them against apoptosis, thereby enhancing insulin secretion.

Cyclic nucleotide phosphodiesterase 3B is connected to osteopontin and protein kinase CK2 in pancreatic <i>β</i>-cells

Islets from RIP-PDE3B mice, exhibiting β-cell specific overexpression of the cAMP/cGMP-degrading enzyme phosphodiesterase 3B (PDE3B) and dysregulated insulin secretion, were subjected to microarray analysis. We show that osteopontin (OPN) mRNA is increased in a dose-dependent manner in islets from RIP-PDE3B mice, as compared to wild-type islets. In addition, in silico analysis shows that PDE3B and OPN are interacting. Furthermore, OPN interacts with protein kinase CK2 ina distinct submodule of the protein-protein interaction network. We studied PDE3B and OPN proteins and, in some cases, also PDE1B and PDE4C, under conditions of relevance for insulin secretion. In the presence of forskolin, PDE inhibitors, insulin, or a protein kinase CK2 inhibitor, similar alterations in protein levels of PDE3B and OPN are shown. In summary, results from using a number of strategies demonstrate a connection between PDE3B and OPNas well as a role for protein kinase CK2 inpancreatic β-cells.

Chronic alcohol consumption potentiates the development of diabetes through pancreatic β-cell dysfunction

Chronic ethanol consumption is well established as a major risk factor for type-2 diabetes(T2D), which is evidenced by impaired glucose metabolism and insulin resistance. However, the relationships between alcoholconsumption and the development of T2 D remain controversial. In particular, the direct effects of ethanol consumption on proliferation of pancreatic β-cell and the exact mechanisms associated with ethanolmediated β-cell dysfunction and apoptosis remain elusive. Although alcoholism and alcohol consumption are prevalent and represent crucial public health problems worldwide, many people believe that low-tomoderate ethanol consumption may protect against T2 D and cardiovascular diseases. However, the J- or U-shaped curves obtained from cross-sectional and large prospective studies have not fully explained the relationship between alcohol consumption and T2 D. This review provides evidence for the harmful effects of chronic ethanol consumption on the progressive development of T2 D, particularly with respect to pancreatic β-cell mass and function in association with insulin synthesis and secretion. This review also discusses a conceptual framework for how ethanolproduced peroxynitrite contributes to pancreatic β-cell dysfunction and metabolic syndrome.

Pancreatic islet transplantation

Type 1 diabetes mellitus is an autoimmune disease,which results in the permanent destruction of β-cells of the pancreatic islets of Langerhans.While exogenous insulin therapy has dramatically improved the quality of life,chronic diabetic complications develop in a substantial proportion of subjects and these complications generally progress and worsen over time.Although intensive insulin therapy has proven effective to delay and sometimes prevent the progression of complications such as nephropathy,neuropathy or retinopathy,it is difficult to achieve and maintain long term in most subjects.Reasons for this diff iculty include compliance issues and the increased risk of severe hypoglycemic episodes,which are generally associated with intensification of exogenous insulin therapy.Clinical studies have shown that transplantation of pancreas or purified pancreatic islets can support glucose homeostasis in type 1 diabetic patients.Islet transplantation carries the special advantages of being less invasive and resulting in fewer complications compared with the traditional pancreas or pancreas-kidney transplantation.However,islet transplantation efforts have limitations including the short supply of donor pancreata,the paucity of experienced islet isolation teams,side effects of immunosuppressants and poor long-term results.The purpose of this article is to review recent progress in clinical islet transplantation for the treatment of diabetes.

ISLET FORMATION AND REGENERATION

Objective To explore the mechanisms of differentiation and development of pancreatic endocrine cells as well as pancreatic regeneration.Methods Human embryonic pancreatic tissue at 7-14 weeks of gestation was collected.Diabetes mellitus rat model was induced with 65 mg/kg of streptozotocin.Insulin, glucagon, somatostatin, nestin, and cytokeratin 19 (CK19) of pancreatic tissues were observed by immunohistochemistry.Results At 9 weeks of gestation, pancreatic epithelial cells began to co-express insulin, glucagon, somatostatin, and CK19 before migration.Islet cells gradually congregated along with the increase of aging, and at 14 weeks of gestation histological examination showed islet formation.At 12 weeks of gestation, nestin-positive cells could be seen in the pancreatic mesenchyme.During early embryogenesis, islet cells of pancreatic ducts co-expressed insulin, glucagon, and somatostatin.During pancreatic regeneration after damage, nestin expression of islet cells increased.Conclusion In the early stage of embryogenesis, islet cells of primary pancreatic ducts can be differentiated to multipotential endocrine cells before migration.During tissue regeneration, pancreatic stem cells may differentiate and proliferate to form pancreatic islet.

Regenerative medicine of pancreatic islets

The pancreas became one of the first objects of regenerative medicine,since other possibilities of dealing with the pancreatic endocrine insufficiency were clearly exhausted.The number of people living with diabetes mellitus is currently approaching half a billion,hence the crucial relevance of new methods to stimulate regeneration of the insulin-secretingβ-cells of the islets of Langerhans.Natural restrictions on the islet regeneration are very tight;nevertheless,the islets are capable of physiological regeneration viaβ-cell self-replication,direct differentiation of multipotent progenitor cells and spontaneousα-toβ-orδ-toβ-cell conversion(trans-differentiation).The existing preclinical models ofβ-cell dysfunction or ablation(induced surgically,chemically or genetically)have significantly expanded our understanding of reparative regeneration of the islets and possible ways of its stimulation.The ultimate goal,sufficient level of functional activity ofβ-cells or their substitutes can be achieved by two prospective broad strategies:β-cell replacement andβ-cell regeneration.The“regeneration”strategy aims to maintain a preserved population ofβ-cells through in situ exposure to biologically active substances that improveβ-cell survival,replication and insulin secretion,or to evoke the intrinsic adaptive mechanisms triggering the spontaneous non-β-toβ-cell conversion.The“replacement”strategy implies transplantation ofβ-cells(as non-disintegrated pancreatic material or isolated donor islets)orβ-like cells obtained ex vivo from progenitors or mature somatic cells(for example,hepatocytes orα-cells)under the action of small-molecule inducers or by genetic modification.We believe that the huge volume of experimental and clinical studies will finally allow a safe and effective solution to a seemingly simple goal-restoration of the functionally activeβ-cells,the innermost hope of millions of people globally.

Applications of stem cells and bioprinting for potential treatment of diabetes

Currently, there does not exist a strategy that can reduce diabetes and scientists are working towards a cure and innovative approaches by employing stem cellbased therapies. On the other hand, bioprinting technology is a novel therapeutic approach that aims to replace the diseased or lost β-cells, insulin-secreting cells in the pancreas, which can potentially regenerate damaged organs such as the pancreas. Stem cells have the ability to differentiate into various cell lines including insulinproducing cells. However, there are still barriers that hamper the successful differentiation of stem cells into β-cells. In this review, we focus on the potential applications of stem cell research and bioprinting that may be targeted towards replacing the β-cells in the pancreas and may offer approaches towards treatment of diabetes. This review emphasizes on the applicability of employing both stem cells and other cells in 3 D bioprinting to generate substitutes for diseased β-cells and recover lost pancreatic functions. The article then proceeds to discuss the overall research done in the field of stem cell-based bioprinting and provides future directions for improving the same for potential applications in diabetic research.

Recent progress in pancreatic islet transplantation

Diabetes mellitus remains a major burden.More than 200 million people are affected worldwide,which represents 6%of the world’s population.Type 1 diabetes mellitus is an autoimmune disease,which induces the permanent destruction of theβ-cells of the pancreatic islets of Langerhans.Although intensive insulin therapy has proven effective to delay and sometimes prevent the progression of complications such as nephropathy,neuropathy or retinopathy,it is difficult to achieve and maintain long term in most subjects.The successes achieved over the last few decades by the transplantation of whole pancreas and isolated islets suggest that diabetes can be cured by the replenishment of deficientβcells.However,islet transplantation efforts have various limitations,including the limited supply of donor pancreata,the paucity of experienced islet isolation teams,side effects of immunosuppressants and poor long term results.The purpose of this article is to review the recent progress in clinical islet transplantation for the treatment of diabetes and to describe the recent progress on pancreatic stem/progenitor cell research,which has opened up several possibilities for the development of new treatments for diabetes.

Recent advances in stem cell research for the treatment of diabetes

The success achieved over the last decade with islet transplantation has intensified interest in treating diabetes,not only by cell transplantation,but also by stem cells.The formation of insulin-producing cells from pancreatic duct,acinar,and liver cells is an active area of investigation.Protocols for the in vitro differentiation of embryonic stem(ES)cells based on normal developmental processes,have generated insulin-producing cells,though at low efficiency and without full responsiveness to extracellular levels of glucose.Induced pluripotent stem cells,which have been generated from somatic cells by introducing Oct3/4,Sox2,Klf4,and c-Myc,and which are similar to ES cells in morphology,gene expression,epigenetic status and differentiation,can also differentiate into insulin-producing cells.Overexpression of embryonic transcription factors in stem cells could efficiently induce their differentiation into insulin-expressing cells. The purpose of this review is to demonstrate recent progress in the research for new sources ofβ-cells, and to discuss strategies for the treatment of diabetes.

Transcriptome Profiles and Gene Expression of Min6 Cells Are Altered by Pancreatic Stellate Cells

Aim: To identify the influence of pancreatic stellate cell (PSCs) secretions on gene expression profiles of Min6 cells by whole transcriptome sequencing. Methods: Pancreatic stellate cells (PSCs) were isolated from C57BL6J mice and propagated in vitro to acquire the activated phenotype. Total RNA was isolated from monocultured (MC) and PSC cocultured (CC) Min6 cells to prepare cDNA libraries, which were subjected to whole transcriptome sequencing for identifying differential expression of β-cell transcription factors (Pdx-1, Rfx6 and NeuroD1) related to insulin gene transcription and GSIS related genes such as Glut2, Gck, Abcc8, Kcnj11 and L-type Ca2+ channels (Cacnb2, Cacna1c). qRT-PCR was used to validate the gene expression. GSIS of Min6 cells was examined by estimating insulin levels in response to high glucose challenge. Results: Transcriptome analysis of discovery set revealed that coculture of Min6 cells with PSCs caused increased expression of β-cell specific genes (Ins1, Rfx6 and NeuroD1) concomitant with decreased expression of Pdx-1, MafA and Nkx2-2. Expression of GSIS associated genes (Glut2, Gck, Abcc8, Kcnj11 and Cacnb2) was decreased in such conditions. Validation by qRT-PCR in Min6 cells cocultured with PSCs revealed increased significant expression of Ins1 (2.1 ± 0.22 folds;p ≤ 0.001), Rfx6 (1.68 ± 0.23 folds;p ≤ 0.002) and NeuroD1 (0.96 ± 0.11 folds;p ≤ 0.01), accompanied by downregulation of Cacnb2 (-0.93 ± 0.57 folds;p ≤ 0.05). PSC secretions did not restore the GSIS from glucose unresponsive higher passage Min6 cells (MC: 1.33 ± 0.42;CC: 1.55 ± 0.72 pmol/mg protein;p = ns) upon high glucose stimulation. However, glucose responsive higher passage Min6 cells cocultured with PSCs presented increased insulin secretion (MC: 7.025 ± 0.64;CC: 14.84 ± 1.01 pmol/mg protein;p ≤ 0.04) concomitant with marginal increase of insulin contents. Conclusion: PSC secretions increase Ins1, Rfx6 and NeuroD1 gene expression, GSIS from glucose responsive Min6 cells, but do not restore the GSIS from glucose unresponsive Min6 cells.

Depletion of insulin receptors leads to β-cell hyperplasia in zebrafish

Hyperglycemia in type 2 diabetes results from an inability of insulin to regulate gluconeogenesis.To characterize the role of the insulin/insulin receptor pathway in glycometabolism and type 2 diabetes,we created a zebrafish model in which insulin receptors a and b(insra and insrb) have been ablated.We first observed that insra and insrb were both expressed abundantly during embryonic development and in various adult tissues.Increased expression of insulin and number of b-cells were observed in insra-/-/-insrb-/-fish together with higher glucose in insra-/-,insrb-/-,or insra-/-/-insrb-/-fish,indicating that insra and insrb were knocked out effectively.However,compared to the wild-type fish,insra-/-/-insrb-/-fish died between 5 and 16 days post-fertilization(dpf) with severe pericardial edema and increased level of cell apoptosis,which was not induced by increased total body glucose content.Increased gluconeogenesis and decreased glycolysis were also observed in both single and double knockout fish,but no mortality or malformation was observed in single knockout fish.Given the importance of insulin receptors in glucose homeostasis and embryonic development,transcriptome analysis was used to provide an important model of defective insulin signaling and to study its developmental consequences in zebrafish.The results indicated that both insra and insrb played a pivotal role in glucose metabolism and embryonic development,and insra was more critical than insrb in the insulin signaling pathway.

The role of fatty acid metabolism and lipotoxicity in pancreatic β-cell injury:Identification of potential therapeutic targets

Over the last 20 years,intensive research has been focused on the specific mechanisms mediating the pancreatic β-cell injury.Both the decreased viability and the dysfunction of β-cells have become the key factors in the development of dia betes mellitus.Thus,it is of utmost importance to elucidate the discrete pathological changes in pancreatic β-cells within the context of the various lipotoxicity models.The goal of these studies is to generate evidence to improve not only the clinical treatment for dia betics,but also modulate the diet and activities of groups at high risk for diabetes.In this regard,we review the role of lipotoxicity in pancreatic β-cell injury and identify potential therapeutic targets in this cell model.

Muscadine or amla extracts standardized to ellagic acid content ameliorate glucolipotoxicity associatedβ-cell dysfunction via inhibition of IL-1βand improved insulin secretion

Glucolipotocixity induces IL-1βsecretion which impairs pancreaticβ-cell insulin secretion.Ellagic acid and urolithin A have strong anti-inflammatory effect on cells.Muscadine and amla are very good sources of ellagic acid.The present study examined the effect of ellagic acid,ellagic acid-rich muscadine or amla extract,or urolothin A on inflammation inβcells under glucolipotoxic conditions.Rat NIT-1βcells were incubated in glucolipotoxic conditions(33.3 mM glucose,250μM palmitic acid or 33.3 mM glucose+250μM palmitic acid with or without ellagic acid,ellagic acid-rich muscadine or amla extracts standardized to its ellagic acid content,or urolithin A).Inflammatory status was evidenced by ELISA analysis of insulin and IL-1βsecretion.Ellagic acid-rich muscadine or amla extracts dose-dependently stimulated insulin secretion and down-regulated IL-1βbetter than pure ellagic acid,or urolithin A.Urolithin A did not statistically stimulate insulin secretion and did not inhibit IL-1β.

Oral nano-formulation improves pancreatic islets dysfunction via lymphatic transport for antidiabetic treatment

Type 2 diabetes mellitus(T2DM)therapy is facing the challenges of long-term medication and gradual destruction of pancreatic isletβ-cells.Therefore,it is timely to develop oral prolonged action formulations to improve compliance,while restoringβ-cells survival and function.Herein,we designed a simple nanoparticle with enhanced oral absorption and pancreas accumulation property,which combined apical sodiumdependent bile acid transporter-mediated intestinal uptake and lymphatic transportation.In this system,taurocholic acid(TCA)modified poly(lactic-co-glycolic acid)(PLGA)was employed to achieve pancreas location,hydroxychloroquine(HCQ)was loaded to execute therapeutic efficacy,and 1,2-dilauroyl-sn-glycero-3-phosphocholine(DLPC)was introduced as stabilizer together with synergist(PLGA-TCA/DLPC/HCQ).In vitro and in vivo results have proven that PLGA-TCA/DLPC/HCQ reversed the pancreatic islets damage and dysfunction,thus impeding hyperglycemia progression and restoring systemic glucose homeostasis via only once administration every day.In terms of mechanism PLGA-TCA/DLPC/HCQ ameliorated oxidative stress,remodeled the inflammatory pancreas microenvironment,and activated PI3K/AKT signaling pathway without obvious toxicity.This strategy not only provides an oral delivery platform for increasing absorption and pancreas targetability but also opens a new avenue for thorough T2DM treatment.

Generation of insulin-producing β-like cells from human iPS cells in a defined and completely xeno-free culture system

Human induced pluripotent stem （hiPS） cells are considered a potential source for the generation of insulin-producing pancreatic β-ceUs because of their differentiation capacity. In this study, we have developed a five-step xeno-free culture system to efficiently dif- ferentiate hiPS cells into insulin-producing cells in vitro. We found that a high NOGGIN concentration is crucial for specifically inducing the differentiation first into pancreatic and duodenal homeobox-1 （PDX1）-positive pancreatic progenitors and then into neurogenin 3 （NGN3）-expressing pancreatic endocrine progenitors, while suppressing the differentiation into hepatic or intestinal cells. We also found that a combination of 3-isobutyl-l-methylxanthine （IBMX）, exendin-4, and nicotinamide was important for the differentiation into insulin single-positive cells that expressed various pancreatic β-cell markers. Most notably, the differentiated cells contained en- dogenous C-peptide pools that were released in response to various insulin secretagogues and high levels of glucose. Therefore, our results demonstrate the feasibility of generating hiPS-derived pancreatic β-ceUs under xeno-free conditions and highlight their poten- tial to treat patients with type I diabetes.