Mature Beta Cells Research Articles

Mesenchymal cells are required for epithelial duct cell-to-beta cell maturation and function in an injured adult pancreas in the rat

The islet, the endocrine portion of the pancreas − develops from an invagination of the pancreatic duct epithelial cells (PDECs) into the surrounding tissue. The contact of the PDECs with mesenchymal cells (MSCs) may be an essential drive for endocrine cell fate. During pancreatic development, cells that express Neurogenin-3 (Ngn3) biomarker are precursors of insulin- producing beta cells. These precursors have been reported in the neogenesis of islets from adult tissues following the surgical ligation of the main pancreatic duct (PDL). But the capacity of these precursors to induce the appropriate signals to complete the entire neogenesis program has been questioned. We studied the fate of co-culture of PDECs and MSCs from the ligated adult pancreas and established the exact location of adult stem- or progenitor-like cells that give rise to beta cells. PDECs were cultured in direct contact with or without MSCs in serum-containing culture media. The cytomorphology of the cells in co-cultures was determined and the immunocytochemical study of the cells was carried out using anti-Ngn3, anti-insulin and anti-cytokeratin-7 (CK7) antibodies. Both the PDEC/MSC- and PDEC/MSC+ cultures showed out- pocketing from duct epithelium by the end of the second week, which are distinct as cell clusters only in PDEC/MSC+ cells later in week four, exhibiting numerous branching ducts. Co-expression of Ngn3 with insulin was observed in both cultures from the second week. However, characterizations of these Ngn3+ cells in the PDEC/MSC+ culture revealed that these cells also co-expressed a CK7 biomarker. This study provides new evidence of the ductal epithelial nature of beta cells in injured adult pancreata; and that the mesenchymal stromal cells are required to sustain Ngn3 expression for beta cell maturation and function.

ROCKII inhibition promotes the maturation of human pancreatic beta-like cells

Diabetes is linked to loss of pancreatic beta-cells. Pluripotent stem cells offer a valuable source of human beta-cells for basic studies of their biology and translational applications. However, the signalling pathways that regulate beta-cell development and functional maturation are not fully understood. Here we report a high content chemical screen, revealing that H1152, a ROCK inhibitor, promotes the robust generation of insulin-expressing cells from multiple hPSC lines. The insulin expressing cells obtained after H1152 treatment show increased expression of mature beta cell markers and improved glucose stimulated insulin secretion. Moreover, the H1152-treated beta-like cells show enhanced glucose stimulated insulin secretion and increased capacity to maintain glucose homeostasis after transplantation. Conditional gene knockdown reveals that inhibition of ROCKII promotes the generation and maturation of glucose-responding cells. This study provides a strategy to promote human beta-cell maturation and identifies an unexpected role for the ROCKII pathway in the development and maturation of beta-like cells.

Do we really need to differentiate mesenchymal stem cells into insulin-producing cells for attenuation of the autoimmune responses in type 1 diabetes: immunoprophylactic effects of precursors to insulin-producing cells

BackgroundType 1 diabetes (T1D) is a multifactorial autoimmune disorder where pancreatic beta cells are lost before the clinical manifestations of the disease. Administration of mesenchymal stem cells (MSCs) or MSCs differentiated into insulin-producing cells (IPCs) have yielded limited success when used therapeutically. We have evaluated the immunoprophylactic potentials of precursors to insulin-producing cells (pIPCs) and IPCs in nonobese diabetic (NOD) mice to ask a basic question: do we need to differentiate MSCs into IPCs or will pIPCs suffice to attenuate autoimmune responses in T1D?MethodsBone marrow-derived MSCs from Balb/c mice were characterized following the International Society for Cellular Therapy (ISCT) guidelines. MSCs cultured in high-glucose media for 11 to 13 passages were characterized for the expression of pancreatic lineage genes using real-time polymerase chain reaction. Expression of the PDX1 gene in pIPCs was assessed using Western blot and fluorescence-activated cell sorting (FACS). Triple-positive MSCs were differentiated into IPCs using a three-step protocol after sorting them for cell surface markers, i.e. CD29, CD44, and SCA-1. Nonobese diabetic mice were administered pIPCs, IPCs, or phosphate-buffered saline (PBS) into the tail vein at weeks 9 or 10 and followed-up for 29–30 weeks for fasting blood glucose levels. Two consecutive blood sugar levels of more than 250 mg/dl were considered diabetic.ResultsMSCs grown in high-glucose media for 11 to 13 passages expressed genes of the pancreatic lineage such as PDX1, beta2, neurogenin, PAX4, Insulin, and glucagon. Furthermore, Western blot and FACS analysis for PDX-1, a transcription factor necessary for beta cell maturation, confirmed that these cells were precursors of insulin-producing cells (pIPCs). NOD mice administered with pIPCs were better protected from developing diabetes with a protective efficacy of 78.4% (p < 0.009); however, administration of IPCs gave protective efficacy of 55% at the end of 28–30 weeks.ConclusionsPrecursors to insulin-producing cells seem to have better potential to arrest autoimmune response in type 1 diabetes when administered before the onset of the disease in NOD mice. When translated to humans, autologous mesenchymal stem cells grown in high-glucose media for 10 to 13 passages may have beneficial effects in individuals at high risk of developing type 1 diabetes.

MicroRNAs modulate core-clock gene expression in pancreatic islets during early postnatal life in rats.

Evidence continues to emerge detailing a fine-tuning of the regulation of metabolic processes and energy homeostasis by cell-autonomous circadian clocks. Pancreatic beta cell functional maturation occurs after birth and implies transcriptional changes triggered by a shift in the nutritional supply that occurs at weaning, enabling the adaptation of insulin secretion. So far, the developmental timing and exact mechanisms involved in the initiation of the circadian clock in the growing pancreatic islets have never been addressed. Circadian gene expression was measured by quantitative RT-PCR in islets of rats at different postnatal ages up to 3months, and by in vitro bioluminescence recording in newborn (10-day-old) and adult (3-month-old) islets. The effect of the microRNAs miR-17-5p and miR-29b-3p on the expression of target circadian genes was assessed in newborn rat islets transfected with microRNA antisense or mimic oligonucleotides, and luciferase reporter assays were performed on the rat insulin-secreting cell line INS832/13 to determine a direct effect. The global regulatory network between microRNAs and circadian genes was computationally predicted. We found up to a sixfold-change in the 24h transcriptional oscillations and overall expression of Clock, Npas2, Bmal1, Bmal2, Rev-erbα, Per1, Per2, Per3 and Cry2 between newborn and adult rat islets. Synchronisation of the clock machinery in cultured islet cells revealed a delayed cell-autonomous rhythmicity of about 1.5h in newborn compared with adult rats. Computational predictions unveiled the existence of a complex regulatory network linking over 40 microRNAs displaying modifications in their expression profiles during postnatal beta cell maturation and key core-clock genes. In agreement with these computational predictions, we demonstrated that miR-17-5p and miR-29b-3p directly regulated circadian gene expression in the maturing islet cells of 10-day-old rats. These data show that the circadian clock is not fully operational in newborn islets and that microRNAs potently contribute to its regulation during postnatal beta cell maturation. Defects in this process may have long-term consequences on circadian physiology and pancreatic islet function, favouring the manifestation of metabolic diseases such as diabetes.

Raptor regulates functional maturation of murine beta cells

Diabetes is associated with beta cell mass loss and islet dysfunctions. mTORC1 regulates beta cell survival, proliferation and function in physiological and pathological conditions, such as pregnancy and pancreatectomy. Here we show that deletion of Raptor, which is an essential component of mTORC1, in insulin-expressing cells promotes hypoinsulinemia and glucose intolerance. Raptor-deficient beta cells display reduced glucose responsiveness and exhibit a glucose metabolic profile resembling fetal beta cells. Knockout islets have decreased expression of key factors of functional maturation and upregulation of neonatal markers and beta cell disallowed genes, resulting in loss of functional maturity. Mechanistically, Raptor-deficient beta cells show reduced expression of DNA-methyltransferase 3a and altered patterns of DNA methylation at loci that are involved in the repression of disallowed genes. The present findings highlight a novel role of mTORC1 as a core mechanism governing postnatal beta cell maturation and physiologic beta cell mass during adulthood.

Generation of Insulin‐Producing Cells From Human‐Induced Pluripotent Stem Cells Using a Stepwise Differentiation Protocol Optimized With Platelet‐Rich Plasma

Studies on patient-specific human-induced pluripotent stem cells (hiPSCs) as well as a series of autologous growth factors presumably will reveal their many benefits for cell base replacement therapy in type 1 diabetes mellitus (TIDM) patients in the future. For this purpose, we established a multistep protocol by adding platelet-rich plasma (PRP) that induce the hiPSCs into insulin-producing cells (IPCs). We present here a differentiation protocol consisting of five stages in two groups including protocol with PRP and without PRP. Charac-teristics of derived IPCs in both groups were evaluated at the mRNA and protein levels, cell cycle and viability in the end stage of cell differentiation. The in vitro studies indicated the treatment of hiPSCs in the protocol with PRP resulting in differentiated cells with strong characteristics of IPCs including islet-like cells, the expression of mature and functional pancreatic beta cell specific marker genes. In addition to these pancreatic specific markers were detected by immunochemistry and Western blot. Our differentiated cells in two groups secreted insulin and C-peptide in a glucose stimulation test by ELISA showing in vitro functional. The results of the cell cycle assay confirmed that differentiation has been done. We reported for the first time that PRP might be ideal additive in the culture medium to induce pancreatic differentiation in the hiPSCs. This study provides a new approach to investigate the role of PRP in pancreatic differentiation protocols and enhance the feasibility of using patient-specific iPSCs and autologous PRP for future beta cells replacement therapies for T1DM. J. Cell. Physiol. 232: 2878-2886, 2017. © 2016 Wiley Periodicals, Inc.

Virgin Beta Cells Persist throughout Life at a Neogenic Niche within Pancreatic Islets

Postnatal maintenance or regeneration of pancreatic beta cells is considered to occur exclusively via the replication of existing beta cells, but clinically meaningful restoration of human beta cell mass by proliferation has never been achieved. We discovered a population of immature beta cells that is present throughout life and forms from non-beta precursors at a specialized micro-environment or "neogenic niche" at the islet periphery. These cells express insulin, but lack other key beta cell markers, and are transcriptionally immature, incapable of sensing glucose, and unable to support calcium influx. They constitute an intermediate stage in the transdifferentiation of alpha cells to cells that are functionally indistinguishable from conventional beta cells. We thus identified a lifelong source of new beta cells at a specialized site within healthy islets. By comparing co-existing immature and mature beta cells within healthy islets, we stand to learn how to mature insulin-expressing cells into functional beta cells.

A Presenilin/Notch1 pathway regulated by miR-375, miR-30a, and miR-34a mediates glucotoxicity induced-pancreatic beta cell apoptosis.

The presenilin-mediated Notch1 cleavage pathway plays a critical role in controlling pancreatic beta cell fate and survival. The aim of the present study was to investigate the role of Notch1 activation in glucotoxicity-induced beta cell impairment and the contributions of miR-375, miR-30a, and miR-34a to this pathway. We found that the protein levels of presenilins (PSEN1 and PSEN2), and NOTCH1 were decreased in INS-1 cells after treatment with increased concentrations of glucose, whereas no significant alteration of mRNA level of Notch1 was observed. Targeting of miR-375, miR-30a, and miR-34a to the 3′utr of Psen1, Psen2, and Notch1, respectively, reduced the amounts of relevant proteins, thereby reducing NICD1 amounts and causing beta cell apoptosis. Overexpression of NICD1 blocked the effects of glucotoxicity as well as miRNA overabundance. Downregulating the expression of miR-375, miR-30a, and miR-34a restored PSEN1, PSEN2, and NICD1 production and prevented glucotoxicity-induced impairment of the beta cells. These patterns of miRNA regulation of the Notch1 cleavage pathway were reproduced in GK rats as well as in aged rats. Our findings demonstrated that miRNA-mediated suppression of NICD1 links the presenilin/Notch1 pathway to glucotoxicity in mature pancreatic beta cells.

Elucidation of mechanism of human pancreatic beta-cell maturation with hiPSC reporter line

Elucidation of mechanism of human pancreatic beta-cell maturation with hiPSC reporter line

Beta Cell Regeneration in Adult Mice: Controversy Over the Involvement of Stem Cells.

Islet transplantation is an effective therapy for severe diabetes. Nevertheless, the short supply of donor pancreases constitutes a formidable obstacle to its extensive clinical application. This shortage heightens the need for alternative sources of insulin-producing beta cells. Since mature beta cells have a very slow proliferation rate, which further declines with age, great efforts have been made to identify beta cell progenitors in the adult pancreas. However, the question whether facultative beta cell progenitors indeed exist in the adult pancreas remains largely unresolved. In the current review, we discuss the problems in past studies and review the milestone studies and recent publications.

Effective endothelial cell and human pluripotent stem cell interactions generate functional insulin-producing beta cells.

Aims/hypothesisEndothelial cells (ECs) play an essential role in pancreatic organogenesis. We hypothesise that effective in vitro interactions between human microvascular endothelial cells (HMECs) and human pluripotent stem cells (hPSCs) results in the generation of functional pancreatic beta cells.MethodsEmbryoid bodies (EBs) derived from hPSCs were cultured alone (controls) or with ECs in collagen gels. Subsequently, cells were analysed for pancreatic beta cell markers, and then isolated and expanded. Insulin secretion in response to glucose was evaluated in vitro by static and dynamic (perifusion) assays, and in vivo by EB transplantation into immunodeficient mice.ResultsCo-cultured EBs had a higher expression of mature beta cells markers and enhanced insulin secretion in vitro, compared with controls. In mice, transplanted EBs had higher levels of human C-peptide secretion with a significant reduction in hyperglycaemia after the selective destruction of native pancreatic beta cells. In addition, there was significant in vitro upregulation of bone morphogenetic proteins 2 and 4 (BMP-2, 4) in co-cultured cells, compared with controls.Conclusions/interpretationECs provide essential signalling in vitro, such as activation of the BMP pathway, for derivation of functional insulin-producing beta cells from hPSCs.Electronic supplementary materialThe online version of this article (doi:10.1007/s00125-016-4078-1) contains peer-reviewed but unedited supplementary material, which is available to authorised users.

Abstract 478: A mouse model for highly metastatic, nonfunctioning pancreatic neuroendocrine tumors

Abstract Pancreatic neuroendocrine tumors are generally classified as functioning or nonfunctioning. Patients with functioning tumors can have severe hypoglycemia because their tumors express high amounts of insulin as well as chromogranin A; fortunately, these tumors are seldom metastatic. Patients with nonfunctioning tumors express high amounts of chromogranin A but little insulin; unfortunately these tumors are highly metastatic to the liver. Nonfunctioning tumors are more common, and a more severe health issue. The highly studied RT2 B6 mouse, in which the SV40 T antigen is expressed from an insulin promoter in an inbred C57Bl/6 genetic background, is a model for functioning pancreatic neuroendocrine tumors: liver metastasis is uncommon in RT2 B6 mice, which are very hypoglycemic and often die when tumors are very small. We have changed the genetic background and are studying RT2 AB6F1 hybrid mice, which have a C57Bl/6 father and a mother from an A/J inbred background. RT2 AB6F1 mice are much less hypoglycemic, and these mice develop liver metastasis with high frequency. We have mated the hybrid AB6F1 mice to their littermates in order to map metastasis genes by F2 analysis, and have discovered that a locus at mouse chromosome 2qG1 links tightly to liver metastasis. One gene within this locus, InsM1, is less expressed in the primary tumors of mice with liver metastatic disease. InsM1 is also less expressed in primary tumors from humans with liver metastatic disease. Previous studies of InsM1 have shown that this protein maintains the differentiated status of pancreatic beta cells. We find that tumors from mice with low expression of InsM1 also express lower amounts of several distinctive markers of differentiated beta cells, but higher amounts of several markers of liver cells. These data suggest that dedifferentiation to stem cells, which can then redifferentiate to other cell types such as liver cells, promotes metastasis of pancreatic beta cell tumors. Intratumoral expression of liver markers may be a useful predictor of metastatic disease in these patients. Dedifferentiation of mature beta cells is also a mechanism by which type 2 diabetes occurs, so there may be similarities between these two diseases. Citation Format: Tanupriya Contractor, Shinta Kobayashi, Richard Clausen, Edaise da Silva, Laura Tang, Chang Chan, Chris R. Harris. A mouse model for highly metastatic, nonfunctioning pancreatic neuroendocrine tumors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 478.

Targeting insulin-producing beta cells for regenerative therapy.

Pancreatic beta cells differ in terms of glucose responsiveness, insulin secretion and proliferative capacity; however, the molecular pathways that regulate this cellular heterogeneity are unknown. We have identified the Wnt-planar cell polarity (PCP) effector Flattop (FLTP) as a biomarker that identifies mature beta cells in the islets of Langerhans. Interestingly, three-dimensional architecture and Wnt-PCP ligands are sufficient to trigger mouse and human beta cell maturation. These results highlight the fact that novel biomarkers shed light on the long-standing mystery of beta cell heterogeneity and identify the Wnt-PCP pathway as triggering beta cell maturation. Understanding heterogeneity in the islets of Langerhans might allow targeting of beta cell subpopulations for regenerative therapy and provide building principles for stem cell-derived islets. This review summarises a presentation given at the 'Can we make a better beta cell?' symposium at the 2015 annual meeting of the EASD. It is accompanied by two other reviews on topics from this symposium (by Amin Ardestani and Kathrin Maedler, DOI: 10.1007/s00125-016-3892-9 , and by Harry Heimberg and colleagues, DOI: 10.1007/s00125-016-3879-6 ) and a commentary by the Session Chair, Shanta Persaud (DOI: 10.1007/s00125-016-3870-2 ).

Closing in on Mass Production of Mature Human Beta Cells

Human pluripotent stem cell differentiation protocols based on mimicking developmental pathways are getting close to generating fully fledged pancreatic endocrine cells, including insulin-producing beta cells. However, challenges remain in identifying pathways to trigger the attainment of robust glucose responsiveness that occurs postnatally in beta cells.

Transcriptome landmarks of the functional maturity of rat beta-cells, from lactation to adulthood.

Research on the postnatal development of pancreatic beta-cells has become an important subject in recent years. Understanding the mechanisms that govern beta-cell postnatal maturation could bring new opportunities to therapeutic approaches for diabetes. The weaning period consists of a critical postnatal window for structural and physiologic maturation of rat beta-cells. To investigate transcriptome changes involved in the maturation of beta-cells neighboring this period, we performed microarray analysis in fluorescence-activated cell-sorted (FACS) beta-cell-enriched populations. Our results showed a variety of gene sets including those involved in the integration of metabolism, modulation of electrical activity, and regulation of the cell cycle that play important roles in the maturation process. These observations were validated using reverse hemolytic plaque assay, electrophysiological recordings, and flow cytometry analysis. Moreover, we suggest some unexplored pathways such as sphingolipid metabolism, insulin-vesicle trafficking, regulation of transcription/transduction by miRNA-30, trafficking proteins, and cell cycle proteins that could play important roles in the process mentioned above for further investigation.

Current Concepts of Using Pigs as a Source for Beta-Cell Replacement Therapy of Type 1 Diabetes

The global prevalence of insulin-dependent diabetes mellitus is rapidly increasing. In spite of major improvements in insulin treatment regimens and diabetes technology (e.g., artificial pancreas devices), glucose control remains problematic in a substantial proportion of diabetic patients. Those patients may benefit from beta-cell replacement therapies. Allotransplantation of pancreas or isolated pancreatic islets is limited by the small number of organ donors. Thus, alternative sources of beta-cells are being developed and tested. These include endocrine progenitor cells or mature beta-cells derived from pluripotent human stem cells and attempts to derive human pancreas tissue in animal hosts by interspecific chimeric complementation experiments. Xenotransplantation of porcine islets is a realistic alternative option. Immune rejection of xenoislets can be prevented by immunosuppression of the recipient or by encapsulation of the islets in microdevices or macrodevices. Using precise and efficient genetic engineering of donor pigs, immune-protected xenoislets with improved functionality can be generated.

Disallowed and Allowed Gene Expression: Two Faces of Mature Islet Beta Cells.

Glucose homeostasis greatly depends on the match between fluctuating insulin demands and adjusted rates of insulin secretion, which is the function of pancreatic beta cells. Emerging evidence suggests that when neonatal beta cells mature, they acquire two faces of differentiated function: an expected "visible face" that depends on specific beta cell proteins needed for regulated insulin release, but also a "hidden face" that represses ubiquitous proteins to prevent inappropriate beta cell function such as elevated basal hormone secretion or insulin release triggered by exercise. This review highlights this novel concept, and we first propose that hidden faces may also be relevant for other specialized tissue functions, such as ketogenesis in the liver. Next, we discuss three scenarios in which aberrant gene expression causes abnormal glucose-induced insulin release and the epigenetic regulation of the hidden face in beta cells. We conclude with perspectives for new research, including beta cell replacement to cure diabetes.

Breaking dogma for future therapy using stem cell - Where we have reached?

Breaking dogma for future therapy using stem cell - Where we have reached?

REST/NRSF Target Genes in Neuronal and Beta Cells: Pathophysiological and Therapeutic Perspectives for Diabetes and Neurodegenerative Disorders

Pancreatic beta and neuronal cells share numerous similarities, including a key transcriptional mechanism of the differentiation programme. The mechanism involves the decrease or the extinction of the transcriptional repressor RE-1-silencing transcription factor (REST), also called neuron-restrictive silencer factor (NRSF), which leads to the expression of various genes encoding proteins required for mature beta and neuronal cell function. Abnormal expression and genetic variation in some of the REST/NRSF target genes have been reported in diabetes and neurodegenerative disorders, suggesting that common pathogenic mechanisms account for beta-cell decline and neuronal degeneration in the two diseases. In addition, some of the REST/NRSF target genes have been identified as potential therapeutic targets for improvement of beta-cell function in diabetes. This review sheds light on the neuronal and beta-cell REST/NRSF target genes that are potential future drug targets for the treatment of diabetes and neurodegeneration.

Distal Pancreatectomy Without Splenectomy Is Associated With Higher Reoperation Rates: Results of the Nationwide Inpatient Sample Database

RESULTS: Alpha cell lines were converted into insulin expressing cells when they were treated with a GLP-1 agonist exendin-4, or if they were transfected with an adenovirus expressing pdx1 and mafA. Further analysis show an increased expression of proliferation markers, but none of the mature beta cell markers were present. Alpha TC cells, when influenced by AAV, forced expression of transcription factors pdx-1 and mafA, demonstrating a robust expression of insulin and markers of mature beta cells.