Human pluripotent stem cell-derived ß cells: Truly immature islet ß cells for type 1 diabetes therapy?

A century has passed since the Nobel Prize winning discovery of insulin, which still remains the mainstay treatment for type 1 diabetes mellitus (T1DM) to this day. True to the words of its discoverer Sir Frederick Banting, "insulin is not a cure for diabetes, it is a treatment", millions of people with T1DM are dependent on daily insulin medications for life. Clinical donor islet transplantation has proven that T1DM is curable, however due to profound shortages of donor islets, it is not a mainstream treatment option for T1DM. Human pluripotent stem cell derived insulin-secreting cells, pervasively known as stem cell-derived ß cells (SC-ß cells), are a promising alternative source and have the potential to become a T1DM treatment through cell replacement therapy. Here we briefly review how islet ß cells develop and mature in vivo and several types of reported SC-ß cells produced using different ex vivo protocols in the last decade. Although some markers of maturation were expressed and glucose stimulated insulin secretion was shown, the SC-ß cells have not been directly compared to their in vivo counterparts, generally have limited glucose response, and are not yet fully matured. Due to the presence of extra-pancreatic insulin-expressing cells, and ethical and technological issues, further clarification of the true nature of these SC-ß cells is required.

Diagnostic role of multislice spiral computed tomography combined with clinical manifestations and laboratory tests in acute appendicitis subtypes.

The study aimed to investigate the diagnostic role of multislice spiral CT (MSCT) combined with clinical manifestations and laboratory tests in acute appendicitis subtypes. Patients diagnosed with acute appendicitis were included for retrospective analysis and their clinical manifestations and MSCT signs were analyzed. The clinical manifestations of different subtypes of acute appendicitis, including simple appendicitis, suppurative appendicitis and gangrenous appendicitis, were compared. The clinical manifestations were anorexia in 51.1% of patients, nausea and vomiting in 62.0%, shifting right lower abdominal pain in 51.1%, elevated body temperature in 31.2%, right lower quadrant abdominal tenderness in 91.4%, rebound tenderness in 91.4%, increased white cell count in 89.1%, high neutrophil count in 88.2%, increased appendiceal diameter enlargement in 100%, surrounding exudate in 95.0%, fecal stones in 51.6%, appendiceal wall thickening in 94.6%, lymph node in 82.8% and intestinal stasis in 18.6%. There were statistically significant differences in body temperature and neutrophil percentage among the subtypes of appendicitis and they were lowest in simple appendicitis and highest in gangrenous appendicitis. There were statistically significant differences in appendix diameter and the surrounding exudate among the subtypes of appendicitis and they were lowest in simple appendicitis and highest in gangrenous appendicitis. Clinical manifestations and MSCT signs, especially body temperature, percentage of neutrophils and the surrounding exudate, might have significant diagnostic value in acute appendicitis.

Stem cell therapy for insulin-dependent diabetes: Are we still on the road?

In insulin-dependent diabetes, the islet ß cells do not produce enough insulin and the patients must receive exogenous insulin to control blood sugar. However, there are still many deficiencies in exogenous insulin supplementation. Therefore, the replacement of destroyed functional ß cells with insulin-secreting cells derived from functional stem cells is a good idea as a new therapeutic idea. This review introduces the development schedule of mouse and human embryonic islets. The differences between mouse and human pancreas embryo development were also listed. Accordingly to the different sources of stem cells, the important research achievements on the differentiation of insulin-secreting ß cells of stem cells and the current research status of stem cell therapy for diabetes were reviewed. Stem cell replacement therapy is a promising treatment for diabetes, caused by defective insulin secretion, but there are still many problems to be solved, such as the biosafety and reliability of treatment, the emergence of tumors during treatment, untargeted differentiation and autoimmunity, etc. Therefore, further understanding of stem cell therapy for insulin is needed.

Functional maturation of immature ß cells: A roadblock for stem cell therapy for type 1 diabetes.

Type 1 diabetes mellitus (T1DM) is a chronic autoimmune disease caused by the specific destruction of pancreatic islet ß cells and is characterized as the absolute insufficiency of insulin secretion. Current insulin replacement therapy supplies insulin in a non-physiological way and is associated with devastating complications. Experimental islet transplantation therapy has been proven to restore glucose homeostasis in people with severe T1DM. However, it is restricted by many factors such as severe shortage of donor sources, progressive loss of donor cells, high cost, etc. As pluripotent stem cells have the potential to give rise to all cells including islet ß cells in the body, stem cell therapy for diabetes has attracted great attention in the academic community and the general public. Transplantation of islet ß-like cells differentiated from human pluripotent stem cells (hPSCs) has the potential to be an excellent alternative to islet transplantation. In stem cell therapy, obtaining ß cells with complete insulin secretion in vitro is crucial. However, after much research, it has been found that the ß-like cells obtained by in vitro differentiation still have many defects, including lack of adult-type glucose stimulated insulin secretion, and multi-hormonal secretion, suggesting that in vitro culture does not allows for obtaining fully mature ß-like cells for transplantation. A large number of studies have found that many transcription factors play important roles in the process of transforming immature to mature human islet ß cells. Furthermore, PDX1, NKX6.1, SOX9, NGN3, PAX4, etc., are important in inducing hPSC differentiation in vitro. The absent or deficient expression of any of these key factors may lead to the islet development defect in vivo and the failure of stem cells to differentiate into genuine functional ß-like cells in vitro. This article reviews ß cell maturation in vivo and in vitro and the vital roles of key molecules in this process, in order to explore the current problems in stem cell therapy for diabetes.

Claudin 4 in pancreatic ß cells is involved in regulating the functional state of adult islets.

The functional state (FS) of adult pancreatic islets is regulated by a large array of regulatory molecules including numerous transcription factors. Whether any islet structural molecules play such a role has not been well understood. Here, multiple technologies including bioinformatics analyses were used to explore such molecules. The tight junction family molecule claudin 4 (Cldn4) was the highest enriched amongst over 140 structural genes analysed. Cldn4 expression was ~75-fold higher in adult islets than in exocrine tissues and was mostly up-regulated during functional maturation of developing islet cells. Cldn4 was progressively down-regulated in functionally compromised, dedifferentiating insulin-secreting ß cells and in db/db type 2 diabetic islets. Furthermore, the genetic deletion of Cldn4 impaired significantly the FS without apparently affecting pancreas morphology, islet architectural structure and cellular distribution, and secretion of enteroendocrine hormones. Thus, we suggest a previously unidentified role for Cldn4 in regulating the FS of islets, with implications in translational research for better diabetes therapies.

RNA-Seq Analysis of Islets to Characterise the Dedifferentiation in Type 2 Diabetes Model Mice db/db.

Type 2 diabetes (T2D) is a global health issue and dedifferentiation plays underlying causes in the pathophysiology of T2D; however, there is a lack of understanding in the mechanism. Dedifferentiation results from the loss of function of pancreatic ß-cells alongside a reduction in essential transcription factors under various physiological stressors. Our study aimed to establish db/db as an animal model for dedifferentiation by using RNA sequencing to compare the gene expression profile in islets isolated from wild-type, db/+ and db/db mice, and qPCR was performed to validate those significant genes. A reduction in both insulin secretion and the expression of Ins1, Ins2, Glut2, Pdx1 and MafA was indicative of dedifferentiation in db/db islets. A comparison of the db/+ and the wild-type islets indicated a reduction in insulin secretion perhaps related to the decreased Mt1. A significant reduction in both Rn45s and Mir6236 was identified in db/+ compared to wild-type islets, which may be indicative of pre-diabetic state. A further significant reduction in RasGRF1, Igf1R and Htt was also identified in dedifferentiated db/db islets. Molecular characterisation of the db/db islets was performed via Ingenuity analysis which identified highly significant genes that may represent new molecular markers of dedifferentiation.

An overview of type 2 diabetes and importance of vitamin D3-vitamin D receptor interaction in pancreatic ß-cells.

One significant health issue that plagues contemporary society is that of Type 2 diabetes (T2D). This disease is characterised by higher-than-average blood glucose levels as a result of a combination of insulin resistance and insufficient insulin secretions from the ß-cells of pancreatic islets of Langerhans. Previous developmental research into the pancreas has identified how early precursor genes of pancreatic ß-cells, such as Cpal, Ngn3, NeuroD, Ptf1a, and cMyc, play an essential role in the differentiation of these cells. Furthermore, ß-cell molecular characterization has also revealed the specific role of ß-cell-markers, such as Glut2, MafA, Ins1, Ins2, and Pdx1 in insulin expression. The expression of these genes appears to be suppressed in the T2D ß-cells, along with the reappearance of the early endocrine marker genes. Glucose transporters transport glucose into ß-cells, thereby controlling insulin release during hyperglycaemia. This stimulates glycolysis through rises in intracellular calcium (a process enhanced by vitamin D) (Norman et al., 1980), activating 2 of 4 proteinases. The rise in calcium activates half of pancreatic ß-cell proinsulinases, thus releasing free insulin from granules. The synthesis of ATP from glucose by glycolysis, Krebs cycle and oxidative phosphorylation plays a role in insulin release. Some studies have found that the ß-cells contain high levels of the vitamin D receptor; however, the role that this plays in maintaining the maturity of the ß-cells remains unknown. Further research is required to develop a more in-depth understanding of the role VDR plays in ß-cell function and the processes by which the beta cell function is preserved.

Transient Impairment of Islet Architectural Development in Pancreas-Specific Bmpr1a-Deleted Prenatal Mice Involves Reduced Expression of E-Cadherin.

Bone morphogenetic protein (BMP) signaling plays critical roles on the development of a large array of embryonic organs and promotes the in vitro formation of pancreatic cystoid colonies containing insulin-producing cells. However, this signaling and its underlying mechanism on in vivo development of prenatal pancreas have not been clearly understood. To address these questions, we analyzed, with a variety of techniques, the prenatal mouse pancreas after Pdx1 (the pancreas and duodenum homeobox factor 1 gene)-driving deletion of the BMP receptor type 1a gene (Bmpr1a). In this study, we report that the Pdx1-driving deletion of Bmpr1a transiently disrupted only the assembly of architectural structure of prenatal islets. The differentiation of endocrine lineage cells and the development of pancreatic acinar tissue were comparable between Bmpr1a-deleted fetuses and -undeleted Controls throughout the period examined. Molecular studies revealed that among many proteins surveyed, the key cell-cell interaction molecule E-cadherin (E-cad) only was expressed significantly less at both messenger RNA (mRNA) and protein levels in Bmpr1a-deleted than Control fetal endocrine cells. We thus conclude that BMP signaling transiently regulates the expression of E-cad and the establishment of prenatal islet architecture.

Differentiation of Islet Progenitors Regulated by Nicotinamide into Transcriptome-Verified ß Cells That Ameliorate Diabetes.

Developmental stage-specific differentiation of stem or progenitor cells into safe and functional cells is of fundamental importance in regenerative medicine, including ß-cell replacement. However, the differentiation of islet progenitor cells (IPCs) into insulin-secreting ß cells remains elusive. Here, we report that the multifunctional molecule nicotinamide (NIC) is a specific differentiation regulator of mouse IPCs. The differentiated cells regulated by NIC exhibited many characteristics of adult ß cells, including ameliorating preclinical diabetes and a highly comparable transcriptome profile. Gene set enrichment analysis showed that during differentiation, numerous IPC transcription factor genes, including Ngn3, Pax4, Fev, and Mycl1, were all down regulated. Pharmacological, biochemical, and gene knockdown analyses collectively demonstrated that NIC regulated the differentiation via inhibiting Sirt1 (silent information regulator transcript 1). Finally, NIC also regulates human IPC differentiation. Thus, our study advances islet developmental biology and impacts on translational research and regenerative therapies to diabetes and other diseases. Stem Cells 2017;35:1341-1354.

In vitro reprogramming of rat bmMSCs into pancreatic endocrine-like cells.

Islet transplantation provides curative treatments to patients with type 1 diabetes, but donor shortage restricts the broad use of this therapy. Thus, generation of alternative transplantable cell sources is intensively investigated worldwide. We previously showed that bone marrow-derived mesenchymal stem cells (bmMSCs) can be reprogrammed to pancreatic-like cells through simultaneously forced suppression of Rest/Nrsf (repressor element-1 silencing transcription factor/neuronal restrictive silencing factor) and Shh (sonic hedgehog) and activation of Pdx1 (pancreas and duodenal transcription factor 1). We here aimed to reprogram bmMSCs further along the developmental pathway towards the islet lineages by improving our previous strategy and by overexpression of Ngn3 (neurogenin 3) and NeuroD1 (neurogenic differentiation 1), critical regulators of the development of endocrine pancreas. We showed that compared to the previous protocol, the overexpression of only Pdx1 and Ngn3 reprogrammed bmMSCs into cells with more characteristics of islet endocrine lineages verified with bioinformatic analyses of our RNA-Seq datasets. These analyses indicated 2325 differentially expressed genes including those involved in the pancreas and islet development. We validated with qRT-PCR analysis selective genes identified from the RNA-Seq datasets. Thus, we reprogrammed bmMSCs into islet endocrine-like cells and advanced the endeavor to generate surrogate functional insulin-secreting cells.

Insulin-secreting ß cells require a post-genomic concept.

Pancreatic insulin-secreting ß cells are essential in maintaining normal glucose homeostasis accomplished by highly specialized transcription of insulin gene, of which occupies up to 40% their transcriptome. Deficiency of these cells causes diabetes mellitus, a global public health problem. Although tremendous endeavors have been made to generate insulin-secreting cells from human pluripotent stem cells (i.e., primitive cells capable of giving rise to all cell types in the body), a regenerative therapy to diabetes has not yet been established. Furthermore, the nomenclature of ß cells has become inconsistent, confusing and controversial due to the lack of standardized positive controls of developmental stage-matched in vivo cells. In order to minimize this negative impact and facilitate critical research in this field, a post-genomic concept of pancreatic ß cells might be helpful. In this review article, we will briefly describe how ß cells were discovered and islet lineage is developed that may help understand the cause of nomenclatural controversy, suggest a post-genomic definition and finally provide a conclusive remark on future research of this pivotal cell.

Pigment epithelium-derived factor (PEDF) regulates metabolism and insulin secretion from a clonal rat pancreatic beta cell line BRIN-BD11 and mouse islets.

Pigment epithelium-derived factor (PEDF) is a multifunctional glycoprotein, associated with lipid catabolism and insulin resistance. In the present study, PEDF increased chronic and acute insulin secretion in a clonal rat ß-cell line BRIN-BD11, without alteration of glucose consumption. PEDF also stimulated insulin secretion from primary mouse islets. Seahorse flux analysis demonstrated that PEDF did not change mitochondrial respiration and glycolytic function. The cytosolic presence of the putative PEDF receptor - adipose triglyceride lipase (ATGL) - was identified, and ATGL associated stimulation of glycerol release was robustly enhanced by PEDF, while intracellular ATP levels increased. Addition of palmitate or ex vivo stimulation with inflammatory mediators induced ß-cell dysfunction, effects not altered by the addition of PEDF. In conclusion, PEDF increased insulin secretion in BRIN-BD11 and islet cells, but had no impact on glucose metabolism. Thus elevated lipolysis and enhanced fatty acid availability may impact insulin secretion following PEDF receptor (ATGL) stimulation.

Transcriptome of pancreas-specific Bmpr1a-deleted islets links to TPH1-5-HT axis.

Bone morphogenetic protein (BMP) signaling is crucial for the development and function of numerous organs, but its role on the function of pancreatic islets is not completely clear. To explore this question, we applied the high throughput transcriptomic analyses on the islets isolated from mice with a pancreas-specific deletion of the gene, Bmpr1a, encoding the type 1a BMP receptor. Consistently, these pBmpr1aKO mice had impaired glucose homeostasis at 3 months, and were more severely affected at 12 months of age. These had lower fasting blood insulin concentrations, with reduced expression of several key regulators of ß-cell function. Importantly, transcriptomic profiling of 3-month pBmpr1aKO islets and bioinformatic analyses revealed abnormal expression of 203 metabolic genes. Critically among these, the tryptophan hydroxylase 1 gene (Tph1), encoding the rate-limiting enzyme for the production of 5-hydroxytryptamine (5-HT) was the highest over-expressed one. 5-HT is an important regulator of insulin secretion from ß cells. Treatment with excess 5-HT inhibited this secretion. Thus our transcriptomic analysis links two highly conserved molecular pathways the BMP signaling and the TPH1-5-HT axis on glucose homeostasis.

Multipotent pancreas progenitors: Inconclusive but pivotal topic.

The establishment of multipotent pancreas progenitors (MPP) should have a significant impact not only on the ontology of the pancreas, but also for the translational research of glucose-responding endocrine ß-cells. Deficiency of the latter may lead to the pandemic type 1 or type 2 diabetes mellitus, a metabolic disorder. An ideal treatment of which would potentially be the replacement of destroyed or failed ß-cells, by restoring function of endogenous pancreatic endocrine cells or by transplantation of donor islets or in vitro generated insulin-secreting cells. Thus, considerable research efforts have been devoted to identify MPP candidates in the pre- and post-natal pancreas for the endogenous neogenesis or regeneration of endocrine insulin-secreting cells. In order to advance this inconclusive but critical field, we here review the emerging concepts, recent literature and newest developments of potential MPP and propose measures that would assist its forward progression.

Pancreatic stem cells remain unresolved.

Diabetes mellitus is caused by absolute (type 1) or relative (type 2) deficiency of insulin-secreting islet ß cells. An ideal treatment of diabetes would, therefore, be to replace the lost or deficient ß cells, by transplantation of donated islets or differentiated endocrine cells or by regeneration of endogenous islet cells. Due to their ability of unlimited proliferation and differentiation into all functional lineages in our body, including ß cells, embryonic stem cells and induced pluripotent stem cells are ideally placed as cell sources for a diabetic transplantation therapy. Unfortunately, the inability to generate functional differentiated islet cells from pluripotent stem cells and the poor availability of donor islets have severely restricted the broad clinical use of the replacement therapy. Therefore, endogenous sources that can be directed to becoming insulin-secreting cells are actively sought after. In particular, any cell types in the developing or adult pancreas that may act as pancreatic stem cells (PSC) would provide an alternative renewable source for endogenous regeneration. In this review, we will summarize the latest progress and knowledge of such PSC, and discuss ways that facilitate the future development of this often controversial, but crucial research.

In vitro reprogramming of rat bone marrow-derived mesenchymal stem cells into insulin-producing cells by genetically manipulating negative and positive regulators.

Islet cell replacement therapy represents the most promising approach for the cure of type 1 diabetes if autoimmunity to ß cells is under control. However, this potential is limited by a shortage of pancreas donors. To address the donor shortage problem, we determined whether bone marrow-derived mesenchymal stem cells (bmMSCs) can be directly reprogrammed to islet lineages by simultaneously forced suppression and over-expression of key regulator genes that play critical roles during pancreas development. Here, we report that rat bmMSCs were converted in vitro into insulin-producing cells by suppressing two-repressor genes repressor element-1 silencing transcription factor/neuronal restrictive silencing factor (Rest/Nrsf) and sonic hedgehog (Shh) and by over-expressing pancreas and duodenal transcription factor 1 (Pdx1). The reprogrammed bmMSCs expressed both genes and proteins specific for islet cells. These converted cells were capable of releasing insulin in a glucose-responsive manner. Our study suggests that bmMSCs may ultimately be reprogrammed to functional insulin-secreting cells.

Directed differentiation of embryonic stem cells allows exploration of novel transcription factor genes for pancreas development.

Embryonic stem cells (ESCs) have been promised as a renewable source for regenerative medicine, including providing a replacement therapy in type 1 diabetes. However, they have not yet been differentiated into functional insulin-secreting ß cells. This is due partially to the knowledge gap regarding the transcription factors (TFs) required for pancreas development. We hypothesize that, if directed differentiation in vitro recapitulates the developmental process in vivo, ESCs provide a powerful model to discover novel pancreatic TF genes. Guided by knowledge of their normal development and using RT-PCR and immunochemical analyses, we have established protocols for directed differentiation of mouse ESCs into pancreatic progenitors. Microarray analyses of these differentiating ESC cells at days 0, 4, 8 and 15 confirmed their sequential differentiation. By day 15, we found up-regulation of a group of pancreatic progenitor marker genes including Pdx1, Ptf1a, Nkx6.1, Pax4 and Pax6. Consistently, Pdx1-immunoreactive cells were detected on day 15. Most of these Pdx1(+) cells also expressed Nkx6.1. Bioinformatic analyses of sequential datasets allowed identification of over 20 novel TF genes potentially important for pancreas development. The dynamic expression of representative known and novel genes was confirmed by quantitative real time RT-PCR analysis. This strategy may be modified to study novel regulatory molecules for development of other tissue and organ systems.

Pancreatic stem cells: from possible to probable.

Type 1 and some forms of type 2 diabetes mellitus are caused by deficiency of insulin-secretory islet ß cells. An ideal treatment for these diseases would therefore be to replace ß cells, either by transplanting donated islets or via endogenous regeneration (and controlling the autoimmunity in type 1 diabetes). Unfortunately, the poor availability of donor islets has severely restricted the broad clinical use of islet transplantation. The ability to differentiate embryonic stem cells into insulin-expressing cells initially showed great promise, but the generation of functional ß cells has proven extremely difficult and far slower than originally hoped. Pancreatic stem cells (PSC) or transdifferentiation of other cell types in the pancreas may hence provide an alternative renewable source of surrogate ß cells. However, the existence of PSC has been hotly debated for many years. In this review, we will discuss the latest development and future perspectives of PSC research, giving readers an overview of this controversial but important area.

TGF-beta induces telomerase-dependent pancreatic tumor cell cycle arrest.

Recent studies suggest that transforming growth factor beta (TGF-beta) inhibits telomerase activity by repression of the telomerase reverse transcriptase (TERT) gene. In this report, we show that TGF-beta induces TERT repression-dependent apoptosis in pancreatic tumor, vascular smooth muscle, and cervical cancer cell cultures. TGF-beta activates Smad3 signaling, induces TERT gene repression and results in G1/S phase cell cycle arrest and apoptosis. TERT over-expression stimulates the G1/S phase transition and alienates TGF-beta-induced cell cycle arrest and apoptosis. Our data suggest that telomere maintenance is a limiting factor of the transition of the cell cycle. TGF-beta triggers cell cycle arrest and death by a mechanism involving telomerase deregulation of telomere maintenance.

Quantification of insulin gene expression during development of pancreatic islet cells.

OBJECTIVE: Despite great progress in understanding the transcriptional regulation of the development of insulin-secreting beta cells, the quantitative temporal expression of insulin gene(s) remains largely unknown. We here aimed to quantify insulin gene transcripts during development. METHODS: We described bioinformatics algorithms to quantify (insulin) gene transcript abundance in sequential microarray data sets at the global level. Several molecular techniques were used to confirm our analyses. RESULTS: We demonstrated that the expression of insulin genes was up-regulated at approximately 14-fold, 700- to 2000-fold, and 5000- to 6000-fold in Pdx1- and Ngn3-expressing cells and adult islets compared with definitive endodermal or embryonic stem cells, respectively. The expression of multiple genes encoding molecules involved in posttranslational modifications of insulin and glucose sensing was also elevated in the same period. All islet and associated genes determined with microarray data were confirmed not only to be up-regulated by real-time quantitative reverse transcriptase polymerase chain reaction but also that the magnitude of their increase quantified with these 2 methods was statistically highly correlated. Consistent with the above, green fluorescence protein expression under the control of the mouse insulin 1 promoter could be visualized in the pancreas from embryonic day (E) 11.5, increasing progressively through E13.5 to E15.5. CONCLUSION: Our study provides a novel insight into islet developmental biology.