Differentiation of Human-Induced Pluripotent Stem Cell-Derived Endocrine Progenitors to Islet-like Cells Using a Dialysis Suspension Culture System.

The production of functional islet-like cells from human-induced pluripotent stem cells (hiPSCs) is a promising strategy for the therapeutic use and disease modeling for type 1 diabetes. However, the production cost of islet-like cells is extremely high due to the use of expensive growth factors for differentiation. In a conventional culture method, growth factors and beneficial autocrine factors remaining in the culture medium are removed along with toxic metabolites during the medium change, and it limits the efficient utilization of those factors. In this study, we demonstrated that the dialysis suspension culture system is possible to reduce the usage of growth factors to one-third in the differentiation of hiPSC-derived endocrine progenitor cells to islet-like cells by reducing the medium change frequency with the refinement of the culture medium. Furthermore, the expression levels of hormone-secretion-related genes and the efficiency of differentiation were improved with the dialysis suspension culture system, possibly due to the retaining of autocrine factors. In addition, we confirmed several improvements required for the further study of the dialysis culture system. These findings showed the promising possibility of the dialysis suspension culture system for the low-cost production of islet-like cells.

Cell population characterization and discovery using single-cell technologies in endocrine systems.

In the last 15 years, single-cell technologies have become robust and indispensable tools to investigate cell heterogeneity. Beyond transcriptomic, genomic and epigenome analyses, technologies are constantly evolving, in particular toward multi-omics, where analyses of different source materials from a single cell are combined, and spatial transcriptomics, where resolution of cellular heterogeneity can be detected in situ. While some of these techniques are still being optimized, single-cell RNAseq has commonly been used because the examination of transcriptomes allows characterization of cell identity and, therefore, unravel previously uncharacterized diversity within cell populations. Most endocrine organs have now been investigated using this technique, and this has given new insights into organ embryonic development, characterization of rare cell types, and disease mechanisms. Here, we highlight recent studies, particularly on the hypothalamus and pituitary, and examine recent findings on the pancreas and reproductive organs where many single-cell experiments have been performed.

Endocrine Autoimmune Disease as a Fragility of Immune Surveillance against Hypersecreting Mutants.

Some endocrine organs are frequent targets of autoimmune attack. Here, we addressed the origin of autoimmune disease from the viewpoint of feedback control. Endocrine tissues maintain mass through feedback loops that balance cell proliferation and removal according to hormone-driven regulatory signals. We hypothesized the existence of a dedicated mechanism that detects and removes mutant cells that missense the signal and therefore hyperproliferate and hypersecrete with potential to disrupt organismal homeostasis. In this mechanism, hypersecreting cells are preferentially eliminated by autoreactive T cells at the cost of a fragility to autoimmune disease. The "autoimmune surveillance of hypersecreting mutants" (ASHM) hypothesis predicts the presence of autoreactive T cells in healthy individuals and the nature of self-antigens as peptides from hormone secretion pathway. It explains why some tissues get prevalent autoimmune disease, whereas others do not and instead show prevalent mutant-expansion disease (e.g., hyperparathyroidism). The ASHM hypothesis is testable, and we discuss experimental follow-up.

The interoceptive hippocampus: Mouse brain endocrine receptor expression highlights a dentate gyrus (DG)-cornu ammonis (CA) challenge-sufficiency axis.

The primeval function of the mammalian hippocampus (HPC) remains uncertain. Implicated in learning and memory, spatial navigation, and neuropsychological disorders, evolutionary theory suggests that the HPC evolved from a primeval chemosensory epithelium. Deficits in sensing of internal body status ('interoception') in patients with HPC lesions argue that internal sensing may be conserved in higher vertebrates. We studied the expression patterns in mouse brain of 250 endocrine receptors that respond to blood-borne ligands. Key findings are (i) the proportions and levels of endocrine receptor expression in the HPC are significantly higher than in all other comparable brain regions. (ii) Surprisingly, the distribution of endocrine receptor expression within mouse HPC was found to be highly structured: receptors signaling 'challenge' are segregated in dentate gyrus (DG), whereas those signaling 'sufficiency' are principally found in cornu ammonis (CA) regions. Selective expression of endocrine receptors in the HPC argues that interoception remains a core feature of hippocampal function. Further, we report that ligands of DG receptors predominantly inhibit both synaptic potentiation and neurogenesis, whereas CA receptor ligands conversely promote both synaptic potentiation and neurogenesis. These findings suggest that the hippocampus acts as an integrator of body status, extending its role in context-dependent memory encoding from 'where' and 'when' to 'how I feel'. Implications for anxiety and depression are discussed.

The Functional Significance of Endocrine-immune Interactions in Health and Disease.

Hormones are known to influence various body systems that include skeletal, cardiac, digestive, excretory, and immune systems. Emerging investigations suggest the key role played by secretions of endocrine glands in immune cell differentiation, proliferation, activation, and memory attributes of the immune system. The link between steroid hormones such as glucocorticoids and inflammation is widely known. However, the role of peptide hormones and amino acid derivatives such as growth and thyroid hormones, prolactin, dopamine, and thymopoietin in regulating the functioning of the immune system remains unclear. Here, we reviewed the findings pertinent to the functional role of hormone-immune interactions in health and disease and proposed perspective directions for translational research in the field.

Autophagy regulates steroid production by mediating cholesterol trafficking in endocrine cells.

Steroid hormones are made from cholesterol and are essential for many developmental processes and disease conditions. The production of these hormones is nutrient dependent and tightly controlled by mechanisms that involve delivery of the precursor molecule cholesterol stored in lipid droplets (LDs). Recent studies have implicated macroautophagy/autophagy, a process regulated by nutrition, in the degradation of LDs and the mobilization of stored lipids. We recently identified an autophagy-dependent mechanism that regulates steroid production via effects on cholesterol trafficking. Through gain- and loss-of-function studies in Drosophila, we found that essential autophagy-related (Atg) genes are required in steroidogenic cells for normal steroid production. Inhibition of autophagy in these cells by knockdown of Atg genes causes strong accumulation of cholesterol in LDs and reduces steroid production, resembling effects seen in some lipid-storage disorders and steroid-dependent cancer conditions. This autophagy-dependent steroid hormone regulation (ASHR) process is regulated by the wts-yki/Warts-Yorkie tumor-suppressor pathway downstream of nutrition, coupling nutrient intake with steroid-dependent developmental growth. This mechanism potentially contributes to the development of certain cancers and lipid-storage disorders and thus may be of great therapeutic relevance.

Transcriptional analysis of abdominal fat in chickens divergently selected on bodyweight at two ages reveals novel mechanisms controlling adiposity: validating visceral adipose tissue as a dynamic endocrine and metabolic organ.

BACKGROUND: Decades of intensive genetic selection in the domestic chicken (Gallus gallus domesticus) have enabled the remarkable rapid growth of today's broiler (meat-type) chickens. However, this enhanced growth rate was accompanied by several unfavorable traits (i.e., increased visceral fatness, leg weakness, and disorders of metabolism and reproduction). The present descriptive analysis of the abdominal fat transcriptome aimed to identify functional genes and biological pathways that likely contribute to an extreme difference in visceral fatness of divergently selected broiler chickens. METHODS: We used the Del-Mar 14 K Chicken Integrated Systems microarray to take time-course snapshots of global gene transcription in abdominal fat of juvenile [1-11 weeks of age (wk)] chickens divergently selected on bodyweight at two ages (8 and 36 wk). Further, a RNA sequencing analysis was completed on the same abdominal fat samples taken from high-growth (HG) and low-growth (LG) cockerels at 7 wk, the age with the greatest divergence in body weight (3.2-fold) and visceral fatness (19.6-fold). RESULTS: Time-course microarray analysis revealed 312 differentially expressed genes (FDR ≤ 0.05) as the main effect of genotype (HG versus LG), 718 genes in the interaction of age and genotype, and 2918 genes as the main effect of age. The RNA sequencing analysis identified 2410 differentially expressed genes in abdominal fat of HG versus LG chickens at 7 wk. The HG chickens are fatter and over-express numerous genes that support higher rates of visceral adipogenesis and lipogenesis. In abdominal fat of LG chickens, we found higher expression of many genes involved in hemostasis, energy catabolism and endocrine signaling, which likely contribute to their leaner phenotype and slower growth. Many transcription factors and their direct target genes identified in HG and LG chickens could be involved in their divergence in adiposity and growth rate. CONCLUSIONS: The present analyses of the visceral fat transcriptome in chickens divergently selected for a large difference in growth rate and abdominal fatness clearly demonstrate that abdominal fat is a very dynamic metabolic and endocrine organ in the chicken. The HG chickens overexpress many transcription factors and their direct target genes, which should enhance in situ lipogenesis and ultimately adiposity. Our observation of enhanced expression of hemostasis and endocrine-signaling genes in diminished abdominal fat of LG cockerels provides insight into genetic mechanisms involved in divergence of abdominal fatness and somatic growth in avian and perhaps mammalian species, including humans.

Modeling coexistence of oscillation and Delta/Notch-mediated lateral inhibition in pancreas development and neurogenesis.

During pancreas development, Neurog3 positive endocrine progenitors are specified by Delta/Notch (D/N) mediated lateral inhibition in the growing ducts. During neurogenesis, genes that determine the transition from the proneural state to neuronal or glial lineages are oscillating before their expression is sustained. Although the basic gene regulatory network is very similar, cycling gene expression in pancreatic development was not investigated yet, and previous simulations of lateral inhibition in pancreas development excluded by design the possibility of oscillations. To explore this possibility, we developed a dynamic model of a growing duct that results in an oscillatory phase before the determination of endocrine progenitors by lateral inhibition. The basic network (D/N + Hes1 + Neurog3) shows scattered, stable Neurog3 expression after displaying transient expression. Furthermore, we included the Hes1 negative feedback as previously discussed in neurogenesis and show the consequences for Neurog3 expression in pancreatic duct development. Interestingly, a weakened HES1 action on the Hes1 promoter allows the coexistence of stable patterning and oscillations. In conclusion, cycling gene expression and lateral inhibition are not mutually exclusive. In this way, we argue for a unified mode of D/N mediated lateral inhibition in neurogenic and pancreatic progenitor specification.

In vitro evidence in rainbow trout supporting glucosensing mediated by sweet taste receptor, LXR, and mitochondrial activity in Brockmann bodies, and sweet taste receptor in liver.

We previously obtained evidence in rainbow trout peripheral tissues such as liver and Brockmann bodies (BB) for the presence and response to changes in circulating levels of glucose (induced by intraperitoneal hypoglycaemic and hyperglycaemic treatments) of glucosensing mechanisms others than that mediated by glucokinase (GK). There were based on mitochondrial production of reactive oxygen species (ROS) leading to increased expression of uncoupling protein 2 (UCP2), and sweet taste receptor in liver and BB, and on liver X receptor (LXR) and sodium/glucose co-transporter 1 (SGLT-1) in BB. We aimed in the present study to obtain further in vitro evidence for the presence and functioning of these systems. In a first experiment, pools of sliced liver and BB were incubated for 6h at 15°C in modified Hanks' medium containing 2, 4, or 8mM d-glucose, and we assessed the response of parameters related to these glucosensing mechanisms. In a second experiment, pools of sliced liver and BB were incubated for 6h at 15°C in modified Hanks' medium with 8mM d-glucose alone (control) or containing 1mM phloridzin (SGLT-1 antagonist), 20µM genipin (UCP2 inhibitor), 1µM trolox (ROS scavenger), 100µM bezafibrate (T1R3 inhibitor), and 50µM geranyl-geranyl pyrophosphate (LXR inhibitor). The results obtained in both experiments support the presence and functioning of glucosensor mechanisms in liver based on sweet taste receptor whereas in BB the evidence support those based on LXR, mitochondrial activity and sweet taste receptor.

Proinflammatory Cytokines Induce Endocrine Differentiation in Pancreatic Ductal Cells via STAT3-Dependent NGN3 Activation.

A major goal of diabetes research is to develop strategies that replenish pancreatic insulin-producing beta cells. One emerging strategy is to harness pancreatic plasticity-the ability of pancreatic cells to undergo cellular interconversions-a phenomenon implicated in physiological stress and pancreatic injury. Here, we investigate the effects of inflammatory cytokine stress on the differentiation potential of ductal cells in a human cell line, in mouse ductal cells by pancreatic intraductal injection, and during the progression of autoimmune diabetes in the non-obese diabetic (NOD) mouse model. We find that inflammatory cytokine insults stimulate epithelial-to-mesenchymal transition (EMT) as well as the endocrine program in human pancreatic ductal cells via STAT3-dependent NGN3 activation. Furthermore, we show that inflammatory cytokines activate ductal-to-endocrine cell reprogramming in vivo independent of hyperglycemic stress. Together, our findings provide evidence that inflammatory cytokines direct ductal-to-endocrine cell differentiation, with implications for beta cell regeneration.

ßlinc1 encodes a long noncoding RNA that regulates islet ß-cell formation and function.

Pancreatic ß cells are responsible for maintaining glucose homeostasis; their absence or malfunction results in diabetes mellitus. Although there is evidence that long noncoding RNAs (lncRNAs) play important roles in development and disease, none have been investigated in vivo in the context of pancreas development. In this study, we demonstrate that ßlinc1 (ß-cell long intergenic noncoding RNA 1), a conserved lncRNA, is necessary for the specification and function of insulin-producing ß cells through the coordinated regulation of a number of islet-specific transcription factors located in the genomic vicinity of ßlinc1. Furthermore, deletion of ßlinc1 results in defective islet development and disruption of glucose homeostasis in adult mice.

Exocytosis in non-neuronal cells.

Exocytosis is the process by which stored neurotransmitters and hormones are released via the fusion of secretory vesicles with the plasma membrane. It is a dynamic, rapid and spatially restricted process involving multiple steps including vesicle trafficking, tethering, docking, priming and fusion. For many years great steps have been undertaken in our understanding of how exocytosis occurs in different cell types, with significant focus being placed on synaptic release and neurotransmission. However, this process of exocytosis is an essential component of cell signalling throughout the body and underpins a diverse array of essential physiological pathways. Many similarities exist between different cell types with regard to key aspects of the exocytosis pathway, such as the need for Ca(2+) to trigger it or the involvement of members of the N-ethyl maleimide-sensitive fusion protein attachment protein receptor protein families. However, it is also equally clear that non-neuronal cells have acquired highly specialized mechanisms to control the release of their own unique chemical messengers. This review will focus on several important non-neuronal cell types and discuss what we know about the mechanisms they use to control exocytosis and how their specialized output is relevant to the physiological role of each individual cell type. These include enteroendocrine cells, pancreatic ß cells, astrocytes, lactotrophs and cytotoxic T lymphocytes. Non-neuronal cells have acquired highly specialized mechanisms to control the release of unique chemical messengers, such as polarised fusion of insulin granules in pancreatic ß cells targeted towards the vasculature (top). This review discusses mechanisms used in several important non-neuronal cell types to control exocytosis, and the relevance of intermediate vesicle fusion pore states (bottom) and their specialized output to the physiological role of each cell type. These include enteroendocrine cells, pancreatic ß cells, astrocytes, lactotrophs and cytotoxic T lymphocytes. This article is part of a mini review series on Chromaffin cells (ISCCB Meeting, 2015).

Chronology of endocrine differentiation and beta-cell neogenesis.

Diabetes is a chronic and incurable disease, which results from absolute or relative insulin insufficiency. Therefore, pancreatic beta cells, which are the only type of cell that expresses insulin, is considered to be a potential target for the cure of diabetes. Although the findings regarding beta-cell neogenesis during pancreas development have been exploited to induce insulin-producing cells from non-beta cells, there are still many hurdles towards generating fully functional beta cells that can produce high levels of insulin and respond to physiological signals. To overcome these problems, a solid understanding of pancreas development and beta-cell formation is required, and several mouse models have been developed to reveal the unique features of each endocrine cell type at distinct developmental time points. Here I review our understanding of pancreas development and endocrine differentiation focusing on recent progresses in improving temporal cell labeling in vivo.

Postnatal Pancreas of Mice Contains Tripotent Progenitors Capable of Giving Rise to Duct, Acinar, and Endocrine Cells In Vitro.

Postnatal pancreas is a potential source for progenitor cells to generate endocrine ß-cells for treating type 1 diabetes. However, it remains unclear whether young (1-week-old) pancreas harbors multipotent progenitors capable of differentiating into duct, acinar, and endocrine cells. Laminin is an extracellular matrix (ECM) protein important for ß-cells' survival and function. We established an artificial extracellular matrix (aECM) protein that contains the functional IKVAV (Ile-Lys-Val-Ala-Val) sequence derived from laminin (designated aECM-lam). Whether IKVAV is necessary for endocrine differentiation in vitro is unknown. To answer these questions, we cultured single cells from 1-week-old pancreas in semi-solid media supplemented with aECM-lam, aECM-scr (which contains a scrambled sequence instead of IKVAV), or Matrigel. We found that colonies were generated in all materials. Individual colonies were examined by microfluidic reverse transcription-polymerase chain reaction, immunostaining, and electron microscopy analyses. The majority of the colonies expressed markers for endocrine, acinar, and ductal lineages, demonstrating tri-lineage potential of individual colony-forming progenitors. Colonies grown in aECM-lam expressed higher levels of endocrine markers Insulin1, Insulin2, and Glucagon compared with those grown in aECM-scr and Matrigel, indicating that the IKVAV sequence enhances endocrine differentiation. In contrast, Matrigel was inhibitory for endocrine gene expression. Colonies grown in aECM-lam displayed the hallmarks of functional ß-cells: mature insulin granules and glucose-stimulated insulin secretion. Colony-forming progenitors were enriched in the CD133(high) fraction and among 230 micro-manipulated single CD133(high) cells, four gave rise to colonies that expressed tri-lineage markers. We conclude that young postnatal pancreas contains multipotent progenitor cells and that aECM-lam promotes differentiation of ß-like cells in vitro.

Slit/Robo signaling regulates cell fate decisions in the intestinal stem cell lineage of Drosophila.

In order to maintain tissue homeostasis, cell fate decisions within stem cell lineages have to respond to the needs of the tissue. This coordination of lineage choices with regenerative demand remains poorly characterized. Here, we identify a signal from enteroendocrine cells (EEs) that controls lineage specification in the Drosophila intestine. We find that EEs secrete Slit, a ligand for the Robo2 receptor in intestinal stem cells (ISCs) that limits ISC commitment to the endocrine lineage, establishing negative feedback control of EE regeneration. Furthermore, we show that this lineage decision is made within ISCs and requires induction of the transcription factor Prospero in ISCs. Our work identifies a function for the conserved Slit/Robo pathway in the regulation of adult stem cells, establishing negative feedback control of ISC lineage specification as a critical strategy to preserve tissue homeostasis. Our results further amend the current understanding of cell fate commitment within the Drosophila ISC lineage.

EndoNet: an information resource about the intercellular signaling network.

BACKGROUND: In multicellular organisms, an intercellular signaling network communicates information from the environment or distant tissues to defined target cells. Intercellular signaling (mostly mediated by hormones) can affect the metabolic state and the gene expression program of target cells, thereby coordinating development, homeostasis of the organism and its reactions to external stimuli. Knowledge of the components of the intercellular signaling (specifically: the endocrine) network and their relations is an important, though so far a largely neglected part of systems biology. DESCRIPTION: EndoNet is an information resource about the endocrine system in human. The content of this database comprises information about the biological components of the endocrine system, like hormones, receptors and cells, as well as their relations like the secretion or the binding of a hormone to its receptor. All data within EndoNet have been manually annotated from the scientific literature. The web interface of EndoNet provides the content by a detailed page for each component. These pages list information about the component, links to external resources including literature as well as to related entities of EndoNet. The anatomical ontology Cytomer is used, in conjunction with the Ontology Based Answers service (OBA), to query and list related anatomical structures ranging from the level of individual cells to complete organs. While querying the web interface the user can add components to an individual network. This network, or the complete network stored in the database, can be further analyzed in a configurable pipeline or can be exported in various formats. CONCLUSION: EndoNet is an important and unique information resource about the intercellular signaling network. Since the intercellular network is an integral part of systems biology, EndoNet provides essential information for analyzing interaction between different cellular networks.

Pancreas development in humans.

PURPOSE OF REVIEW: We highlight some of the major recent advances in characterizing human pancreas development and endocrine cell differentiation. RECENT FINDINGS: Extensive research efforts have helped to define crucial events in the mouse pancreas organogenesis. Information gained from these studies was used to develop human embryonic stem cell (hESC) differentiation protocols with the goal of generating functional glucose-responsive, insulin-producing human ß-cells. In spite of remarkable progress in hESC differentiation, current protocols based on mouse developmental biology can produce human ß-cells only in vivo. New differentiation markers and recently generated reagents may provide an unprecedented opportunity to develop a high-density expression map of human fetal pancreas and pancreatic islets that could serve as a reference point for in vitro hESC differentiation. SUMMARY: Integrating an increased knowledge of human pancreas development into hESC differentiation protocols has the potential to greatly advance our ability to generate functional insulin-producing cells for ß-cell replacement therapy.

Ovarian and placental morphology and endocrine functions in the pregnant giraffe (Giraffa camelopardalis).

Gross, histological and immunocytochemical examinations carried out on maternal and fetal reproductive tissues from two pregnant giraffes at an estimated 8 and 13.5 months of gestation (term=15 months) revealed a typically ruminant macrocotyledonary placenta with binucleate trophoblast cells scattered sparsely in the placentome where they stained intensely with a prolactin antiserum. Binucleate cells were present in greater numbers in the intercotyledonary allantochorion where they did not stain for prolactin whereas the uninucleate trophoblast still did. A single large corpus luteum of pregnancy and several small luteinised follicles were present in the maternal ovaries while the fetal ovaries at 13.5 months gestation showed an assortment of enlarging antral follicles and partially and completely lutenised follicles, the granulosa and luteal cells of which stained positively for 3ß-hydroxysteroid dehydrogenase (3ß-HSD), 17,20 lyase, prolactin, progesterone receptor and androgen receptor, but negatively for aromatase. The uninucleate trophoblast of the placentome and intercotyledonary allantochorion, the epithelium of the maternal endometrial glands, the seminiferous epithelium in the fetal testis at 8 months of gestation and the zonae fasciculata and reticularis of the fetal adrenal at 13.5 months also stained positively for 3ß-HSD and negatively for aromatase. Endocrinologically, it appears that the giraffe placenta is more similar to that of the sheep than the cow with a placental lactogen as the likely driver of the considerable degree of luteinisation seen in both the maternal and the fetal ovaries.

New findings in pancreatic and intestinal endocrine development to advance regenerative medicine.

PURPOSE OF REVIEW: We highlight some of the major recent advances that have been made towards understanding the mechanisms that control endocrine differentiation and cell identity in the pancreas and intestine. RECENT FINDINGS: Notch signaling plays a complex role in the fate choice between endocrine, duct, and acinar lineages in the developing pancreas. New approaches to dissecting the role of mesenchymal cells in the developing endocrine pancreas reveal inhibitory signals from the endothelium. Epigenetic mechanisms represent another layer of control over pancreatic development and ß cell identity. Further details on the transcriptional control of enteroendocrine cell development have emerged and revealed a surprising role for FoxO1 in restraining insulin expression in the gut. Incremental progress is being made in the field of directed differentiation of embryonic stem cells to pancreatic ß cells and the first reported differentiation of human embryonic stem cells into intestinal organoids containing enteroendocrine cells represents a major breakthrough. SUMMARY: Greater knowledge of the fundamental processes controlling endocrine development in the pancreas and intestine has the potential to advance the field of regenerative medicine by providing a pathway to successfully create cell types of clinical interest.