Characterization of the Secretome, Transcriptome, and Proteome of Human ß Cell Line EndoC-ßH1.

Early diabetes research is hampered by limited availability, variable quality, and instability of human pancreatic islets in culture. Little is known about the human ß cell secretome, and recent studies question translatability of rodent ß cell secretory profiles. Here, we verify representativeness of EndoC-ßH1, one of the most widely used human ß cell lines, as a translational human ß cell model based on omics and characterize the EndoC-ßH1 secretome. We profiled EndoC-ßH1 cells using RNA-seq, data-independent acquisition, and tandem mass tag proteomics of cell lysate. Omics profiles of EndoC-ßH1 cells were compared to human ß cells and insulinomas. Secretome composition was assessed by data-independent acquisition proteomics. Agreement between EndoC-ßH1 cells and primary adult human ß cells was ∼90% for global omics profiles as well as for ß cell markers, transcription factors, and enzymes. Discrepancies in expression were due to elevated proliferation rate of EndoC-ßH1 cells compared to adult ß cells. Consistently, similarity was slightly higher with benign nonmetastatic insulinomas. EndoC-ßH1 secreted 783 proteins in untreated baseline state and 3135 proteins when stressed with nontargeting control siRNA, including known ß cell hormones INS, IAPP, and IGF2. Further, EndoC-ßH1 secreted proteins known to generate bioactive peptides such as granins and enzymes required for production of bioactive peptides. EndoC-ßH1 secretome contained an unexpectedly high proportion of predicted extracellular vesicle proteins. We believe that secretion of extracellular vesicles and bioactive peptides warrant further investigation with specialized proteomics workflows in future studies.

Loss of Human Beta Cell Identity in a Reconstructed Omental Stromal Cell Environment.

In human type 2 diabetes, adipose tissue plays an important role in disturbing glucose homeostasis by secreting factors that affect the function of cells and tissues throughout the body, including insulin-producing pancreatic beta cells. We aimed here at studying the paracrine effect of stromal cells isolated from subcutaneous and omental adipose tissue on human beta cells. We developed an in vitro model wherein the functional human beta cell line EndoC-ßH1 was treated with conditioned media from human adipose tissues. By using RNA-sequencing and western blotting, we determined that a conditioned medium derived from omental stromal cells stimulates several pathways, such as STAT, SMAD and RELA, in EndoC-ßH1 cells. We also observed that upon treatment, the expression of beta cell markers decreased while dedifferentiation markers increased. Loss-of-function experiments that efficiently blocked specific signaling pathways did not reverse dedifferentiation, suggesting the implication of more than one pathway in this regulatory process. Taken together, we demonstrate that soluble factors derived from stromal cells isolated from human omental adipose tissue signal human beta cells and modulate their identity.

Regulated expression and function of the GABAB receptor in human pancreatic beta cell line and islets.

G protein-coupled receptors are seven transmembrane signaling molecules that are involved in a wide variety of physiological processes. They constitute a large protein family of receptors with almost 300 members detected in human pancreatic islet preparations. However, the functional role of these receptors in pancreatic islets is unknown in most cases. We generated a new stable human beta cell line from neonatal pancreas. This cell line, named ECN90 expresses both subunits (GABBR1 and GABBR2) of the metabotropic GABAB receptor compared to human islet. In ECN90 cells, baclofen, a specific GABAB receptor agonist, inhibits cAMP signaling causing decreased expression of beta cell-specific genes such as MAFA and PCSK1, and reduced insulin secretion. We next demonstrated that in primary human islets, GABBR2 mRNA expression is strongly induced under cAMP signaling, while GABBR1 mRNA is constitutively expressed. We also found that induction and activation of the GABAB receptor in human islets modulates insulin secretion.

Stearoyl CoA desaturase is a gatekeeper that protects human beta cells against lipotoxicity and maintains their identity.

AIMS/HYPOTHESIS: During the onset of type 2 diabetes, excessive dietary intake of saturated NEFA and fructose lead to impaired insulin production and secretion by insulin-producing pancreatic beta cells. The majority of data on the deleterious effects of lipids on functional beta cell mass were obtained either in vivo in rodent models or in vitro using rodent islets and beta cell lines. Translating data from rodent to human beta cells remains challenging. Here, we used the human beta cell line EndoC-ßH1 and analysed its sensitivity to a lipotoxic and glucolipotoxic (high palmitate with or without high glucose) insult, as a way to model human beta cells in a type 2 diabetes environment. METHODS: EndoC-ßH1 cells were exposed to palmitate after knockdown of genes related to saturated NEFA metabolism. We analysed whether and how palmitate induces apoptosis, stress and inflammation and modulates beta cell identity. RESULTS: EndoC-ßH1 cells were insensitive to the deleterious effects of saturated NEFA (palmitate and stearate) unless stearoyl CoA desaturase (SCD) was silenced. SCD was abundantly expressed in EndoC-ßH1 cells, as well as in human islets and human induced pluripotent stem cell-derived beta cells. SCD silencing induced markers of inflammation and endoplasmic reticulum stress and also IAPP mRNA. Treatment with the SCD products oleate or palmitoleate reversed inflammation and endoplasmic reticulum stress. Upon SCD knockdown, palmitate induced expression of dedifferentiation markers such as SOX9, MYC and HES1. Interestingly, SCD knockdown by itself disrupted beta cell identity with a decrease in mature beta cell markers INS, MAFA and SLC30A8 and decreased insulin content and glucose-stimulated insulin secretion. CONCLUSIONS/INTERPRETATION: The present study delineates an important role for SCD in the protection against lipotoxicity and in the maintenance of human beta cell identity. DATA AVAILABILITY: Microarray data and all experimental details that support the findings of this study have been deposited in in the GEO database with the GSE130208 accession code.

Development of the Endocrine Pancreas in the Beagle Dog: From Fetal to Adult Life.

Our objectives were to describe, in Beagle dogs, the ontogenesis of beta (insulin-producing) and alpha (glucagon-producing) cells from fetal to early postnatal life and adulthood. In addition, to have some insight into interspecies comparison, Beagle dog pancreases were compared to pancreases from a Labrador and Chow Chow. At midgestation, the epithelium was dense, beta cells scarce, and alpha cells numerous and concentrated in the center of the pancreatic bud. From 36 to 45 days post conception (pc), beta cell numbers increased and the epithelium expanded and branched out. At 55 days pc, large beta cell aggregates were seen. At weaning, the islets were similar to those in adults, with limited alpha cells intermingled with numerous beta cells. Quantification of the Alpha to Beta cells ratio has shown a gradual increase of beta cells proportion throughout development. Similar findings were obtained in the two other breeds. In conclusion, in the fetal Beagle dog beta cells emerge from the pancreatic bud at midgestation, but the endocrine structure is mature only in early postnatal life. The ontogenesis of the endocrine pancreas demonstrated in dogs resembles that reported in rats and mice. In contrast, human beta cells appear earlier, at the beginning of the second trimester of gestation. Our study provides a detailed morphological description of pancreatic development in dogs but supplies no information on alpha- or beta-cell function during fetal life. The morphological data reported here provide a foundation for building physiological studies. Anat Rec, 300:1429-1438, 2017. © 2017 Wiley Periodicals, Inc.

Aggregation of Engineered Human ß-Cells Into Pseudoislets: Insulin Secretion and Gene Expression Profile in Normoxic and Hypoxic Milieu.

Innovative treatments to cure type 1 diabetes are being actively researched. Among the different strategies, the replacement of ß-cells has given promising results. Classically, islets from cadaveric donors are transplanted into diabetic patients, but recently phase I clinical trials that use stem cell-derived ß-cells have been started. Such protocols require either an immunosuppressive treatment or the macroencapsulation of the ß-cells. They involve cell aggregation and the exposure of the cells to hypoxia. Using an engineered human ß-cell, we have addressed these two problems: a novel human ß-cell line called EndoC-ßH3 was cultured as single cells or aggregated clusters. EndoC-ßH3 cells were also cultured at normal atmospheric oxygen tension (pO2 = 21%) or hypoxia (pO2 = 3%) in the presence or absence of modulators of the hypoxia-inducible factor 1α (HIF1α) pathway. Cell aggregation improved glucose-stimulated insulin secretion, demonstrating the benefit of cell-cell contacts. Low oxygen tension decreased ß-cell viability and their sensitivity to glucose, but did not alter insulin production nor the insulin secretion capacity of the remaining cells. To investigate the role of HIF1α, we first used a HIF stabilizer at pO2 = 21%. This led to a mild decrease in cell viability, impaired glucose sensitivity, and altered insulin secretion. Finally, we used a HIF inhibitor on EndoC-ßH3 pseudoislets exposed to hypoxia. Such treatment considerably decreased cell viability. In conclusion, aggregation of the EndoC-ßH3 cells seems to be important to improve their function. A fraction of the EndoC-ßH3 cells are resistant to hypoxia, depending on the level of activity of HIF1α. Thus, these cells represent a good human cell model for future investigations on islet cell transplantation analysis.

A human beta cell line with drug inducible excision of immortalizing transgenes.

OBJECTIVES: Access to immortalized human pancreatic beta cell lines that are phenotypically close to genuine adult beta cells, represent a major tool to better understand human beta cell physiology and develop new therapeutics for Diabetes. Here we derived a new conditionally immortalized human beta cell line, EndoC-ßH3 in which immortalizing transgene can be efficiently removed by simple addition of tamoxifen. METHODS: We used lentiviral mediated gene transfer to stably integrate a tamoxifen inducible form of CRE (CRE-ERT2) into the recently developed conditionally immortalized EndoC ßH2 line. The resulting EndoC-ßH3 line was characterized before and after tamoxifen treatment for cell proliferation, insulin content and insulin secretion. RESULTS: We showed that EndoC-ßH3 expressing CRE-ERT2 can be massively amplified in culture. We established an optimized tamoxifen treatment to efficiently excise the immortalizing transgenes resulting in proliferation arrest. In addition, insulin expression raised by 12 fold and insulin content increased by 23 fold reaching 2 µg of insulin per million cells. Such massive increase was accompanied by enhanced insulin secretion upon glucose stimulation. We further observed that tamoxifen treated cells maintained a stable function for 5 weeks in culture. CONCLUSIONS: EndoC ßH3 cell line represents a powerful tool that allows, using a simple and efficient procedure, the massive production of functional non-proliferative human beta cells. Such cells are close to genuine human beta cells and maintain a stable phenotype for 5 weeks in culture.

Human fucci pancreatic Beta cell lines: new tools to study Beta cell cycle and terminal differentiation.

Regulation of cell cycle in beta cells is poorly understood, especially in humans. We exploited here the recently described human pancreatic beta cell line EndoC-ßH2 to set up experimental systems for cell cycle studies. We derived 2 populations from EndoC-ßH2 cells that stably harbor the 2 genes encoding the Fucci fluorescent indicators of cell cycle, either from two vectors, or from a unique bicistronic vector. In proliferating non-synchronized cells, the 2 Fucci indicators revealed cells in the expected phases of cell cycle, with orange and green cells being in G1 and S/G2/M cells, respectively, and allowed the sorting of cells in different substeps of G1. The Fucci indicators also faithfully red out alterations in human beta cell proliferative activity since a mitogen-rich medium decreased the proportion of orange cells and inflated the green population, while reciprocal changes were observed when cells were induced to cease proliferation and increased expression of some beta cell genes. In the last situation, acquisition of a more differentiated beta cell phenotype correlates with an increased intensity in orange fluorescence. Hence Fucci beta cell lines provide new tools to address important questions regarding human beta cell cycle and differentiation.

Development of a conditionally immortalized human pancreatic ß cell line.

Diabetic patients exhibit a reduction in ß cells, which secrete insulin to help regulate glucose homeostasis; however, little is known about the factors that regulate proliferation of these cells in human pancreas. Access to primary human ß cells is limited and a challenge for both functional studies and drug discovery progress. We previously reported the generation of a human ß cell line (EndoC-ßH1) that was generated from human fetal pancreas by targeted oncogenesis followed by in vivo cell differentiation in mice. EndoC-ßH1 cells display many functional properties of adult ß cells, including expression of ß cell markers and insulin secretion following glucose stimulation; however, unlike primary ß cells, EndoC-ßH1 cells continuously proliferate. Here, we devised a strategy to generate conditionally immortalized human ß cell lines based on Cre-mediated excision of the immortalizing transgenes. The resulting cell line (EndoC-ßH2) could be massively amplified in vitro. After expansion, transgenes were efficiently excised upon Cre expression, leading to an arrest of cell proliferation and pronounced enhancement of ß cell-specific features such as insulin expression, content, and secretion. Our data indicate that excised EndoC-ßH2 cells are highly representative of human ß cells and should be a valuable tool for further analysis of human ß cells.

A genetically engineered human pancreatic ß cell line exhibiting glucose-inducible insulin secretion.

Despite intense efforts over the past 30 years, human pancreatic ß cell lines have not been available. Here, we describe a robust technology for producing a functional human ß cell line using targeted oncogenesis in human fetal tissue. Human fetal pancreatic buds were transduced with a lentiviral vector that expressed SV40LT under the control of the insulin promoter. The transduced buds were then grafted into SCID mice so that they could develop into mature pancreatic tissue. Upon differentiation, the newly formed SV40LT-expressing ß cells proliferated and formed insulinomas. The resulting ß cells were then transduced with human telomerase reverse transcriptase (hTERT), grafted into other SCID mice, and finally expanded in vitro to generate cell lines. One of these cell lines, EndoC-ßH1, expressed many ß cell-specific markers without any substantial expression of markers of other pancreatic cell types. The cells secreted insulin when stimulated by glucose or other insulin secretagogues, and cell transplantation reversed chemically induced diabetes in mice. These cells represent a unique tool for large-scale drug discovery and provide a preclinical model for cell replacement therapy in diabetes. This technology could be generalized to generate other human cell lines when the cell type-specific promoter is available.

A new strategy to generate functional insulin-producing cell lines by somatic gene transfer into pancreatic progenitors.

BACKGROUND: There is increasing interest in developing human cell lines to be used to better understand cell biology, but also for drug screening, toxicology analysis and future cell therapy. In the endocrine pancreatic field, functional human beta cell lines are extremely scarce. On the other hand, rodent insulin producing beta cells have been generated during the past years with great success. Many of such cell lines were produced by using transgenic mice expressing SV40T antigen under the control of the insulin promoter, an approach clearly inadequate in human. Our objective was to develop and validate in rodent an alternative transgenic-like approach, applicable to human tissue, by performing somatic gene transfer into pancreatic progenitors that will develop into beta cells. METHODS AND FINDINGS: In this study, rat embryonic pancreases were transduced with recombinant lentiviral vector expressing the SV40T antigen under the control of the insulin promoter. Transduced tissues were next transplanted under the kidney capsule of immuno-incompetent mice allowing insulinoma development from which beta cell lines were established. Gene expression profile, insulin content and glucose dependent secretion, normalization of glycemia upon transplantation into diabetic mice validated the approach to generate beta cell lines. CONCLUSIONS: Somatic gene transfer into pancreatic progenitors represents an alternative strategy to generate functional beta cell lines in rodent. Moreover, this approach can be generalized to derive cells lines from various tissues and most importantly from tissues of human origin.

NAD+ released during inflammation participates in T cell homeostasis by inducing ART2-mediated death of naive T cells in vivo.

Mono ADP-ribosyltransferase 2 (ART2) is an ectoenzyme expressed on mouse T lymphocytes, which catalyze the transfer of ADP-ribose groups from NAD(+) onto several target proteins. In vitro, ADP-ribosylation by ART2 activates the P2X7 ATP receptor and is responsible for NAD(+)-induced T cell death (NICD). Yet, the origin of extracellular NAD(+) and the role of NICD in vivo remain elusive. In a model of acute inflammation induced by polyacrylamide beads, we demonstrate release of NAD(+) into exudates during the early phase of the inflammatory response. This leads to T cell depletion in the draining lymph nodes from wild-type and, more severely, from mice lacking the CD38 NAD(+) glycohydrolase, whereas no effect is observed in ART2-deficient animals. Intravenous injection of NAD(+) used to exacerbate NICD in vivo results in fast and dramatic ART2- and P2X7-dependent depletion of CD4+ and CD8+ T lymphocytes, which can affect up to 80% of peripheral T cells in CD38(-/-) mice. This affects mainly naive T cells as most cells surviving in vivo NAD+ treatment exhibit the phenotype of recently activated/memory cells. Consistently, treatment with NAD(+) abolishes primary Ab response to a T-dependent Ag in NICD-susceptible CD38(-/-) mice but has no effect on the secondary response when given several days after priming. Unexpectedly NAD+ treatment improves the response in their wild-type BALB/c counterparts. We propose that NAD(+) released during early inflammation facilitates the expansion of primed T cells, through ART2-driven death of resting cells, thus contributing to the dynamic regulation of T cell homeostasis.

Convergent alteration of granulopoiesis, chemotactic activity, and neutrophil apoptosis during mouse selection for high acute inflammatory response.

Neutrophil homeostasis was investigated in two mouse lines, AIRmax and AIRmin, genetically selected for high or low acute inflammatory response (AIR) and compared with unselected BALB/c mice. Mature neutrophil phenotype and functions appeared similar in the three mouse lines. However, an unprecedented phenotype was revealed in AIRmax animals characterized by a high neutrophil production in bone marrow (BM), a high number of neutrophils in blood, a high concentration of chemotactic agents in acrylamide-induced inflammatory exudates, and an increased resistance of locally infiltrated neutrophils to spontaneous apoptosis. In vitro, BM production of neutrophils and eosinophils was accompanied by an unusual high up-regulation of cytokine receptors as assessed by antibodies to CD131, which bind the common beta chain of receptors to interleukin (IL)-3, IL-5, and granulocyte macrophage-colony stimulating factor. An accelerated neutrophil maturation was also observed in response to all-trans retinoic acid. Several candidate genes can be proposed to explain this phenotype. Yet, more importantly, the results underline that genetic selection, based on the degree of AIR and starting from a founding population resulting from the intercross of eight inbred mouse lines, which display a continuous range of inflammatory responses, can lead to the convergent selection of alleles affecting neutrophil homeostasis. Similar gene combinations may occur in the human with important consequences in the susceptibility to inflammatory or infectious diseases and cancer.