Development of a conditionally immortalized human pancreatic β cell line

International audienceDiabetic patients exhibit a reduction in β 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 β cells is limited and a challenge for both functional studies and drug discovery progress. We previously reported the generation of a human β cell line (EndoC-βH1) that was generated from human fetal pancreas by targeted oncogenesis followed by in vivo cell differentiation in mice. EndoC-βH1 cells display many functional properties of adult β cells, including expression of β cell markers and insulin secretion following glucose stimulation; however, unlike primary β cells, EndoC-βH1 cells continuously proliferate. Here, we devised a strategy to generate conditionally immortalized human β cell lines based on Cre-mediated excision of the immortalizing transgenes. The resulting cell line (EndoC-βH2) 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 β cell–specific features such as insulin expression, content, and secretion. Our data indicate that excised EndoC-βH2 cells are highly representative of human β cells and should be a valuable tool for further analysis of human β cells

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

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

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-βH1 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-βH1 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 GABA B receptor in human pancreatic beta cell line and islets

International audienceG 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

Immunohistochemical analysis of grafts developed in Scid mice.

<p>A–C: Staining for insulin (red), SV40T (green) and DAPI (blue); D–F: Staining for insulin (red), Pdx1 (green) and DAPI (blue); G–I: Staining for insulin (red), BrdU (green) and DAPI (blue). Scale bars: 25 µm</p

Immunocytochemical Characterization of RYAS41 cells.

<p>A: Schematic representation of the culture procedure used to derive the RYAS41 cell line. P represents passage number. Surface of the culture well is indicated below the time line. B: Coexpression of insulin (red) and SV40T (green); insulin (red) and c-peptide (green), insulin (red) and Pdx1 (green); and double staining for insulin (red) and BrdU (green). Scale bars: 25 µm.</p

Gene expression profile in RYAS41 and glucose-stimulated insulin secretion.

<p>A: Semi-quantitative RT PCR comparison between RYAS41, lung (negative control) and pancreas (positive control) from E17 rat embryos. PCR products after 40 amplification cycles are analyzed on a 2% agarose gel. B: CT (threshold cycle) value are normalized to cyclophilin and presented as fold increase compared to 832/13 INS-1 cells. Values are means+/−S.E.M. of Q-PCR performed in duplicates from 3 independent RNA extractions. C: RYAS41 secrete insulin in response to glucose stimulation. Insulin secreted into the medium is presented as % of insulin content secreted per hour. Values are means+/−S.E.M. of three independent cell cultures.</p

Transplanted RYAS41 cells restore normoglycemia in diabetic mice.

<p>Fourteen <i>scid</i> mice were injected with streptozotocin. Two days later, 3-weeks lasting insulin capsules were subcutaneously implanted to hyperglycemic mice. Two weeks later, half of the mice were transplanted under the kidney capsule with 10⁶ RYAS41 cells. Grafted cells were removed by nephrectomy at day 66. Values are means+/−S.E.M.</p