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

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

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