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Development of a conditionally immortalized human pancreatic β cell line
Raphaël Scharfmann, … , Paul Czernichow, Philippe Ravassard
Raphaël Scharfmann, … , Paul Czernichow, Philippe Ravassard
Published March 25, 2014
Citation Information: J Clin Invest. 2014;124(5):2087-2098. https://doi.org/10.1172/JCI72674.
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Technical Advance Endocrinology

Development of a conditionally immortalized human pancreatic β cell line

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Abstract

Diabetic 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.

Authors

Raphaël Scharfmann, Severine Pechberty, Yasmine Hazhouz, Manon von Bülow, Emilie Bricout-Neveu, Maud Grenier-Godard, Fanny Guez, Latif Rachdi, Matthias Lohmann, Paul Czernichow, Philippe Ravassard

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Figure 1

Strategy to produce reversely immortalized cell line and immunofluorescence analysis of EndoC-βH2 cells.

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Strategy to produce reversely immortalized cell line and immunofluoresce...
(A) Schematic representation of excisable lentiviral vector used to produce the EndoC-βH2 reversely immortalized cell line. (B) Immunofluorescence analysis of EndoC-βH2 cells. EndoC-βH2 cells stained positive for insulin (INS) and coexpressed C-peptide (C-PEPT), CHGA, PDX1, NKX6.1, SV40 LT, and Ki67. The cells rarely express somatostatin (SST) and do not express glucagon (GCG), amylase (AMY), carboxipeptidase-A (CPA), or SOX9. Nuclei were stained with Hoechst 33342 fluorescent stain (blue). Photographs of all insulin stainings were taken using a 700 ms acquisition time to obtain an unsaturated signal. Scale bars: 50 μm.

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