Abstract
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-βH2 to set up experimental systems for cell cycle studies. We derived 2 populations from EndoC-βH2 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.
Highlights
The cell cycle consists in four phases: G1, S, G2 and M
EndoC-bH2 Fucci cell lines Among various attempts to generate human pancreatic beta cell lines, the recently described EndoCbH1 cells offer the most compelling functional characterization [28]. This cell line was obtained by transduction of human fetal pancreatic cells with two lentiviral vectors encoding the large T antigen of the SV40 virus (SV40LT) and the human telomere reverse transcriptase enzyme, both of them being controlled by the by the rat insulin promoter (RIP)
To design human Fucci beta cells, we started from the closely related cell line, termed EndoC-bH2 [12] that has been generated in the same way as EndoCbH1, with one important difference providing more experimental flexibility: the two immortalizing transgenes, SV40LT and human telomere reverse transcriptase enzyme (hTERT) are flanked by Lox sequences
Summary
The cell cycle consists in four phases: G1, S, G2 and M. The most performant is the so-called Fucci system (Fluorescent Ubiquitination-based Cell Cycle Indicator) It combines two distinct fluorescent markers, namely human CDT1 (Cdc dependent transcript 1) fused to an orange fluorescent protein (monomeric Kusabira Orange, mKO2) and human GEMININ fused to a green fluorescent protein (monomeric Azami Green, mAG) [7]. Several cell cycle regulators or transcription factors, either alone or in combinations, appear to stimulate the proliferation of human adult beta cells [20,21,22,23] These studies provide questionable conclusions as being mostly based on vast (adenovirusmediated) overexpression. New methods and tools to study human beta proliferation would be highly desirable to overcome these limits Toward this goal, we set up here human beta cell lines stably expressing the Fucci cell cycle indicators. We provide here several lines of evidence indicating that these new Fucci cell lines are reliable and convenient tools to decipher the control of cell cycle and differentiation of human pancreatic beta cells
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