Phenotypic and functional characterization of glucagon-positive cells derived from spontaneous differentiation of D3-mouse embryonic stem cells

Abstract

Background Glucagon expression is being considered as a definitive endoderm marker in protocols aiming to obtain insulin-secreting cells from embryonic stem cells. However, it should be considered that in vivo glucagon is expressed both in definitive endoderm- and neuroectoderm-derived cells. Therefore, the true nature and function of in vitro spontaneously differentiated glucagon-positive cells remains to be established.

Methods D3 and R1 mouse embryonic stem cells as well as α-TC1-9 cells were cultured and glucagon expression was determined by real-time PCR and immunocytochemistry. Functional analyses regarding intracellular calcium oscillations were performed to further characterize glucagon+ cells.

Results Specifically, 5% of D3 and R1 cells expressed preproglucagon, with a small percentage of these (<1%) expressing glucagon-like peptide 1. The constitutive expression of protein convertase 5 supports the expression of both peptides. Glucagon+ cells co-expressed neurofilament middle and some glucagon-like peptide-1+ cells, glial fibrillary acidic protein, indicating a neuroectodermic origin. However, few glucagon-like peptide-1+ cells did not show coexpression with glial fibrillary acidic protein, suggesting a non-neuroectodermic origin for these cells. Finally, glucagon+ cells did not display Ca2+ oscillations typical of pancreatic α-cells.

Discussion These results indicate the possible nondefinitive endodermal origin of glucagon-positive cells spontaneously differentiated from D3 and R1 cell lines, as well as the presence of cells expressing glucagon-like peptide-1 from two different origins.