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Title: Phenotypic and functional characterization of glucagon-positive cells derived from spontaneous differentiation of D3-mouse embryonic stem cells
Authors: Vicente-Salar, Nestor
Santana, Alfredo 
Juan-Picó, Pablo
Reig, Juan A.
Roche, Enrique
UNESCO Clasification: 32 Ciencias médicas
241003 Citología humana
Keywords: Cell culture
Definitive endoderm
Pancreatic hormones
Stem cell
Issue Date: 2013
Journal: Cytotherapy 
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 Ca(2+) 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.
ISSN: 1465-3249
DOI: 10.1016/j.jcyt.2012.08.002
Source: Cytotherapy [ISSN 1465-3249], v. 15, p. 122-131
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