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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 Neuroectoderm 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. | URI: | http://hdl.handle.net/10553/46089 | 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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