Morphological, genetic and protein characterization in the differentiation of embryonic stem cells to early pancreatic cells

DOI : http://dx.doi.org/10.4067/S0717-95022013000400044
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Vázquez-Zapién, G. J.; Sánchez Monroy, V.; Chirino López, Y. I., & Mata-Miranda, M. M.

Summary

Due to the boom in regenerative medicine, Stem Cells (SC) represent a source of cell replacement to any tissue, we decided to undertake this research with the objective of differentiating mouse embryonic stem cells (mESC) to early pancreatic cells, developing their genetic and morphological characterization. Initially Mouse embryonic fibroblasts (MEF) were grown and arrested in their cell cycle with mitomycin, subsequently mouse embryonic SC (mESC) were expanded and subjected in to a pancreatic cell differentiation protocol of 21 days. During differentiation, morphology and the relative expression of sox-17, pdx-1, Ins-1 and Ins-2 genes were assessed, also the production of insulin and glucagon proteins was determinated by fluorescence microscopy and flow cytometry. Embryoid bodies (EBs) were obtained from mESC, with different morphological characteristics according to their differentiation, which expressed endodermal germ line genes (sox-17 y pdx-1) at days 0, 11 and 17 of differentiation, an inductor gene of embryonic pancreas development (pdx-1) was detected at day 11 of differentiation. Pancreas genes (ins-1 e ins-2) were expressed at day 17 and 21 of differentiation. Finally the production of insulin and glucagon proteins was detected on the EBS at day 21 of differentiation. In conclusion, the mESC differentiation was achieved. The morphological analysis evidenced three-dimensional cell clusters corresponding to EBs. Analysis of the gene expression patterns in the differentiation process, cells initially showed genetic characteristics of endoderm and thereafter from day 17 of differentiation characteristics of early pancreatic cells which by day 21 of differentiation expressed insulin and glucagon proteins. The above results suggest the need to continue with the study of differentiation protocols to determine the ESC behavior under differentiation and their potential therapeutic use by implementing differentiated cells in animal models.

KEY WORDS: Stem cell; Stem cell differentiation; Differentiated cells; Embryoid bodies; Insulin-producers cells.

How to cite this article

VÁZQUEZ-ZAPIÉN, G. J.; SÁNCHEZ MONROY, V.; CHIRINO LÓPEZ, Y. I. & MATA-MIRANDA, M. M. Morphological, genetic and protein characterization in the differentiation of embryonic stem cells to early pancreatic cells. Int. J. Morphol., 31(4):1421- 1429, 2013.