Pancreatic Differentiation of Sox 17 Knock-in Mouse Embryonic Stem Cells in Vitro
الموضوعات :
1 - Department of Biology of Reproduction, Instituteof AnimalScience, Pratelstvi 815, 10400 Prague, Czech Republic
الکلمات المفتاحية: insulin, definitive endoderm, Differentiation, embryonic stem cells, progenitors,
ملخص المقالة :
The way to overcome current limitations in the generation of glucose-responsive insulin-producing cells is selective enrichment of the number of definitive endoderm (DE) progenitor cells. Sox17 is the marker of mesendoderm and definitive endoderm. The aim of the present research was to study the potential of Sox17 knock-in CGR8 mouse embryonic stem (ES) cells to differentiate into insulin producing cells in vitro by detection of specific markers at different stages of three-step protocol of differentiation. At day 5+9 cells strongly expressed Sox17 as well as CxCr4 and N-cadherine, while staining for Oct-4 (octamer-binding transcription factor 4) was faint. At day 5+16 the expression of Sox17 gradually disappeared and cells were positive for Neurogenin 3 (NGN3), Islet-1 and C-peptide. At last stage of differentiation protocol (day 5+23) Sox17 knock-in CGR8 ES derived cells showed insulin and C-peptide expression. We conclude that Sox17 knock-in mouse ES cells are equally suitable for differentiation into insulin producing cells as commonly used CGR8 ES cells. Genetically modified ES cells allow the selection of Sox17-expressing cells and the selective enrichment of DE progenitors.
Blyszczuk P., Czyz J., Kania G., Wagner M., Roll U., St-Onge L. and Wobus A.M. (2003).Expression of Pax4 in embryonic stem cells promotes differentiation of nestin-positive progenitor and insulin-producing cells.Proc. Natl. Acad. Sci.USA. 100, 998-1003.
Blyszczuk P., Asbrand C., Rozzo A., Kania G., St-Onge L., Rupnik M. and Wobus A.M. (2004).Embryonic stem cells differentiate into insulin-producing cells without selection of nestin-expressing cells. Int. J. Dev. Biol.48, 1095-1104.
D'Amour K.A., Bang A.G., Eliazer S., Kelly O.G., Agulinick A.D., Smart N.G., Moorman M.A., Kroon E., Carpenter M.K. and Baetge E.E. (2006). Production of pancreatic hormone-expressing endocrine cells from human embryonic stem cells. Natl. Biotechnol. 24, 1392-1401.
GuG., Wells J.M., Dombkowski D., Preffer F., Aronow B. and Melton D.A. (2004). Global expression analysis of gene regulatory pathways during endocrine pancreatic development. Development. 131, 165-179.
Kanai-Azuma M., Kanai Y., Gad J.M., Tajima Y., Taya C,. Kurohmaru M., Sanai Y., Yonekawa H., Yazaki K., Tam P.P and Hayashi Y. (2002). Depletion of definitive gut endoderm in Sox17-null mutant mice. Development. 129, 2367-2379.
Rolletschek A., Schroeder I.,Schulz H., Hummel O., Huebner N. and Wobus A.M. (2010). Characterization of mouse embryonic stem cell differentiation into the pancreatic lineage in vitro by transcriptional profiling, quantitative RT-PCR and immunocytochemistry.Int. J. Dev. Biol. 54, 41-54.
SchroederI., Rolletschek A., Blyszczuk P., Kania G. and Wobus A.M. (2006). Differentiation of mouse embryonic stem cells to insulin-producing cells.Nature Prot.1, 495-507.
Tada S., Era T., Furusawa C., Sakurai H., Niahikawa S., Kinoshita M., Nakao K., Chiba T. and Nishikawa S. (2005). Characterization of mesendoderm: a diverging point of the definitive endoderm and mesoderm in embryonic stem cell differentiation culture. Development. 132, 4363-4374.
Wobus A.M. and Boheler K.R. (2005). Embryonic stem cells: prospects for developmental biology and cell therapy. Physiol. Rev.85, 635-678.
