naa a22 4500 510739547 CHVBK 20180411083023.0 cr unu---uuuuu 180411e20131001xx s 000 0 eng 10.1007/s12015-012-9416-1 doi (NATIONALLICENCE)springer-10.1007/s12015-012-9416-1 MicroRNA Target Sites as Genetic Tools to Enhance Promoter-Reporter Specificity for the Purification of Pancreatic Progenitor Cells from Differentiated Embryonic Stem Cells [Elektronische Daten] [Ulf Diekmann, Matthias Elsner, Jan Fiedler, Thomas Thum, Sigurd Lenzen, Ortwin Naujok] Pluripotent cells hold great promise for cell replacement therapies in regenerative medicine. All known protocols for directed in vitro differentiation of pluripotent cells did not yield pure populations complicating the characterization of the derived cells. In addition, the risk of tumor formation due to residual undifferentiated cells is a serious unresolved problem. In the present study the tissue-specific mouse Pdx1 promoter was used to control the expression of the reporter gene GFP2 in mouse ES cells in order to purify them via FACS during in vitro differentiation. The background fluorescence of transduced ES cells hampered the purification of Pdx1-positive cells due to a contaminating population of partially undifferentiated cells. MicroRNAs (mir) are important regulators of gene expression and were used to enhance promoter specificity during differentiation towards pancreatic progenitor cells. The mouse mmu-mir-294 was found to be mainly expressed during pluripotency, whereas the expression of the mir-302 cluster was increased during early differentiation. Integration of a microRNA target site for the mmu-mir-294 into the lentiviral vector reduced the background fluorescence specifically during pluripotency and permitted re-occurrence of GFP2 expression upon differentiation. A combination of the microRNA target site with the Pdx1 promoter fragment allowed the purification of pancreatic progenitors from differentiated ES cells. This population reflected an early pancreatic progenitor population without other contaminating cell lineages. In conclusion, microRNA target sites are efficient regulatory elements to control transgene expression and to enhance tissue specificity as presented in this study facilitating the sorting and purification of Pdx1-positive pancreatic progenitor cells. Springer Science+Business Media New York, 2012 Embryonic stem cells nationallicence Differentiation nationallicence microRNA nationallicence Pancreatic progenitors nationallicence Pdx1 nationallicence Purification nationallicence Diabetes mellitus nationallicence Diekmann Ulf Institute of Clinical Biochemistry, Hannover Medical School, 30625, Hannover, Germany aut Elsner Matthias Institute of Clinical Biochemistry, Hannover Medical School, 30625, Hannover, Germany aut Fiedler Jan Institute of Molecular and Translational Therapeutic Strategies (IMTTS), Hannover Medical School, Hannover, Germany aut Thum Thomas Institute of Molecular and Translational Therapeutic Strategies (IMTTS), Hannover Medical School, Hannover, Germany aut Lenzen Sigurd Institute of Clinical Biochemistry, Hannover Medical School, 30625, Hannover, Germany aut Naujok Ortwin Institute of Clinical Biochemistry, Hannover Medical School, 30625, Hannover, Germany aut Stem Cell Reviews and Reports Springer US; http://www.springer-ny.com 9/5(2013-10-01), 555-568 1550-8943 9:5<555 2013 9 12015 https://doi.org/10.1007/s12015-012-9416-1 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1007/s12015-012-9416-1 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Diekmann Ulf Institute of Clinical Biochemistry, Hannover Medical School, 30625, Hannover, Germany aut NATIONALLICENCE 700 1- Elsner Matthias Institute of Clinical Biochemistry, Hannover Medical School, 30625, Hannover, Germany aut NATIONALLICENCE 700 1- Fiedler Jan Institute of Molecular and Translational Therapeutic Strategies (IMTTS), Hannover Medical School, Hannover, Germany aut NATIONALLICENCE 700 1- Thum Thomas Institute of Molecular and Translational Therapeutic Strategies (IMTTS), Hannover Medical School, Hannover, Germany aut NATIONALLICENCE 700 1- Lenzen Sigurd Institute of Clinical Biochemistry, Hannover Medical School, 30625, Hannover, Germany aut NATIONALLICENCE 700 1- Naujok Ortwin Institute of Clinical Biochemistry, Hannover Medical School, 30625, Hannover, Germany aut NATIONALLICENCE 773 0- Stem Cell Reviews and Reports Springer US; http://www.springer-ny.com 9/5(2013-10-01), 555-568 1550-8943 9:5<555 2013 9 12015 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer