title: MicroRNA target sites as genetic tools to enhance promoter-reporter specificity for the purification of pancreatic progenitor cells from differentiated embryonic stem cells.
abstract: 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.
authors.affiliation: Institute of Clinical Biochemistry, Hannover Medical School, 30625, Hannover, Germany.
authors.forename: Ulf
authors.initials: U
authors.lastname: Diekmann
authors.forename: Matthias
authors.initials: M
authors.lastname: Elsner
authors.forename: Jan
authors.initials: J
authors.lastname: Fiedler
authors.forename: Thomas
authors.initials: T
authors.lastname: Thum
authors.forename: Sigurd
authors.initials: S
authors.lastname: Lenzen
authors.forename: Ortwin
authors.initials: O
authors.lastname: Naujok
mesh_terms: D016475:3T3 Cells; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002454:Cell Differentiation / Q000235:genetics; D045744:Cell Line, Tumor; D019070:Cell Lineage / Q000235:genetics; D002478:Cells, Cultured; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism*; D018507:Gene Expression Regulation, Developmental; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D018398:Homeodomain Proteins / Q000235:genetics; D006801:Humans; D016086:Lentivirus / Q000235:genetics; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D035683:MicroRNAs / Q000235:genetics* / Q000378:metabolism; D008856:Microscopy, Fluorescence; D010179:Pancreas / Q000166:cytology / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013234:Stem Cells / Q000166:cytology / Q000378:metabolism*; D015534:Trans-Activators / Q000235:genetics; D000097573:Red Fluorescent Protein
chemical_list: D049452:Green Fluorescent Proteins; D018398:Homeodomain Proteins; D008164:Luminescent Proteins; D035683:MicroRNAs; D015534:Trans-Activators; D000097573:Red Fluorescent Protein; C556303:MIRN294 microRNA, mouse; C558265:MIRN302 microRNA, mouse; C084010:pancreatic and duodenal homeobox 1 protein