title: Functional genetic targeting of embryonic kidney progenitor cells ex vivo.
abstract: The embryonic mammalian metanephric mesenchyme (MM) is a unique tissue because it is competent to generate the nephrons in response to Wnt signaling. An ex vivo culture in which the MM is separated from the ureteric bud (UB), the natural inducer, can be used as a classic tubule induction model for studying nephrogenesis. However, technological restrictions currently prevent using this model to study the molecular genetic details before or during tubule induction. Using nephron segment-specific markers, we now show that tubule induction in the MM ex vivo also leads to the assembly of highly segmented nephrons. This induction capacity was reconstituted when MM tissue was dissociated into a cell suspension and then reaggregated (drMM) in the presence of human recombinant bone morphogenetic protein 7/human recombinant fibroblast growth factor 2 for 24 hours before induction. Growth factor-treated drMM also recovered the capacity for organogenesis when recombined with the UB. Cell tracking and time-lapse imaging of chimeric drMM cultures indicated that the nephron is not derived from a single progenitor cell. Furthermore, viral vector-mediated transduction of green fluorescent protein was much more efficient in dissociated MM cells than in intact mesenchyme, and the nephrogenic competence of transduced drMM progenitor cells was preserved. Moreover, drMM cells transduced with viral vectors mediating Lhx1 knockdown were excluded from the nephric tubules, whereas cells transduced with control vectors were incorporated. In summary, these techniques allow reproducible cellular and molecular examinations of the mechanisms behind nephrogenesis and kidney organogenesis in an ex vivo organ culture/organoid setting.
authors.affiliation: Biocenter Oulu, Infotech Oulu, Center for Cell Matrix Research, Faculty of Biochemistry and Molecular Medicine, University of Oulu, Oulu, Finland;
authors.forename: Sanna
authors.initials: S
authors.lastname: Junttila
authors.affiliation: Biocenter Oulu, Infotech Oulu, Center for Cell Matrix Research, Faculty of Biochemistry and Molecular Medicine, University of Oulu, Oulu, Finland;
authors.forename: Ulla
authors.initials: U
authors.lastname: Saarela
authors.affiliation: Biocenter Oulu, Infotech Oulu, Center for Cell Matrix Research, Faculty of Biochemistry and Molecular Medicine, University of Oulu, Oulu, Finland;
authors.forename: Kimmo
authors.initials: K
authors.lastname: Halt
authors.affiliation: Biocenter Oulu, Infotech Oulu, Center for Cell Matrix Research, Faculty of Biochemistry and Molecular Medicine, University of Oulu, Oulu, Finland;
authors.forename: Aki
authors.initials: A
authors.lastname: Manninen
authors.affiliation: Biocenter Oulu, Infotech Oulu, Center for Cell Matrix Research, Faculty of Biochemistry and Molecular Medicine, University of Oulu, Oulu, Finland;
authors.forename: Heikki
authors.initials: H
authors.lastname: Pärssinen
authors.affiliation: Functional Genomics Center Zurich, University of Zurich/ETH Zurich, Zurich, Switzerland;
authors.forename: M Rita
authors.initials: MR
authors.lastname: Lecca
authors.affiliation: Walter-Brendel-Centre of Experimental Medicine, Ludwig-Maximilians-University of Munich, Munich, Germany; and.
authors.forename: André W
authors.initials: AW
authors.lastname: Brändli
authors.affiliation: Rangos Research Center, Children's Hospital of Pittsburgh, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania.
authors.forename: Sunder
authors.initials: S
authors.lastname: Sims-Lucas
authors.affiliation: Biocenter Oulu, Infotech Oulu, Center for Cell Matrix Research, Faculty of Biochemistry and Molecular Medicine, University of Oulu, Oulu, Finland;
authors.forename: Ilya
authors.initials: I
authors.lastname: Skovorodkin
authors.affiliation: Biocenter Oulu, Infotech Oulu, Center for Cell Matrix Research, Faculty of Biochemistry and Molecular Medicine, University of Oulu, Oulu, Finland; seppo.vainio@oulu.fi.
authors.forename: Seppo J
authors.initials: SJ
authors.lastname: Vainio
mesh_terms: D000818:Animals; D055419:Bone Morphogenetic Protein 7; D016222:Fibroblast Growth Factor 2; D051858:Forkhead Transcription Factors / Q000378:metabolism; D018390:Gene Targeting; D018014:Gene Transfer Techniques; D007668:Kidney / Q000196:embryology*; D008648:Mesoderm / Q000502:physiology*; D051379:Mice; D013234:Stem Cells / Q000502:physiology*
chemical_list: C525819:BMP7 protein, human; D055419:Bone Morphogenetic Protein 7; D051858:Forkhead Transcription Factors; C489562:Foxd1 protein, mouse; D016222:Fibroblast Growth Factor 2
keywords: ex vivo organogenesis; kidney development; nephron segmentation; organ; reconstruction; renal primary cells; viral RNAi