title: Nonviral genetic modification mediates effective transgene expression and functional RNA interference in human mesenchymal stem cells.
abstract: BACKGROUND
  Human mesenchymal stem cells (hMSC) are increasingly the focus of both basic and clinical research due to their ability to strike a balance between self-renewal and commitment to mesodermal differentiation. However, the promising therapeutic utility of hMSC in regenerative medical approaches requires detailed knowledge about their molecular characteristics. Therefore, genetic modification of hMSC provides a powerful tool to understand their complex molecular regulation mechanisms.
  
  
  METHODS
  Here we describe a proof of concept approach of separate and combined gene transfer and gene silencing by nonviral DNA transfection of enhanced green fluorescent protein (EGFP) and EGFP-targeted small interfering RNAs (siRNAs) in hMSC. For optimization of nonviral DNA and siRNA transfer different liposomal-based transfection strategies were validated.
  
  
  RESULTS
  The highest fraction of EGFP-expressing hMSC was obtained using Lipofectamine 2000 (50%) which also mediated the highest transfection rates of siRNAs into hMSC (>or=92%). Stably EGFP-expressing hMSC maintained their proliferation capacity paired with the ability to differentiate into different mesodermal lineages (bone, cartilage, and fat) without loss of transgene expression. Based on our nonviral nucleic acid delivery technique we showed efficient, functional, and long-term RNA interference (RNAi) in hMSC by gene specific knock-down of transiently and stably expressed EGFP (88-98%).
  
  
  CONCLUSIONS
  This is the first demonstration of efficient nonviral transfer of both nucleic acids (DNA and siRNA) into hMSC, exhibiting the potential of targeted modification of hMSC. In particular, the combination of these techniques represents a powerful gene transfer/silencing strategy, thus facilitating detailed genetic approaches to study regulatory networks in stem cell differentiation processes.
authors.affiliation: Chirurgisches Forschungslabor, Chirurgische Klinik und Poliklinik-Innenstadt, Ludwig-Maximilians-Universität München, Germany.
authors.forename: Juergen
authors.initials: J
authors.lastname: Hoelters
authors.forename: Marisa
authors.initials: M
authors.lastname: Ciccarella
authors.forename: Marei
authors.initials: M
authors.lastname: Drechsel
authors.forename: Claudia
authors.initials: C
authors.lastname: Geissler
authors.forename: Hülya
authors.initials: H
authors.lastname: Gülkan
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Böcker
authors.forename: Matthias
authors.initials: M
authors.lastname: Schieker
authors.forename: Marianne
authors.initials: M
authors.lastname: Jochum
authors.forename: Peter
authors.initials: P
authors.lastname: Neth
mesh_terms: D018929:Cell Culture Techniques; D002454:Cell Differentiation; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D019793:Fluorescein; D005456:Fluorescent Dyes; D015870:Gene Expression / Q000187:drug effects*; D020868:Gene Silencing; D018014:Gene Transfer Techniques; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000502:physiology*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D019076:Transgenes / Q000187:drug effects*
chemical_list: D005456:Fluorescent Dyes; D034741:RNA, Small Interfering; D049452:Green Fluorescent Proteins; D019793:Fluorescein