title: Quantitative polymerase chain reaction as a reliable method to determine functional lentiviral titer after ex vivo gene transfer in human mesenchymal stem cells.
abstract: BACKGROUND
  Human mesenchymal stem cells (hMSCs) are a promising target for ex vivo gene therapy and lentiviruses are excellent gene transfer vehicles in hMSCs since they achieve high transduction rates with long-term gene expression. Nevertheless, senescence of hMSCs may limit therapeutic applications due to time-consuming cell selection and viral titration. Here, we describe a fast and reliable method to determine functional lentiviral titer by quantitative polymerase chain reaction (qPCR) after highly efficient ex vivo gene transfer in hMSCs.
  
  
  METHODS
  Lentivirus production was tested with different types of packaging systems. Using p24 ELISA remaining viral particles were detected in the cell culture supernatant. The lentiviral gene transfer efficiency was quantified by FACS analysis. Lentiviral titers were determined by qPCR of expressed transgenes.
  
  
  RESULTS
  Third-generation self-inactivating vectors showed highly efficient gene transfer in hMSCs. No viral antigen was detected in the cell culture supernatant after four media changes, suggesting the absence of infectious particles after 4 days. We observed a linear correlation between virus dilution and level of transgene expression by qPCR analysis, therefore allowing viral titering by quantification of transgene expression. Finally, we demonstrated that transduced hMSCs retained their stem cell character by differentiation towards adipogenic, osteogenic and chondrogenic lineages.
  
  
  CONCLUSIONS
  Quantification of transgene copy numbers by qPCR is a fast and reliable method to determine functional lentiviral titer after ex vivo gene transfer in hMSCs.
authors.affiliation: Experimental Surgery and Regenerative Medicine, Department of Surgery, Ludwig-Maximilians-University, Munich, Germany.
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Böcker
authors.forename: Oliver
authors.initials: O
authors.lastname: Rossmann
authors.forename: Denitsa
authors.initials: D
authors.lastname: Docheva
authors.forename: Georg
authors.initials: G
authors.lastname: Malterer
authors.forename: Wolf
authors.initials: W
authors.lastname: Mutschler
authors.forename: Matthias
authors.initials: M
authors.lastname: Schieker
mesh_terms: D000956:Antigens, Viral / Q000032:analysis; D002454:Cell Differentiation / Q000187:drug effects; D002478:Cells, Cultured; D004351:Drug Resistance / Q000187:drug effects; D004797:Enzyme-Linked Immunosorbent Assay; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006583:Hexadimethrine Bromide / Q000494:pharmacology; D006801:Humans; D016086:Lentivirus / Q000235:genetics* / Q000302:isolation & purification*; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism* / Q000821:virology*; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods*; D011760:Pyrrolidinones / Q000494:pharmacology; D014161:Transduction, Genetic; D019076:Transgenes; D019065:Virus Assembly / Q000187:drug effects
chemical_list: D000956:Antigens, Viral; D011760:Pyrrolidinones; C420726:blasticidin A; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D006583:Hexadimethrine Bromide