Morphological analysis and lentiviral transduction of fetal monkey bone marrow-derived mesenchymal stem cells

Charles C Lee, Donald B. Kohn, Jason E. Ekert, Alice F Tarantal

Research output: Contribution to journalArticle

41 Scopus citations

Abstract

We explored the transduction kinetics of HIV-1-derived lentiviral vectors containing the CMV, EF1α, or PGK promoter expressing EGFP in fetal rhesus monkey bone marrow-derived mesenchymal stem cells (rhMSC). Studies included the effects of transduction (MOI 0-100) on growth, cell cycle, and differentiation toward an osteogenic lineage. Flow cytometric analysis indicated an approximate 8- to 10-fold greater quantity of EGFP-expressing rhMSC when cells were transduced with the CMV or EF1α promoter compared to PGK, although quantitative PCR revealed no differences at the DNA level. The CMV promoter initially expressed 10- to 100-fold higher levels of EGFP compared to EF1α or PGK, respectively, at increasing MOI, although a significant decline in transgene expression was observed posttransduction and with advancing passage (P < 0.01), whereas a significant increase in the level of expression was observed over time with the EF1α promoter. At an MOI of 100, a transient arrest at the S phase of the cell cycle was observed for both vector constructs. Transduced rhMSC differentiated toward an osteogenic lineage comparable to untransduced rhMSC and showed equivalent levels of alkaline phosphatase activity. These findings suggest that the SIN HIV-1-derived lentiviral vectors used in these studies can efficiently transduce rhMSC in vitro (CMV > EF1α > PGK) without inhibiting differentiation potential, although the cell cycle was transiently altered at high MOI.

Original languageEnglish (US)
Pages (from-to)112-123
Number of pages12
JournalMolecular Therapy
Volume9
Issue number1
DOIs
StatePublished - Jan 2004

Keywords

  • Cell cycle
  • EGFP
  • Lentiviral vector
  • MSC
  • Rhesus monkey

ASJC Scopus subject areas

  • Molecular Biology

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