Lentiviral Vector-Mediated Transduction of Human Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) represent a promising cell source for cell-based and gene therapeutic strategies. To study differentiation of mesenchymal stem cells, effective introduction of specific genes of interest into stem cells is a critical step. Objectives: To determine the efficiency of gene transduction of various mesemchymal stem cells using a lentiviral vector. Methods: MSCs of different tissue origins -- human bone marrow (hBMMSCs), dental pulp (hDPSCs), periodontal ligament (hPDLSCs), apical papilla (hSCAP), as well as ferret dental pulps (fDPSCs), were isolated and grown in appropriate culture media. Expression of CD14, CD34, CD45, CD73, CD90 and CD105 were examined with specific antibodies and flow cytometry analysis. NIH3T3 and human gingival fibroblasts (hGFs) were also used for comparison. The lentiviral vector pFUGW carrying the enhanced green fluorescent protein (EGFP) reporter cDNA, the packaging plasmid pCMVR8.2DVPR and the VSV-G protein expression plasmid (pHCMV-G)  were used to produce virus according to our previous studies (Yin et al, 2006). Various MSCs at passages 2-3 were infected with lentiviral vectors (~10⁸ infectious units/ml). Results: CD14, CD34 and CD45 were not expressed by these cells except weakly on hDPSCs and hGFs, whereas CD73, CD90 and CD105 were moderately to highly expressed on these cells except that CD73 was not detected on hPDLSCs and no CD73 and CD90 on fDPSCs. All cell types infected by pFUGW expressed EGFP of various intensities observable 2 d after infection. Qualitative analysis showed the following relative EGFP intensities: hDPSCs>hBMMSCs=hPDLSCs=hSCAPs>fDPSCs=hGFs>NIH3T3. The transduction rates were 87-99% and the percentage of cells expressing high intensity of EGFP varied. There was minimal or no loss of high EGFP expressing cells after cell passaging. Transduced cells also maintained their differentiation capacities. Conclusions: Lentiviral vectors appear to be efficient in transducing various MSCs while not affecting their stem cell properties. (Support: AAE Foundation and NIAMS IRP)