Improved transduction efficiency of human amniotic mesenchymal stem cells using optimized lentiviral vectors

Background: Amniotic mesenchymal stem cells (A-MSC)
are excellent candidates for regenerative medicine since they
are multipotent, easy to isolate, expandable and seem to be
immunoprivileged. In rodents, stable genetic modification with
viral vectors improves A-MSC function. Unfortunately, a single
cycle transduction with standard viral vectors does not achieve
high efficiency in human A-MSC. On the other hand, multiple
transduction cycles or antibiotic-based selection methods may alter the cell phenotype. We hypothesized that the use of lentiviral vectors containing specific regulatory sequences may result in improved transduction efficiency in human A-MSC. Accordingly, we compared two types of third generation lentiviral vectors, one of which containing the optimized sequences for Polypurine Tract (cPPT) and Woodchuck Posttranscriptional Regulatory Element (WPRE).
Methods: Human A-MSC were isolated from amniotic membranes
of human term placenta. The immunophenotype of the cells was determined by fluorescence-activated cell sorting (FACS). To confirm their mesenchymal origin, the ability of A-MSC to
differentiate into adipocytes and osteocytes was tested by RT-PCR
and cytochemistry. The production of both lentiviral vectors was carried out following standard protocols. The cPPT and WPRE sequences were cloned between the LTR sequences. It is known that the cPPT enhances the lentivirus production, whereas the WPRE stabilizes the unspliced RNAs during the transfection. In both vectors, a fluorescent tag (green fluorescent protein – GFP) and a gene encoding for a protein of interest (Insulin-like growth factor I – IGF-I) were also cloned. The number of GFP positive cells was quantified by FACS following a single cycle of transduction. Finally, the expression of IGF-I was assessed with Western blot analysis.
Results: A-MSC were successfully isolated and displayed the
antigen profile typical of bone marrow-derived MSC. Specifically,
they were positive for CD90, CD105, CD73, HLA-ABC e CD117
and negative for HLA-DR, CD34, CD45, CD14, CD80, CD31 and
CD133. Furthermore, A-MSC efficiently differentiated into osteocytes and adipocytes. In particular, at the end of the differentiation protocol into osteocytes, the A-MSC expressed the typical osteogenic genes secreted phosphoprotein 10, cathepsin K and bone sialoprotein and stained positive for Alkaline Phosphatase activity and Von Kossa. After the differentiation protocol into adipocytes, the A-MSC expressed the typical adipogenic genes adipocyte differentiation related protein and peroxisome proliferator-activated receptor gamma and stained positive for Oil Red-O. The transduction efficiency with the standard lentivirus was only 15%. Conversely, with the optimized vector we obtained transduction efficiency as high as 50%. Accordingly, the A-MSC transduced with the optimized vector expressed higher levels of IGF-I protein compared with the A-MSC genetically modified with the standard vector.
Conclusions: We have demonstrated for the first time that, using
lentiviral vectors containing the cPPT and WPRE sequences, it is
possible to efficiently transduce human A-MSC with a single cycle
of transduction.