354. Genetic Modification of Murine Stromal- Derived Cells (MSC) in Suspension Culture as a Model for the Rapid Generation of Transplantable Cytoreagents
Authors: Yung-Wei Pan, Tammy Tien Luoh, Peter Kurre
Journal: Molecular Therapy (2006)

Bone marrow-derived mesenchymal stromal cells (MSC) can contribute to tissue repair after cardiovascular injury, and appear to have immunomodulatory activity in some experimental models. Convenient recovery, propagation, and extensive proliferation characteristics recommend them for a range of cellular therapies, some of which require their stable genetic modification. Others have previously demonstrated that HIV-derived lentiviral vectors efficiently transduce human mesenchymal cells (Zhang XY et al., Mol Ther. 2002 May;5:555–65). However, adherence and ongoing subculture of MSC risk differentiation and may increase phenotypic heterogeneity within the cell product. Our studies explored whether stromal-derived cells can be efficiently transduced without adherence and subculture requirements. Murine (m-) MSC were established from C57BL/6 animals and characterized to ascertain differentiation capacity along adipogenic and osteogenic phenotypes. Cells were transduced with 3rd generation lentiviral vectors expressing GFP from an elongation factor 1 alpha promoter and pseudotyped with vesicular stomatitis virus G (VSV-G) protein. We trypsinized and washed mMSC from subconfluent primary cultures and transduced them in suspension or adherent after replating with subsequent determination of gene transfer, fluorescence intensity, and proviral copy numbers. Results demonstrated that gene transfer in suspension is sensitive to changes in vector- and fibronectin concentration, and routinely exceeded transfer to mMSC exposed in standard adherent culture. We were able to transduce up to 58% of cells after a single 1 hour incubation with vector at multiplicity of infection (MOI) of 10. Consistent with studies in murine fibroblasts, fluorescence intensity tended to increase with MOI. This was mirrored by a rapid rise in proviral copy number (up to 16 copies at MOI 10), determined by real-time PCR. We conducted experiments in mMSC (5th passage) immediately upon thawing, again showing gene transfer in over 50% of target cells. We further confirmed gene transfer rates of up to 35% in mMSC progenitors from day 14 initiation culture - commonly termed CFU fibroblast (CFU-F)- after a single vector exposure (MOI 10) of murine whole bone marrow immediately upon harvest. Gene transfer and CFU-F clonogenicity after transduction in suspension decreased at MOI greater than 3, suggesting VSV-G Env protein toxicity, previously described by others. In conclusion, our studies demonstrated rapid (1 hour), efficient HIV-lentiviral transduction of mMSC in suspension and evaluated important parameters governing the efficiency of gene transfer under these conditions. We suggest that this protocol will be useful in the context of cellular therapy using MSC, other stromal-derived, or endothelial cells as cytoreagents for the production of secreted proteins - as recently described (Bigger BW et al., Gene Ther. 2006 Jan;13(2):117–26.) for coagulation factor IX released from primary hematopoietic cells.