2.2. Transduction of rDPSCs with DsRed

To facilitate transduction of rDPSC cultures, lentivirus encoding DsRed was generated. Briefly, plasmids pRSV/Rev (Addgene #12253, Watertown, MA, USA), pMDLg/pRRE (Addgene #12251, Watertown, MA, USA), pMD2.g (Addgene #12259, Watertown, MA, USA), and pLenti CMV Neo Dest (Addgene #17392, Watertown, MA, USA) encoding a DsRed open reading frame were mixed at a 1.25:1.25:6.25:6.25 ratio along with polyethylenimine (PEI) (MW, Polysciences, Inc, Warrington, PA, USA) at a 3:1 (PEI:Plasmids) ratio in unmodified DMEM media for 20 min. Media containing the DNA/PEI complexes and supplemented with 5% FBS was then added to 2.5 × 10⁷ HEK293 T/17 cells (ATCC, Manasses, VA, USA) at 24 h post-plating. Cells were incubated with the transfection media for 4 h at 37 °C and 5% CO₂, before being replaced with DMEM media containing 5% FBS. Forty-eight hours after transfection, lentivirus-rich media was removed and centrifuged at 600× g then passed through a 0.45-μm filter to clear cellular debris. To concentrate the lentivirus, the solution was added to Oak Ridge centrifuge tubes, underlaid with 20% sucrose/PBS, and spun for 4 h at 20,000× g [42]. The resulting viral pellet was resuspended in PBS, aliquoted, and stored at −80 °C.

To titer the lentivirus, 2 × 10⁵ HEK293 T/17 cell suspensions in DMEM media containing 5% FBS were mixed with 8 μg/mL hexadimethrine bromide (polybrene) (Sigma-Aldrich, St. Louis, MO, USA) and varying volumes of concentrated lentivirus, before plating to a 6-well tissue culture plate and incubating for 48 h. After media replacement and a further 48 h of culturing, cells were enzymatically harvested with trypsin and fixed in 4% PFA/PBS for 10 min. Transduction efficiency was then determined via flow cytometry, using the excitation/emission (556/586 nm) characteristics of DsRED. The infection titer was calculated for wells displaying 5–30% positive fluorescent population based on established formulaic methods [43].

Transduction of DMEM/F12-cultured rDPSC populations was conducted by exposing 1 × 10⁶ cells suspended in growth media containing 10% FBS and 1% penicillin/streptomycin to 8 μg/mL polybrene and DsRED lentivirus at a multiplicity of infection (MOI) of 10, based on previous efficiency data for rat mesenchymal stem cells (MSCs). Cells were then plated to a T175 culture flask and incubated for 48 h before replacing the media with fresh growth media supplemented with 20% FBS. Cell culture expansion and cryopreservation were then carried out as earlier described. Fluorescent imaging of transduced populations, hence forth designated as rDPSC-R, was performed and overlaid with phase contrast captures to verify population percentages expressing the DsRed.